CN106755100A - The genomes of controllability HIV 1 target editing system and its targeting carrier system - Google Patents

Abstract

The present invention relates to a kind of genomes of controllability HIV 1 targeting editing system, including the multiple gRNA expression plasmids of spCAS9 enzymes collocation；Expression of the pUC pUC to spCAS9 with the addition of control element；Targeting sequences of the multiple gRNA for the encoder block genome sequence design of the genomes of HIV 1 and/or CCR5.To improve its targeting, the liposome element auxiliary that above-mentioned targeting editor pUC pUC has installed multiple target spot additional is delivered to the positive cell of CD4, CCR5 and/or CXCR4；To reduce the Non-specific integration to other cells, the plasmid of the system only exists in HIV 1 and can be integrated when active；Moreover, it is achieved that the expression of the CAS9 controlled by TAT protein and R element could only start in the presence of the live viruses of HIV 1, expression is then closed after virus lays dormant or inactivation, reduce the potentially danger to target cell.

Description

Controllability HIV-1 genomes target editing system and its targeting carrier system

Technical field

The invention belongs to genome editing technique field, and in particular to the genome editor of HIV-1 mediations and pDNA medicines Targeted delivery system.

Background technology

Now with the progress of the targeting operating technology of gene, remove external source infection viral genome or be adapted from body The dream of interior some deleterious genes is increasingly becoming reality, such as TALEN technologies and CRISPR technologies.The two respectively has advantage and disadvantage, than As TALEN technology miss rates are very low, the DNA element for being limited to targeting is larger, beyond the capacity of majority carrier, thus is difficult to Multiple target practice；And CRISPR technologies carry multiple gRNA only with a nucleic acid cleaving enzymatic CAS9, it is possible to achieve many target position are cut Cut, but at present there is a shortcoming in CRISPR, that is, and effect of missing the target, the cutting to non-specific sites sequence can cause larger Potential danger.SpCAS9 is bacterium (streptococcus pyogenes) albumen, and its long-term expression in human body cell will be made to body genome Into the risk that the immunizing antigen inside certain threat and active cell is offered.Or needed when gene target medicine is made To express in short-term, or realizing controlled expression, and the strategy expressed in short-term be realized, it is possible to use plasmid DNA vectors technology, in machine Make circles expression in vivo, expression time is short in the cell for the plasmid of the method, the cutting effect of spCAS9 can be influenceed, to this Need to improve plasmid RT in the cell.Although occurring in that some episome (episome) technologies have this energy at present Power, but the episome for being used at present both from virus, it is necessary to express some virus proteins to maintain episomal duplication, but These virus proteins have larger potential hazard to cell, such as, with certain carcinogenic potential, greatly limit it clinical Use scope.There is the expression of stabilization for this integrated plasmid, if realizing that controllability is expressed, CAS9 cutting effects will be solved Not enough shortcoming.But integrated plasmid generally requires specific enzyme to mediate, be typically transferred to using virus-mediated at present and Integrate, using viral carrying DNA to intracellular, there is efficient stable, but there is also cell nonspecific infection, base Lack because the antigenicity of a group random integration, virus is high, viral infection cell-specific is low, viral production is cumbersome, be difficult accumulating etc. Point.Expression of the virus of random integration to neighbouring gene is interfered, and causing the expression of gene increases, is mutated or inactivates, and is facing Certain risk is faced on bed, limitation threshold is of a relatively high.Such as adeno-associated virus AAV is imitated to the site-directed integration in AAVS1 sites Rate is very low, and other are viral, such as slow virus, the characteristics of do not have site-directed integration substantially.

In view of the carrying shortcoming of above-mentioned pDNA medicines, targeting modification and cutting for some special genes, existing skill Art cannot realize site-directed integration, then we can contemplate the medicine loading and integration for reducing non-specific cell, only will be special Property integrate be confined to specific cells group, be so greatly lowered occur Non-specific integration cell context, reduce correlation The danger of medicine.Such as, for the targeting CRISPR elements of AIDS virus HIV-1, if realized only in the thin of virus infection Integrated in born of the same parents, then can just substantially reduce the harm to non-infected cell.Virus-mediated is selectively targeted, at present can be right The capsid protein of virus makees the modification of targeting engineering, it is had affinity to some cell surface receptors, right so as to realize The cell for expressing special receptor is integrated.But the capsid protein gp120 that HIV-1 is combined to CD4, CCR5 and CXCR4 is in patient In be possible to that neutralizing antibody occurs, and the coated virion of capsid protein of HIV-1 itself is more fragile, is grasped intolerant to purifying Make and preserve, it is limited to a certain extent is used for the virus-mediated DNA inputs of targeting and integrates.Therefore people employ Capsid protein VSVG from bubble type Stomatovirus, not only increases stability and also enhances appeal and security, but by This is lost to the specific of CD4 positive cells infection and greatly reduces the lymphoid stem cell positive to some CXCR4 Dip-dye ability, and can have certain dip-dye energy to above-mentioned T cell using the coated slow virus of measles virus MV capsid proteins Power, but there is larger Immunoresistance to the viral capsid proteins in crowd, limit its extensive use.And suitable one Part HIV-1 viruses can enter lymphatic system, infection CXCR4 positive T lymphoid progenitor cells, especially to the later stage bounce-back rank of infection Section, it is hiding and constantly to blood supplement and releasing virus particle shelter it that these CXCR4 positive lymphocyte turns into virus Ground, and traditional chemicals is also unable to do what one wishes to this.In this regard, the CRISPR targetings of the genome targeting cutting for HIV-1 Element, develops a kind of cell that can only integrate those HIV-1 positives and security carrier technique again high, will be with important Application value.

In addition to the pDNA targeting inputs to specific cells monoid, also there is the expression of controllability further to increase its peace Quan Xing.In this regard, people devise the expression control element of various controllabilitys or inductivity at present, such as induced using tetracycline Expression system, estrogen inducible expression, interferon-induced expression system, moulting hormone inducible expression, Cumate are lured Expression system, FK506/ rapamycins inducible expression and RU486 inducible expressions etc. are led, above-mentioned inducible system respectively has excellent Shortcoming.Only biocompatibility is preferable, the less inducible system of toxic and side effect just has a Clinical practice value, such as, tetracycline is lured Expression system is led, is induced using tetracycline and the like, the expression to gene can be just realized in relatively low concentration range (Tet-ON) or (Tet-OFF) is closed, and the tetracycline of low concentration and the like is to the biocompatibility and peace of human body Full property is higher, and the system has larger clinical practice potentiality.In addition, new, the safe inducible expression of exploitation also has There is important application value, the inducible expression of TAT protein and its R control element such as HIV-1, to HIV-1 sun Property cell gene target operation have certain application value.R element is to be present in viral genome LTR (long terminal repeat, LTR) the preceding paragraph DNA sequence dna, the RNA of the sequential coding is exactly in its rna plymerase ii Initiation complex faces position, can form a kind of special higher structure TAR, what the special RNA structures can be encoded with HIV-1 Albumen TAT is combined, and controls the translation of viral gene to extend, using the DNA control elements and TAT protein, it is possible to achieve to gene Controlling expression, be applied to the gene expression of HIV-1 positive cells.

Because DNA or RNA etc. belongs to macromolecular substances, and the means of traditional small-molecule drug delivering then cannot be accurate Efficiently be delivered to it is intracellular, if without virus-mediated channel genes, needing the new non-viral carrying technology of exploitation to use In Intracellular delivery, this is accomplished by special carrying carrier and targeted molecular technology, and this is a key point of such medicine. And the delivering of pDNA/RNA medicines is carried out using liposome nanometer technology or non-liposomal cation nanometer technology, presently, there are The shortcoming that targeting efficiency is low, cell absorptivity is low, circles DNA/RNA half-life shorts, expression efficiency are low, especially cation Nano particle technology, also has the shortcomings that liver renal toxicity higher, it is necessary to make technical further raising and optimization.But its Have the advantages that safe, cost of manufacture relatively it is viral it is relatively low, beneficial to industrially scalable manufacture, accumulating can be freezed, because of this A little medicines have the advantages that clinical threshold is low, room for improvement is big, easy large-scale commercial production with respect to viral vector.

It is main using virus, liposome and nanometer polymerization currently for the conveying of the mcroorganism molecule such as DNA, RNA and albumen The means such as thing.The good characteristic that wherein good biocompatibility of liposome, useful load are big, toxicity is low, technology is more ripe, at present Develop various liposomes targeting carrying methods, including temperature sensitive type, immunologic pattern, pH responsive types, PEG modification long circulating liposome, Flexible lipidosome etc..The above-mentioned type liposome respectively has its advantage, and reality is increased using often several technical methods are used in combination The good characteristic of stuffing plastid.The cell-targeting that the liposome delivery technique is applied to the macromoleculars such as the DNA of non-viral mediation is defeated Send, with larger technology development space and application potential, be expected to be used for the targeting conveying of related drugs molecule.

The content of the invention

Regarding to the issue above, the present invention is intended to provide one kind can realize specific cutting, controlled expression, safely and effectively Multiple gene targeting system, is that the gene therapy of the virus types such as AIDS infectious disease lays the foundation.

To achieve the above object, present invention firstly provides a kind of controllability HIV-1 genomes targeting editing system.

A kind of controllability HIV-1 genomes targeting editing system, including utilization high specific spCAS9 (spCAS9-HF1, SpCAS9 containing N497A/R661A/Q695A/Q926A mutation) (Nature.2016Jan 28；529(7587):490-5.doi: 10.1038/nature16526.) or other spCAS9 editing enzymeses modified version expression plasmid, the control system can answer For other editing enzymeses, such as cpf1 classes, others C2C2 classes, C2C1 classes, the editing enzymes of CASx and CASy classes, collocation gRNA expression The combination of plasmid, to reduce potential Non-specific integration；It is potential non-specific to reduce using the gRNA technologies of 17-19nt Property targeting cutting power (Nat Biotechnol.2014Mar；32(3):279-84.doi:10.1038/nbt.2808.).

The targeting editing system can integrator gene group, its integration to genome depends on the HIV-1 of cell infection Live virus；

Expression of the targeting editing system to spCAS9/spCAS9-HF1 with the addition of control element, make the table of Cas9 enzymes Up to or by HIV-1 promoter and TAT protein regulating and expressing, or by the strong promoter and R element of built-in mammal The chimeric promoters of composition control it to express, or by built-in strong mammalian promoters and TetO/TetR systems constitute it is embedding Promoter control table is closed to reach；

Expression of the targeting editing system to multiple gRNA uses the promoter of mammal；The multiple gRNA refers to The encoder block genome sequence for being directed to HIV-1 genomes and/or CCR5 design targeting sequence.

SpCAS9 the or spCAS9-HF1 expression plasmids will start containing HIV-1 genomes controlling element, HIV-1 expression The fragment of element, genome conformity element and spCAS9/spCAS9-HF1 expression cassettes is inserted into carrier；The Insert Fragment according to It is secondary to contain 5 '-LTR, packaging signal Ψ, spCAS9/spCAS9-HF1 expression cassette, RRE, cPPT and 3 '-LTR elements；Described 5 '- LTR elements have been sequentially connected FIV U3 elements, R element and U5 elements, the 3 '-LTR elements be sequentially connected FIV U3 elements, R element and U5 elements；

Be inserted into fragment containing multiple gRNA expression cassettes in carrier by the multiple gRNA expression plasmids；The insertion piece Section is the one kind in following two：One, Insert Fragment contains 5 '-LTR, packaging signal Ψ, RRE, cPPT, multiple gRNA tables successively Up to frame and Sin3 ' LTR elements；Two, Insert Fragment contains ITR, multiple gRNA expression cassettes, ITR, REP expression cassette successively；It is described 5 '-LTR elements have been sequentially connected FIV U3 elements, R element and U5 elements；The multiple gRNA expression cassettes contain and are directed to respectively The gRNA expression cassettes in the U3 areas, protease P ro code areas and REV protein-coding regions of HIV-1, each gRNA expression cassette is by difference Promoter control respectively；Sin3 ' the LTR elements have been sequentially connected Δ U3 elements, R element and U5 elements, the Δ U3 units Part is the U3 areas for losing transcripting starting ability.

Further, spCAS9/spCAS9-HF1 and gRNA is expressed respectively on two kinds of expression plasmids, or in same table It is co-expressed on up to plasmid, the co-expression plasmid is to insert the fragment of spCAS9/spCAS9-HF1 and multiple gRNA expression cassettes Between unique 5 '-LTR and 3 '-LTR on to same carrier；The U3 that the 5 '-LTR originate containing FIV, the 3 '-LTR contain The U3 or Δ U3 in FIV sources.

Further, in spCAS9/spCAS9-HF1 expression plasmids and/or gRNA expression plasmids or co-expression plasmid also One or several in elements below can be added：RRE, cPPT, wPRE element；Eukaryotic promoter can with R element composition Regulation and control chimeric promoters, the controllable chimeric promoters of eukaryotic promoter and TetO sequences composition, TetR expression cassettes, S/MAR units Part and insulator Ins elements.

Further, the eukaryotic promoter includes that EF1a promoters, actin promoters, Ubc promoters, PGK start Son.

Further, in the sequence containing Seq ID No.1-5 in ordered list in the multiple gRNA expression vectors At least 2 kinds.

Further, the spCAS9 expression plasmids include following plasmid：p1、p7；The gRNA expression plasmids are included such as Lower plasmid：p5；The spCAS9, multiple gRNA co-expression plasmids include following plasmid：P6-1, p6-2 and p6-3, described p6- Seq ID No.21 in 35 heavy gRNA coded sequences such as sequence table；Seq ID No.7 institutes in the sequence of the p1 such as sequence table Show, the sequence of p5 as shown in Seq ID No.20 in sequence table, the sequence of p6-1 as shown in Seq ID No.8 in sequence table, p7 Sequence as shown in Seq ID No.9 in sequence table.

Above-mentioned targeting editing system can be infected with liposome, cationic nano-grain or lentivirus mediated to HIV-1 Cell in delivered, the delivering of wherein lentivirus mediated can be the slow virus of HIV-1 integrase inactivation types, such as Yin Jiyin The HIV-1 integrases that the site such as missing or D64/D116/E152 occurs point mutation and inactivates, it is to avoid non-specific gene group is whole Close, only to the genome conformity containing HIV-1 live virus cells.

Thus, present invention also offers a kind of targeting carrier system for conveying above-mentioned targeting editing system, the load Fortune system is liposome or slow virus.

The slow virus is that the targeting editing system is arranged in pairs or groups with the second generation or third generation slow virus packaging system, Carry out slow virus packaging；The integrase inactivation of the slow virus system is lacked, and its integration to genome depends on cell The HIV-1 live viruses for being infected；Described packaging virus capsid protein is VSVG, or is measles virus (Measles Virus) the capsid protein in source, or be sindbis alphavirus (Sindbis Virus) and the capsid of other viral sources Albumen.The targeting editing system of the lentivirus mediated can be used to prevent the genome conformity of the cell to being uninfected by HIV-1, and energy Enough integration that genome is carried out under the auxiliary of HIV-1 infection viruses.

A kind of liposome for conveying above-mentioned targeting editing system, is mainly prepared from the following components：

DSPC (DSPC)：Cholesterol：Polypeptide-PEG- phosphatide mass ratio=20-70:10-30: 0.0001-2；

Or, DPPC (DPPC)：DSPC (DSPC)：1,2- dioleoyl phosphorus Phosphatidylcholine (DOPC)：Cholesterol：Polypeptide-PEG- phospholipid molar ratio=5:0.5-2:0.5-3:0.5-2:0.0001-0.5.

Further, the polypeptide includes anti-CD4 polypeptides (being named as aCD4), anti-CCR5 polypeptides (being named as aCCR5), resists One or more combination in CXCR4 polypeptides (being named as aCXCR4).Due to HIV-1 viruses be generally divided into CCR5 preferendums and CXCR4 preferendums, wherein infection late viral often CXCR4 preferendums occurs after morphing, cause virus to appear in the CXCR4 positives Cell, such as T lymphocyte stem cells, myeloid cell and macrophage, therefore it is directed to the targeting of the positive cells of HIV-1 in design , it is necessary to consider targeting of the design for CXCR4 when target.Therefore the targeting point for CD4, CCR5 and CXCR4 is devised Son.

Further, anti-CD4 polypeptides are one section of amino acid sequence of the variable region from antiCD4 mAb ST40, by excellent Change transformation to obtain, sequence is Seq ID No.10 in sequence table：ARRPKFYRAPYVKNHPNVWGPWVAYGP.

Further, anti-CCR5 polypeptides come from gp120V2 areas, can combine CCR5, and sequence is：ASTTTNYT.

Further, anti-CXCR4 small peptides change structure from T140, and containing alpha-non-natural amino acid modification, sequence is Seq in sequence table ID No.11：H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂, wherein 2 cysteines carry out cyclisation treatment in building-up process.

Further, polypeptides in combination mode is that the series connection of two or more polypeptides is synthesized into a double target spot polypeptides or many targets Point polypeptide；Combination including aCD4 and aCCR5：aCD4_CCR5、aCCR5_CD4；The combination of aCD4 and aCXCR4：aCD4_ CXCR4、aCXCR4_CD4；The combination of aCCR5 and aCXCR4：aCCR5_CXCR4、aCXCR4_CCR5；

The sequence of the aCD4_CCR5 is Seq ID No.12 in sequence table：H- ARRPKFYRAPYVKNHPNVWGPWVAYGPSASTTTNYT-NH₂, wherein its amino can be put into left-hand end or right-hand end,

The sequence of the aCCR5_CD4 is Seq ID No.13 in sequence table：H- ASTTTNYTSARRPKFYRAPYVKNHPNVWGPWVAYGP-NH₂；

The sequence of the aCD4_CXCR4 is Seq ID No.16 in sequence table：Ala-Arg-Arg-Pro-Lys-Phe- Tyr-Arg-Ala-Pro-Tyr-Val-Lys-Asn-His-Pro-Asn-Val-Trp-Gly-Pro-Trp-Val-Ala-Tyr- Gly-Pro-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂,

The sequence of the aCXCR4_CD4 is Seq ID No.17 in sequence table：H-Arg-Arg-Nal-Cys-Tyr-Cit- Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg-Ala-Arg-Arg-Pro-Lys-Phe-Tyr-Arg-Ala-Pro- Tyr-Val-Lys-Asn-His-Pro-Asn-Val-Trp-Gly-Pro-Trp-Val-Ala-Tyr-Gly-Pro-NH₂,

The aCCR5_CXCR4 polypeptide sequences are Seq ID No.14 in sequence table：H-Arg-Arg-Nal-Cys-Tyr- Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg-Ser-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr- NH₂,

The sequence of the aCXCR4_CCR5 is Seq ID No.15 in sequence table：Ala-Ser-Thr-Thr-Thr-Asn- Tyr-Thr-Ser-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂；

The polypeptide aCD4, aCCR5, aCXCR4, aCD4_CCR5, aCCR5_CD4, aCD4_CXCR4, aCXCR4_CD4, ACCR5_CXCR4, aCXCR4_CCR5 and PEG-PE conjugate may be used alone or in combination when liposome is made and uses；It is preferred that It is applied in combination, to target more HIV-1 infected cells.

The polypeptide aCD4, aCCR5, aCXCR4, aCD4_CCR5, aCCR5_CD4, aCD4_CXCR4, aCXCR4_CD4, ACCR5_CXCR4, aCXCR4_CCR5 are using artificial synthesized.

Further, polypeptide is recombinant polypeptide, and expressed sequence inserted into expression vector, and expressed in Host Strains, extracted, Purifying.

Further, the recombinant polypeptide includes：Anti- CD4 recombinates scFV recombinant polypeptides (abbreviation aCD4scFV), its nucleic acid Coding expressed sequence be sequence table in Seq ID No.18, its encoding amino acid sequence be sequence table in Seq ID No.21 (contain E.coli Pel B guide peptide).

Further, aCCR5 expressed sequences are added in the upstream of aCD4scFV, obtains aCCR5_CD4scFV fusions many Peptide, the nucleic acid of the coding expressed sequence is Seq ID No.19 in sequence table, and amino acid sequence is Seq ID in sequence table No.22.The sequence can also add (His) in C-terminal₆Label.

Further, the coupling that polypeptide can be between PEG molecules is using the one kind in following methods：(1) carboxyl-ammonia is utilized Condensation reaction between base, the PE-PEG-COOH molecules that will contain are in catalyst n-HOSu NHS and carbodiimide (EDC) under catalytic reaction and containing-NH₂The polypeptide condensation reaction of group, forms amido link；(2) processed using maleimide Polypeptide and PE-PEG-SH react, or with the maleimide-PEG-PE with maleimide base group and carry in turn The polypeptide of sulfydryl modification carries out the conjugate that Michael addition reaction forms sulfide based structural；(3) using band p-nitrophenyl carbonic ester The PE-PEG-pNP of base (p-nitrophenylcarbonyl, pNP) reacts in the basic conditions with the amino of polypeptide, reacts shape Into the mephenesin Carbamate key conjugate of stabilization；Above-mentioned active group is on PEG molecules, therefore polypeptide is directly coupled at PEG points On son.

Further, selected from one or more in phosphatidyl-ethanolamine (PE) or its derivative, described spreads out phosphatide Biology includes DSPE (DSPE), DOPE (DOPE) and two palmitic acid phosphatidyl second Hydramine (DPPE).

Further, the molecular weight of the PEG (polyethylene glycol) is 200-10000, preferably 200-3000.Use PEG pairs Surface of liposome carries out modification and can improve the half-life period of liposome, increases the stability of epicyte protein liposome, reduces and adjusts Reason is acted on, and reduces the phagocytosis of reticuloendothelial system.

Further, penetrating peptide can be also coupled on PEG, be formed " penetrating peptide-PEG- phosphatide ", with target spot polypeptide-PEG-PE Collocation is used；Penetrating peptide-PEG- phosphatide is 0.0001-2 with the mol ratio of other polypeptides-PEG- phosphatide:1.The penetrating peptide contains There are the 6-12 amino acid residue of positively charged, such as sequence：H-TyrGlyArgLysLysArgArgGlnArgArgArg-NH₂, with Above-mentioned target spot polypeptide-PEG- phosphatide collocation is used.Penetrating peptide increased the fusion faculty of liposome and cell.

Further, also add the phosphatide of hyaluronic acid in liposome components, the phosphatide of the hyaluronic acid and its The mol ratio of his polypeptide-PEG- phosphatide is 0.00001-1：1, the phosphatide of the hyaluronic acid is by maleic acid base group modification Phosphatide (Maleimide- phosphatide) or maleimide-PEG- phosphatide react and to be formed " thoroughly with the hyaluronic acid with sulfydryl modification Bright matter acid-phosphatide " or " hyaluronic acid-PEG- phosphatide ", for above-mentioned target spot polypeptide-PEG- phosphatide and penetrating peptide-PEG- phosphorus Fat collocation is used.The addition of hyaluronic acid-phosphatide or hyaluronic acid-PEG- phosphatide can reduce the immunogenicity of liposome and improve Biocompatibility.

Further, above-mentioned functions polypeptide, can also directly with the coupling of the liposome molecule covalent such as PE, for directly whole It is incorporated into liposome membrane molecular layer.

Further, can be anti-using film dispersion method, reverse evaporation, gradient using above-mentioned liposome components embedding plasmid To stowage and double emulsion etc..

Further preferred film dispersion method, comprises the following steps that：

(1) by DSPC：Cholesterol：Polypeptide-PEG2000-DSPE 20-70 in mass ratio:10-30:1 is made uniform mixing Liquid, or by DPPC, DSPC, DOPC, cholesterol, polypeptide-PEG2000-DSPE in molar ratio 5:1:2:1:0.0001-0.5 is made Uniform mixed liquor；

(2) chloroform is prepared：Methanol solution, chloroform is 1-3 with the volume ratio of methyl alcohol:1；

(3) by one of above two mixed liquor and the 5-500 times of chloroform of volume：Methyl alcohol (1-2:1) solution is mixed, in rotation Evaporation solvent in evaporator, forms thin layer；

(4) by above-mentioned dry thin layer rehydration and plasmid is added, 5-50mM HEPES or Tris-Cl is contained wherein in plasmid (pH7.5-8.5), the concentration of plasmid is in 200ng/ μ l-8000ng/ μ l；

(5) 40-65 DEG C of heating is vortexed and processes 2-6 minutes, repeats 2-5 times, obtains embedding the liposome film bubble of plasmid.

Further, after the completion of film dispersion method prepares liposome membrane bubble, the liposome for obtaining is again by polymeric membrane mistake The methods such as filter, extruding, remove the liposome of greater particle size, obtain the smaller liposome of particle diameter.The specific method of extruding is：By fat Plastid squeezes through the cellulose acetate sheets or polycarbonate membrane in 200nm apertures at 40-65 DEG C, is repeated 5-20 times, with The aperture for reducing liposome and the uniform particle sizes' degree for improving liposome, the unilamellar liposome film bubble for being obtained are dialysed through semi-permeable membrane Overnight purify, or purified by SephadexG-50 posts or similar gels post, to remove free non-integral protein and miscellaneous Matter.Compare the making of liposome, in addition to without cell membrane extract, other steps, technique and content all same.

Further, DSPC：Cholesterol：The mass ratio of polypeptide-PEG2000-DSPE is 50:15:1；DPPC、DSPC、 DOPC, cholesterol, the mol ratio of polypeptide-PEG2000-DSPE are 5:1:2:1:0.02.

Further, also fled from and carefully with improving the endocytosis body of plasmid containing auxiliary ingredients in the plasmid of the step (4) Karyon conveying capacity, the auxiliary ingredients are polyethyleneimine, protamine sulfate, positively charged amino acid polypeptide or cell Promote one or more in fusogenic peptide, wherein promote fusogenic peptide be divided into viral source, bacterial origin and it is artificial change structure source, be divided into pH The polypeptide of sensitive and pH non-sensitive types, the preferably pH sensitive rush fused polypeptide of the present invention.

Beneficial effects of the present invention are：The present invention will coding spCAS9 (or high specific spCAS9-HF1), multiple gRNA Deliver into positive intracellular of CD4, CCR5 and/or CXCR4, rely on the integrase of wild HIV-1, protease, reverse transcriptase, REV and TAT etc. carries out genome conformity and the expression of target cell, it is to avoid the non-selectable gene group of lentivirus mediated is integrated and not Controllability is expressed, and the especially expression of spCAS9 enzymes has obtained controllable.Its control system is divided into R element-TAT protein control system Or Tet-ON control systems, the spCAS9 of controllability avoids the potentially danger to target cell, wherein R element-TAT protein control The spCAS9 of system just lowers or closes the advantage of expression automatically after HIV genomes inactivation.These plasmids can lose with integrase Plasmid collocation living, packs integrase inactivation type slow virus, for the gene delivery of lentivirus mediated.Invention also uses many Weight gRNA technologies, Mutiple Targets cut the key gene group sequence of HIV-1, greatly reduce HIV-1 genomes because single target spot is mutated And produce the possibility of resistance.Designed multiple gRNA have the genome height phase with various domestic common HIV-1 strains Like property, the potential ability with broad spectrum activity.For the Mutiple Targets target polypeptide and Pegylation of CD4, CCR5 and CXCR4 (PEG) coupling of-PE, strengthens to X4 and the targeting of R5 biting property HIV-1 virus infected cells, beneficial to follow-up frozen dried, Reduce immunogenicity.The integration collocation of the PE of the polypeptide-Pegylation of above-mentioned target spot targeting and ratio, do not influence and cell membrane Fusion, increase selectively targeted effect.The PE for adding the hyaluronic acid of proper proportion is conducive to improving the biology of liposome The immunogenicity of compatibility and reduction liposome.

Brief description of the drawings

Fig. 1：G1 sequences and 4 kinds of sequence alignments of HIV-1 strains；

Fig. 2：G2 sequences and 4 kinds of sequence alignments of HIV-1 strains；

Fig. 3：G3 reverse complementary sequences and 4 kinds of sequence alignments of HIV-1 strains；

Fig. 4：G4 reverse complementary sequences and 4 kinds of sequence alignments of HIV-1 strains；

Fig. 5：The composition schematic diagram of plasmid p1-p5 Insert Fragments；

Fig. 6：The plasmid map of plasmid p1；

Fig. 7：The plasmid map of plasmid p5；

Fig. 8：The composition schematic diagram of plasmid p6-1, p6-2 and p6-3 Insert Fragment；

Fig. 9：The plasmid map of plasmid p6-1；

Figure 10：The composition schematic diagram of plasmid p7-p12 Insert Fragments；

Figure 11：The plasmid map of plasmid p7；

Figure 12：The plasmid map of plasmid p9；

Figure 13：The composition schematic diagram of plasmid p13, p14 Insert Fragment；

Figure 14：The plasmid map of plasmid p14；

Figure 15：The composition schematic diagram of plasmid p15 Insert Fragments；

Figure 16：The composition schematic diagram of plasmid p16, p17 Insert Fragment；

Figure 17：Uciferase activity after the cationic liposomal transfection of embodiment 6；

Figure 18：Uciferase activity after the slow-virus transfection of embodiment 6；

Figure 19：Uciferase activity after the cationic liposomal transfection of embodiment 7；

Figure 20：Uciferase activity after the slow-virus transfection of embodiment 7；

Figure 21：The uciferase activity of embodiment 9；

Figure 22：The uciferase activity of embodiment 10；

Figure 23：The uciferase activity of embodiment 12；

Figure 24：The uciferase activity of embodiment 13；

Figure 25：The genome conformity efficiency of embodiment 14；

Figure 26：The virus-mediated genome conformity efficiency of the integrase Inactivating mutations packaging system of embodiment 15 packaging.

Specific embodiment

With reference to specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following implementation Example.In the present invention unless otherwise noted, the commercially available acquisition of raw material, method is the conventional method of this area.

Following instrument, material and method have been applied in embodiments of the invention：

1st, instrument plasmid pLVX_CD4, pLVX_CCR5 and pLVX_CXCR4：For effective analysis of liposomes targeting element Targeting validity, constructs plasmid pLVX_CD4, pLVX_CCR5 and pLVX_CXCR4.Clone CD4 (GenBank：NM_ 000616)、CCR5(GenBank：) and CXCR4 (GenBank NM_000579：NM_001008540 cDNA sequence), inserts respectively Enter pLVX-acGFPN1 carriers (TAKARA, precious biological), and containing terminator codon, to eliminate the expression of acGFP, only maintain insertion The expression of gene.It is steady to turn or transiently transfect Hela cells, Hela cells is obtained the expression of associated receptor, it is easy to detection related Acceptor promotes ability to the targeted fusion of its target liposomes.

2nd, instrument plasmid pLVX_Luc, pTAT, pCCR5_P2A_Luc：Due to pLVX-acGFPN1 carriers contain it is complete The 5 ' of HIV-1 and 3 ' LTR elements, with the potential ability sheared by target practice plasmid, the luciferase of built-in expression can be used for Inspection shear effect.In order to detect the control action of TAT gene pairs R elements, luciferase is inserted pLVX- by us In acGFPN1 carriers, containing terminator codon, terminate the expression of acGFP, structure obtains pLVX_Luc plasmids.By the outer of TAT protein After aobvious subsequence splicing, in insertion pcDNA3.1 plasmids, in the control of CMV promoter and bovine growth hormone gene termination signal sequence The lower expression of system, for corotation, structure obtains plasmid pTAT.In order to detect that CCR5gRNA targets the shear effect of element, our structures PCCR5_P2A_Luc is built, the plasmid is built-up on the basis of pcDNA3.1+ carriers, by the CCR5cDNA encoder blocks of people Downstream fusion P2A autotomys peptide sequence and luciferase, and three forms a reading frame, occurs after CCR5 above is sheared There is frameshift mutation in frameshift mutation, P2A and Luc below, so as to lose the activity of luciferase.

3rd, the cell platform construction of target practice efficiency is detected：In order to simulated infection HIV-1 lymphocyte and be easy to quantify point Analysis, building plasmid p16 and p17 is used to simulate HIV-1 full-length genomes.HIV-1 genomes are inserted into Seq ID in sequence table On the plasmid backbone of No.6, and by encode gp120 most of DNA sequence dna be replaced into luciferase sequence (plasmid p16) or EGFP sequences (plasmid p17), such as Figure 16.Retain whole gp41 sequences in p16 and p17, it is therefore an objective to retain the RRE for overlapping Sequence, the design can eliminate the capacity packing of HIV-1 viruses, eliminate its packaging and appeal, it is ensured that the safety of experimental implementation Property.And marker genes encoding luciferase luciferase (being abbreviated as Luc) and green fluorescent protein eGFP of insertion can be with The quantitative of gene targeting efficiency, qualitative detection are provided.P16 or p17 plasmids are made into slow virus, Hela cells are infected, base is obtained Because of group stable expression cell an integrated clone, for the detection cell platform of target practice efficiency.

The multiple steady structures for turning cell of p16-CD4-CCR5/p17-CD4-CCR5：Slow disease of the packaging containing CD4 sequence plasmids The slow virus of poison, packaging containing CCR5 sequence plasmids is contaminated p16/p17 and surely turns Hela cells and obtain.Further sense on this basis The slow virus of dye packaging CXCR4, can obtain p16-CD4-CCR5-CXCR4/p17-CD4-CCR5-CXCR4 stable cell strains. The puromycin of 0.5-1 μ g/ml is maintained in cell culture fluid, cell total rna, reverse transcription, RT-PCR identifications CD4, CCR5 is extracted With the expression of CXCR4；The Hela cells of non-infection virus are used to identify positive expression cell line as control.It is steady to turn cell choosing Select the standard of cell clone：Vitro growth rates, the vigor of cell and cellular morphology are close with normal Hela, amplify culture and are formed One Cell subline of the stabilization above-mentioned factor of expression.

4th, the packaging of slow virus makes：Plasmid (p1-p17) and the second generation (three pUC pUCs) constructed by the present invention or The third generation (four pUC pUCs) slow virus packaging system cotransfection 293T cells, slow virus packaging step routinely is carried out, and is turned Transfection reagent is lipofectamine 2000, and the culture supernatant containing virus, warp are collected in transfection respectively after 48 hours and 72 hours Cross Purification by filtration, determine the potency of virus, packing freeze -80 DEG C it is standby.

5th, cell culture：Hela cells use the culture of DMEM high glucose mediums (containing dual anti-), the 5- of addition activated carbon treatment 10% high-quality business hyclone, 37 DEG C, 5%CO₂Lower culture.The cracking of cell, the measure of luciferase are according to Promega The explanation of the Luciferase Assay Reagent box of company is operated.Cell transfecting is divided into cationic liposomal transfection method and self-control fat Plasmids method, cationic liposomal transfection uses lipofectamine 2000, and operating procedure is carried out by reagent specification；From The transfection ratio of liposome processed, general 48 orifice plate uses 0.5-2 μ l liposomes/hole.

6th, cationic-liposome：Lipofectamine2000 (Life Technology, article No. 11668-019).

7th, Luciferase Assay Reagent box (Promega, article No. E1910).

Design of the embodiment 1 for AIDS virus HIV-1 genomes and the gRNA sequences of CCR5

The design standard of sequence is as follows：Selection 19-28 nt of length, with the various AIDS strains in China's Mainland prevalence With homology higher, the similarity with human genome is low, at least 4 base differences of more than nt, prevents non-specific Property cutting generation.Selection in addition is directed to the crucial DNA element of HIV-1, including LTR areas, Pol code areas, Rev protein-coding regions Deng position.Wherein there is the sequences such as integration and the rna plymerase ii promoter of important genome in LTR areas, containing a U3 area, RQu He U5 areas, because integrated plasmid needs the presence of RQu He U5 areas, therefore when gRNA is designed, focal selection U3 areas.In It is to devise 2 difference gRNA for HIV-1U3 areas, sequence is respectively：In sequence table in Seq ID No.2 and sequence table Seq ID No.3.A gRNA is also devised otherwise for Rev albumen, sequence is Seq ID No.1 in sequence table.In addition also The gRNA of the protease P ro for Pol code areas is devised, sequence is Seq ID No.4 in sequence table, and the sequence is located at should Pol albumen upstream, once it is mutated and frameshit occurs, it will cause Pro, Pol and the expression cassette of integrase that frameshift mutation occurs. We devise 4 kinds of gRNA (Seq ID No.1-4 in sequence table) altogether, carry out Mutiple Targets for the genome to HIV-1 and beat Target is sheared.We are named as a gRNA targeting sequence is devised near the 186th amino acid codes of CCR5 in addition G5, for targetting the CCR5 genes in editor inactivation target cell, blocking HIV-1 viruses continue the infection and invasion to cell.

Fig. 1, Fig. 2, Fig. 3 and Fig. 4 are respectively the different sub- of 4 kinds of common AIDS virus strains of 4 gRNA sequences and China The genome sequence of strain is compared, wherein bThai, b ' Thai, crf01, crf07 and crf08 be the popular AIDS in 5 kinds of China's Mainlands Virus stain, the character after strain name is clone's number of strain, it was demonstrated that related gRNA sequences are that various country are common Conserved sequence common to AIDS virus, therefore with the potential ability of the targeting various HIV-1 virus stains genomes of cutting.

Seq ID No.1 in sequence table：G1,5 '-GGAATCGAAGAAGGAGG-3 '

Seq ID No.2 in sequence table：G2,5 '-AAACTACACACCAGGGCC-3 '

Seq ID No.3 in sequence table：G3,5 '-GATAGACCCATAAATCA-3 '

Seq ID No.4 in sequence table：G4,5 '-GCCAAAGAGTGATTTGA-3 '

Seq ID No.5 in sequence table：G5,5 '-GAATTGATACTGACTGTA-3 '

(intracellular HIV-1 virus-mediated plasmid is whole for the double-plasmid expression system of the use of embodiment 2 p1, p2, p3, p4, p5 Close and the mediation of TAT protein/R element the isogenic expression plasmid systems of spCAS9) structure

Plasmid backbone is built first, and its sequence is shown in Seq ID No.6 in sequence table.The plasmid backbone is built upon changing structure On pUC19 plasmid basics, the MCS of structure pUC19 is changed again, remain original Ori replication orgins and penicillin Resistant gene.All spCAS9 expression plasmids and multiple gRNA expression plasmids are set on the basis of the plasmid backbone in the present invention Meter.

The MCS of Seq ID No.6 is in plasmid backbone sequence table：

The design of spCAS9 or spCAS9-HF1 expression plasmids p1, p2 and p3：The pUC pUC is used for genes within cells group Integration, it is necessary to host cell in exist activity HIV-1 virus, virus mediation under, genome conformity is carried out, in virus TAT protein and the R element collective effect of its own under, start spCAS9 or spCAS9-HF1 expression.In order to prevent for U3 The gRNA in area is cut to the U3 areas of itself, and U3 areas are replaced with the U3 areas of feline immunodeficiency virus FIV.HIV-1 is also carried in addition The original U5 sequences of virus and packaging signal Ψ, by REV response elements RRE (234nt), cPPT (central polypurine Tract) (124nt) is inserted into the downstream of spCAS9 encoder blocks, obtains plasmid p1.Can also be to increase WPRE elements in plasmid (Woodchuck hepatitis virus post-transcriptional regulatory element, WPRE), is used for Strengthen the expression of gene, enter core and core output, such as plasmid p3.In order to improve the time that the plasmid before integration exists in the cell, Can also be to S/MAR sequences (coming from the genes of people IFN-beta 1) be added in plasmid, to improve its half-life period in the cell, such as Plasmid p2, p3.Plasmid p1, p2 and p3 be coding spCAS9 albumen can integration vector, vector insert composition schematic diagram As shown in figure 5, wherein the sequence of p1 is Seq ID No.7 in sequence table, plasmid map is shown in Fig. 6.

The building process of plasmid p1, p2 and p3 is as follows：Synthesis FIV5 ' Duan U3 areas, then carry out weight with HIV-15 ' ends U3 Folded PCR amplifications, amplification Chong Die with the U3 downstreams of HIV-1 obtains chimeric 5 '-LTR U3 sequences.Will chimeric 5 '-LTR U3 and R- U5- Ψ, RRE fragment mix, and whole FIV is fitted together to 5 '-LTR-R-U5- by the primer with two ends respectively with EcoRI, BamHI site Ψ amplifications turn into a fragment, are inserted into EcoRI, BamHI site of plasmid backbone.Then PCR amplifications two ends carry NLS signals SpCAS9-HF1, insert plasmid backbone AgeI and KpnI sites.Over-lap PCR expand RRE and cPPT sequences, insertion KpnI and XbaI sites；3 '-LTR sequences are identical with 5 '-LTR sequences, then by sequence insertion XbaI and PacI sites, obtain plasmid p1.Plasmid p2 is obtained in the 3 '-LTR downstreams insertion S/MAR sequences of p1 plasmids.Inserted between the cPPT and 3 '-LTR of p1 plasmids WPRE regulating and controlling sequences obtain plasmid p3.The sequence of above-mentioned plasmid and following each plasmids can also all using artificial synthesized Method builds.

The design of multiple gRNA expression plasmids p4 and p5：The pUC pUC is also that the HIV-1 by infecting helps be incorporated into place In key-gene group.The plasmid devises internal promoter, therefore we remove the U3 areas in 3 '-LTR areas, (are lost labeled as Δ U3 Go the autonomous ability for starting transcription), related 3 '-LTR sequence marks for Sin3 ' LTR, 5 '-LTR still using FIV-U3 and The R-U5 sequences of HIV itself.RRE, cPPT with the addition of wPRE and increase using original sequence of virus HIV-1 in multiple gRNA areas Strong element, but in order to prevent the reinforcing element to inserting the interference of point gene, increased insulator sequence Ins (from chicken β- Globin locus HS4 genes), upstream and downstream has been made the insulation processing of correlation.HU6 (the U6 genes from people) and hH1 (the H1 genes from people) promoter is respectively started the expression of g1 and g2, and mU6 (the U6 genes from mouse) and h7SK (come from people SnRNA 7SK genes promoter) promoter is respectively started the expression of g3 and g4.Other hH1-g2 and h7SK-g4 makees respectively Reverse insertion, prevent that expression cassette from causing on same positive-sense strand or antisense strand is interfering with each other.Related plasmids composition is illustrated P4 and p5 in figure such as Fig. 5, wherein p5 are the p4 versions for increasing S/MAR sequences.Fig. 7 is the plasmid map of plasmid p5.

The building process of plasmid p4 and p5 is as follows：By what is be close to after FIV U3-R-U5 sequences and the-LTR of HIV genomes 5 ' Between 1517bp sequences insertion EcoRI and XhoI sites, by between 225bp insulator sequences insertion XhoI and BamHI sites, institute The plasmid for obtaining between hU6-g1-hH1-g2 insertion BamHI and AgeI sites, obtains plasmid process by BamHI and AgeI digestions AgeI and KpnI digestions, are connected into mU6-g3 and h7SK-g4, by between wPRE sequences and ins sequences insertion KpnI and XbaI sites, Sin3 '-LTR are then inserted between XbaI and SpeI sites, identify direction of insertion, choose positive insertion plasmid, are built and are obtained p4 matter Grain.The genome of PCR methods amplification human archeocyte obtains S/MAR sequences, inserts between PacI the and SpeI sites of p4 plasmids, builds Obtain p5 plasmids.

When genome editor is applied to, the plasmid that will encode spCAS9 (or spCAS9-HF1) and coding gRNA is taken two-by-two With using, such as one or more in p1, p2 or p3, with plasmid p4 or/and p5 matched combined.

The structure of the plasmid p6 expression systems of embodiment 3 (spCAS9 or spCAS9-HF1, gRNA are co-expressed in same plasmid) Build

The design of p6 pUC pUCs：The pUC pUC is in same plasmid by spCAS9 and multiple gRNA co expressions On, by intracellular active HIV-1 mediated integrations genome.Wherein spCas9 changes and is started by eukaryotic promoters such as EF1a, wherein Eukaryotic promoter can also be from the isogenic strong promoter of actin, Ubc, PGK, the growth hormone gene of user plus Tail signal.The R element of HIV is added as expression control element, after HIV is inactivated, the iris action of R element will make spCAS9 Expression minimize level be even switched off expression.In order to prevent the expression from viral LTR from disturbing, employ Sin3 ' LTR and set Meter.For Enhanced expressing, wPRE elements are with the addition of, to prevent wPRE elements after integrating from, to the conversion risk of body cell, adding Come from chicken HS4 gene insulator sequence Ins, such as p6-1 plasmids.Simple substance grain sets in the design and implementation example 2 in multiple gRNA areas Meter is identical.In addition, in order to reduce because the long influence caused to viral packaging efficiency of Insert Fragment, Ins and wPRE elements can Removal, such as plasmid p6-2；Multiple gRNA can also extend and increase, such as increase the gRNA for CCR5 genes：G5, is placed in silk floss Under sheep 7SK gene promoters sh7SK, such as plasmid p6-3, the expressed sequence such as Seq ID No.21 of 5 weight gRNA.P6 inserts piece The schematic diagram of section is shown in Fig. 8.

The structure of plasmid p6-1：The structure of 5 ' FIV-U3-LTR, Ψ, RRE and cPPT is identical with p1, SA (splice Acceptor) sequence is removed, and inserts EcoRI the and XhoI sites of plasmid backbone.CHS4 insulator sequences PCR introduce XhoI and BamHI sites, insert XhoI the and BamHI sites of plasmid backbone after digestion.Over-lap PCR expands EF1a-R sequences, inserts BamHI With AgeI restriction enzyme sites.High-fidelity PCR amplification spCAS9-HF1, inserts AgeI the and KpnI restriction enzyme sites of plasmid backbone.hU6- G1, hH1-g2 obtain fragment by the method for synthesizing, and insert KpnI the and XbaI sites of plasmid backbone.Synthesis mU6-g3 and H7SK-g4 fragments, insert XbaI the and SpeI sites of plasmid backbone.Fusion DNA vaccine expands wPRE-Ins-Sin3 ' LTR, inserts matter Between SpeI the and PacI sites of grain skeleton.The sequence of p6-1 is Seq ID No.8 in sequence table, and plasmid map is shown in Fig. 9.P6-2 It is then to eliminate 2 Ins and wPRE sequences, p6-3 plasmids are then that a sh7SK-g5 is increased on the basis of p6-2, this 5 kinds The expressed sequence of gRNA such as Seq ID No.21.

When genome editor is applied to, due to p6 pUC pUCs can express simultaneously spCAS9 (or spCAS9-HF1) and GRNA, therefore can be used alone.

Embodiment 4 uses double-plasmid expression system (HIV-1 mediated integrations, the Tet-ON of p7, p8, p9, p10, p11, p12 Control table reach double-plasmid expression system) structure

The design of expression plasmid p7, p8, p9, p10, p11, p12：The pUC pUC also uses double-mass model pattern and HIV- The genome conformity of 1 mediation, but spCAS9 expression plasmids employ Tet-on inducible systems and carry out spCAS9 or spCAS9-HF1 Control table reach.It is non-viral in eukaryotics such as EF1a or a TetO sequence of 40nt is added in downstreams of viral promotors, and Add the termination signal sequence BGHpA of bovine growth hormone gene.In addition under the startup of the eukaryotic promoters such as PGK, a knot is expressed The albumen TetR of identification TetO is closed, the TetO sequences to the gene of spCAS9 (or spCAS9-HF1) carry out inhibition combination, and Add the termination signal Glo-pA of people's beta-globin genes.PGK, TetR and Glo-pA have made reverse insertion, prevent expression cassette That is caused on same positive-sense strand or antisense strand is interfering with each other.The plasmid equally with the addition of RRE, cPPT, WPRE and Ins insulation Subcomponent, used Sin3 ' the LTR sequences that security is higher, it is to avoid the interference to internal promoters such as EF1a.Obtain plasmid p7.Because the length of TetR protein expression frames is larger, cause the length of plasmid p7 excessive, it is contemplated that to remove TetR expression cassettes, from And obtain plasmid p8.And TetR expression cassettes are put into multiple gRNA expression plasmids and get on to express, such as the expression cassette is inserted into In p4 plasmids.S/MAR elements can be also respectively added on p7, p8 and p9 plasmid in addition, the plasmid before integrating is improved in the cell Holdup time, respectively obtain plasmid p10, p11 and p12.The composition of plasmid p7, p8, p9, p10, p11, p12 Insert Fragment shows Intention is shown in Figure 10.

The building process of plasmid p7 is as follows：The structure of 5 ' FIV-U3-LTR, RRE and cPPT is identical with p1, SA (splice Acceptor) sequence is removed, and inserts EcoRI the and XhoI sites of plasmid backbone.Ins and EF1a-TetO sequences are by overlapping PCR methods are cloned, and two ends are connected into XhoI and BamHI sites.High-fidelity PCR amplification spCAS9, two ends carry NLS coded sequences, insert Enter AgeI the and KpnI sites of plasmid backbone.PGK-TetR-GlobinpA is expanded by fusion DNA vaccine method, upstream insertion NotI Site, downstream introduces KpnI restriction enzyme sites, reversely the KpnI and NotI plasmid enzyme restrictions site of insertion plasmid backbone.BGH-pA is upper and lower Trip introduces KpnI sites, inserts the KpnI sites of plasmid backbone, identifies direction, the positive insertion person of selection.wPRE-Ins- Sin3 ' LTR are expanded by over-lap PCR and connected, and two ends introduce SpeI and PacI sites respectively, insert plasmid backbone SpeI and PacI restriction enzyme sites, sequencing identification.Its sequence is Seq ID No.9 in sequence table, and plasmid map is as shown in figure 11.

The building process of plasmid p9 is as follows：Synthesis FIV5 ' Duan U3 areas, then carry out over-lap PCR expansion with HIV-15 ' ends U3 Increase, amplification Chong Die with the U3 downstreams of HIV-1 obtains chimeric 5 '-LTR U3 sequences.Will chimeric 5 '-LTR U3 and R-U5- Ψ, Whole FIV is fitted together to 5 '-LTR-R- by the PCR fragment mixing of RRE and cPPT, the primer with two ends respectively with EcoRI, XhoI site U5- Ψ, RRE and cPPT amplification turn into a fragment, insert EcoRI, XhoI site of plasmid backbone.Then with being respectively provided with The primer of XhoI and BamHI expands the cHS4 insulators (Ins) of 225bp, and be inserted respectively into plasmid backbone XhoI and BamHI sites.HU6-g1 and hU1-g2 fusion fragments insert BamHI the and AgeI sites of plasmid backbone by PCR method.g3- The sequence of mU6 and h7SK-g4 introduces restriction enzyme site by artificial synthesized using PCR, inserts the AgeI and KpnI of plasmid backbone Site.The expression cassette of TetR, containing PGK, TetR and beta-globin PolyA signal sequences, the method by over-lap PCR synthesizes It is a complete encoder block sequence, inserts KpnI the and NotI restriction enzyme sites of plasmid backbone.WPRE, Ins and Sin3 '-LTR then leads to Cross two ends and be respectively provided with the primer in SpeI and PacI sites and directly expand p4 the or p5 plasmids that have connected and obtain, then insert Enter SpeI the and PacI sites of plasmid backbone.Plasmid p9 confirms that without mutation plasmid map is as shown in figure 12 by sequencing.

Plasmid p10,11,12 are obtained by inserting S/MAR sequences on the basis of plasmid p7, p8 and p9 respectively.

When genome editor is applied to, the plasmid that will encode spCAS9 (or spCAS9-HF1) and coding gRNA is taken two-by-two With using, such as one or more in p7, p8, p10 or p11, with plasmid p9 or/and p12 matched combined；P7 or/and p10, With p4 or/and p5 matched combineds.Combinations thereof is capable of achieving tetracycline inducible expression, i.e., without tetracycline and its novel analogs When, TetR suppresses spCAS9 expression, and when there is certain density tetracycline or its new analog, can release TetR To the expression inhibiting of spCAS9, start expression.Taken when by p7 and p9 collocation, p7 and p12 collocation, p10 and p9 collocation, p10 and p12 Timing, can improve the expression quantity of TetR albumen.

Other above-mentioned p7, p8, p9, p10, p11, p12 can also collocation be used mutually with p1, p2, p3, p4, p5, p6-1, Need to only be followed in different collocation and contain at least one spCAS9 expression plasmids and at least one multiple gRNA expression plasmids. Whether different collocation involves the R element-TAT system controllability and measurabilities or tetracycline induction controllability that can realize spCAS9.

Embodiment 5 uses double-plasmid expression system (HIV mediated integrations, the TAT/R induction spCAS9 tables of p13, p14, p15 Up to mediating multiple gRNA to integrate with AAV, Tet-ON induced expression double-mass models system) structure

The design of expression plasmid p13, p14：The pUC pUC also uses double-mass model and expresses spCAS9 and gRNA respectively. The genome conformity that the expression plasmid of spCAS9 is mediated using HIV-1, the expression of spCAS9 is melted using R element and EF1a promoters Control is closed, the induction releasing R element for TAT checks effect to EF1a promoters.And BGHpA is added, use Sin3 ' LTR Element, RRE, cPPT, wPRE and Ins, such as p13 plasmids.The plasmid for expressing multiple gRNA can be using adeno-associated virus (AAV) Integrated mechanism carries out the integration of genome, but needs to express the REP albumen of AAV, due to the protein on cells toxic side effect, because This needs to carry out the expression of REP albumen the control of Tet-ON elements, such as p14, and needs for expressing gene to be inserted into 2 Between set terminal repetitive sequence (Inverted Terminal Rpeats, ITR).AAV viruses etc. can increase to AAVS1 sites Site-directed integration, reduces Non-specific integration.Plasmid p13, p14 Insert Fragment composition schematic diagram is shown in Figure 13.

The building process of plasmid p14 is as follows：

Left side ITR-hU6-g1-g2-hH1 is inserted XhoI the and BamHI sites of plasmid backbone by over-lap PCR, then MU6-g3-g4-h7SK-ITR is inserted BamHI the and AgeI restriction enzyme sites of plasmid backbone by over-lap PCR.By over-lap PCR By the fusion of EF1a and TetO short-movie sections, AgeI the and XbaI sites of plasmid backbone are inserted.PCR expands the REP gene codes of AAV-2 Frame, inserts XbaI the and NotI sites of plasmid backbone.PCR methods expand BGH poly A, insert the NotI and AgeI of plasmid backbone Site, sequencing identification.

On an other empty carrier, the sense primer with PacI and the anti-sense primer pair with KpnI, PCR amplifications PGK promoters, PacI the and KpnI sites of insertion vector.There is the primer PCR of KpnI and downstream with HindIII to expand with upstream belt Increase TetR, KpnI the and HindIII sites of insertion vector.Upstream is expanded with the primer PCR of HindIII and downstream with EcoRI Increase S/MAR sequences, insertion HindIII and EcoRI sites.Then with the sense primer with EcoRI and with EcoRI and SpeI The anti-sense primer of (closing on inner side in EcoRI) expands beta-globin PolyA signal sequences to PCR, inserts EcoRI Point, identifies forward and reverse, takes positive insertion person.SpeI and PacI digestion carriers are finally used, fragment two ends carry SpeI and PacI Point, on the plasmid built before insertion, sequencing identification.Its plasmid map is as shown in figure 14.

After two sequences of plasmid merge, using SpeI and PacI digestions, small fragment gel extraction is constructed before insertion SpeI the and PacI restriction enzyme sites of plasmid, sequencing identification excludes mutation.

In addition, devising gRNA target practice sequence g5 for CCR5 in embodiment 1, started by sh7SK promoters and expressed, used In shearing CCR5 sequences, 4 kinds of gRNA with anti-HIV-1 are shared, and constitute 5 heavy gRNA, and 5 effects of stressing defense are played to AIDS, are contained There is the plasmid of the sequence in addition to p6-3, also one plasmid from double-mass model system is named as p15, Insert Fragment composition Schematic diagram is shown in Figure 15, and the multiple gRNA expressed sequences of insertion are Seq ID No.21.Plasmid p15 can be carried with any of above Encoding plasmids (plasmid p1, p2, p3, p7, p8, p10, the p11 and p13) collocation of spCAS9 is used.

The HIV-1 mediated integrations of embodiment 6, shearing of the TAT induced expression double-mass model systems to HIV-1 genomes

Hela cells (surely turning p16) is transfected using commercial cationic liposome lipofectamine 2000, after 36 hours, Detect the activity (Promega kits) of luciferase.Result such as Figure 17, display p1-p4 combination, p1-p5 combinations, p2-p5 groups Close and p3-p5 combines the activity that can reduce the luciferase that p16 is encoded, and singly turning p1, p2, p3, p4 and p5 can not change P16 surely turns the activity of the intracellular luciferases of Hela, and wherein blank is idle running liposome.

P1, p3, p4, p5 and the p1-p4 packed using slow virus are combined, p3-p4 is combined, p3-p5 is combined and infected respectively P16- surely turns Hela cells, wherein the virus for combining infection is by infecting the slow virus that shown plasmid is packed, 2 subinfections respectively Interval 12-24 hours.6th day cell lysis after virus infection, detect the uciferase activity of cell.Figure 18 shows, single matter The infection of grain can not change the activity of luciferase, can be very after two kinds of virus combinations (p1-p4, p3-p4 or p3-p5) of infection Significant reduction even eliminates the expression of luciferase to close to background level.

Shearing of the Tet-ON inductivities double-plasmid expression system of embodiment 7 to HIV-1 genomes

P7 and p8 etc. expresses the plasmid of spCAS9, and promoter is built-in eukaryotic promoter, and the promoter is subject to TetO-TetR Control, when cell tetracycline and the like induction after, combinations of the TetR to TetO elements can be released, eliminate its expression Inhibitory action.P7, p8 plasmid can be used with the collocation of the plasmid such as p4, p5, p9 and p15.In p16 surely turns Hela cells, single turn Or the corresponding plasmid of corotation, cell is divided into 2 groups, and one group is processed without Doxycycline (abbreviation Dox), another set 40ng/ Ml Dox are processed 36 hours, and 2 transfections are spaced 12 hours, and the molecular number ratio of plasmid is 1 in many plasmid combinations:1, last time Cell lysis after transfecting 36 hours, determine the activity of luciferase.Experiment display, is not added with Dox treatment, no matter turn in wink simple substance or Double-mass model, can not substantially eliminate the expression (Figure 19) of the luciferase of Hela cells (surely turning p16).And with Dox process it is thin In born of the same parents, double Pignus pignoris grain (p7-p4, p7-p9 or p8-p9) significantly reduce the table of the luciferase of Hela cells (surely turning p16) Up to (p<0.05) (T inspections).

P7 plasmids and p9 plasmids pack slow virus respectively, and single virus or Geminivirus infection p16 surely turn Hela cells, point Into 2 groups, one group is not added with Dox, and one group adds Dox (treatment 5 days), and virus determines the activity of luciferase for metainfective 6th day.Figure 20 It has been shown that, adding the double-mass model p7-p9 of Dox groups can effectively eliminate the expression of luciferase.

The expression control of the spCAS9 that 8 R elements of embodiment/TAT is started to built-in promoter

P6-1 plasmids are that spCAS9 and gRNA are incorporated on a plasmid, can so use table by a plasmid Reach.It is limited to the capacity packing of slow virus, this Large plasmid cannot be controlled table using some elements of more HIV-1 viruses Reach.Therefore the built-in promoter EF1a that R element is cloned into spCAS9 is up, with built-in eukaryotic promoter co- controlling spCAS9 Expression.Eliminated when by HIV genomes and other genetic constitutions, and automatically after elimination TAT, you can prevent the expression of spCAS9. P13 plasmids are also to follow the thinking and build.To verify behind EF1a promoters plus whether R element is to the table of spCAS9 Experimental verification is turned using wink up to control action is played.Plasmid is combined using pLVX_Luc, pTAT and p6-1 or p13-p4 for building to be total to Turn, it is found that p6-1 simple substance grain can suppress the expression of luciferase of pLVX_Luc, the plasmid group of p13 and p4 in the presence of pTAT Conjunction also has certain inhibitory action in the presence of pTAT, and each experimental group for lacking pTAT plasmids can not effectively suppress fluorescence The expression (Figure 19) of plain enzyme, p6 is the abbreviation of plasmid p6-1 in figure.

Control actions of the TAT of embodiment 9 to built-in promoter-R element chimeric promoters

P6-1 and p13 plasmids are using the spCAS9 inside the chimeric promoters control of built-in promoter EF1a and R element Expression, by turning the above-mentioned plasmid of expression and pTAT encoding plasmids and pLVX-Luc plasmids with complete U3 structures wink, due to U3 areas are present on 5 '-LTR and 3 '-LTR, and targeting shearing easily causes the overall removal of the genome structure on the inside of 2 U3 areas existing As (Sci Rep.2016Jul 7；6:28213.doi:10.1038/srep28213.), such that it is able to detect built-in gene by fluorescence Plain enzyme is sheared inactivation.Single turn of cationic-liposome or corotation pLVX_Luc, pTAT and p6-1 or p13-p4 combination plasmids, its Middle pLVX_Luc plasmids are 1 with the molecular number ratio of other plasmids:3, cell is common Hela, and transfection determines fluorescein after 36 hours The activity of enzyme, p6 is the abbreviation of plasmid p6-1 in figure.Figure 21 display expression pTAT groups effectively reduce the activity of luciferase (p<0.05, T inspection).

The effect that the CCR5 of embodiment 10 targetings gRNA is sheared to CCR5

P15 plasmids increased a gRNA, i.e., for the gRNA of CCR5, if the target practice sequence can cut to CCR5 Cut, it will form the effect of similar CCR5 △ 32 mutation, the mutation causes single open reading frame to misplace, lacked logical with G-protein signal The related extracellular tricyclic structure in road, so as to lose the function that mediation HIV-1 enters cell, HIV-1 senses is prevented so as to play Contaminate the effect of T lymphocytes.Therefore while gRNA of the design for HIV-1 genomes, one is increased for CCR5's GRNA sequences, so as to form 5 heavy gRNA, the plasmid is p15, and the plasmid can be with p1, p2, p3, p7, p8, p10, p11 and p13 Plasmid is arranged in pairs or groups, and constitutes double-mass model targeting system.To verify its effect, p7 and p15 combinations are verified.pCCR5_P2A_Luc The common Hela cells (pCCR5_P2A_ of lipofectamine2000 cotransfections is used in plasmid and p7, p15 or p7-p15 combination respectively Luc plasmids are 1 with the molecular proportion of other plasmids:3).Transfection determines the activity of luciferase after 36 hours, observation CCR5 is sheared Afterwards to the influence of luciferase, as a result such as Figure 22, turn relative to simple substance grain wink, p7 and p15 combinations can effectively cut CCR5, Substantially reduce the activity (p of the luciferase of pCCR5_P2A_Luc<0.01, T inspection).

The film dispersion method of embodiment 11 makes liposome

(1) synthesis of polypeptide-PEG-PE

1. it is respectively synthesized targeted polypeptide with solid-phase synthesis：aCD4、aCCR5、aCXCR4、aCD4_CCR5、aCCR5_ CD4, aCD4_CXCR4, aCXCR4_CD4, aCCR5_CXCR4, aCXCR4_CCR5, sequence are respectively Seq ID in sequence table No.10、ASTTTNYT、Seq ID No.11、Seq ID No.12、Seq ID No.13、Seq ID No.16、Seq ID No.17、Seq ID No.14、Seq ID No.15.Wherein aCXCR4, aCD4_CXCR4, aCXCR4_CD4, aCCR5_CXCR4, The step of aCXCR4_CCR5 also additionally will carry out disulfide bond and is cyclized in building-up process.

2. the anti-CD4 of the anti-CCR5- of targeted polypeptide recombinate scFV recombinant polypeptides (B of Pel containing E.coli guide peptide) (referred to as ACCR5_CD4scFV synthesis)：Wherein anti-CD4scFV fragments are obtained, the table by screening full people's phage antibody displaying storehouse Optimize by bacterium rare codon up to sequence, express nucleic acid sequence be in sequence table Seq ID No.18 (amino acid sequence is Seq ID No.21), expression can obtain aCD4scFV polypeptides.Anti- CCR5 polypeptides sequence is added behind E.coli Pel B guiding peptides Row, sequence is Seq ID No.19 (amino acid sequence is Seq ID No.22) in sequence table, and expression can obtain aCCR5_ CD4scFV polypeptides.The expressed sequence is inserted into sequence table and forms expression vector in Seq ID No.6 plasmid backbones, by this Expression vector obtains mesh after E.coli BL21 expression by conventional concentration, extraction and purifying (crossing ion column and gel column) Polypeptide.

3. penetrating peptide is synthesized with solid-phase synthesis, sequence is：H-TyrGlyArgLysLysArgArgGlnArgArgArg- NH₂。

4. the synthesis of pNP-PEG-PE：Take 240mg PE to be put into flask, the imitative preparation of chlorination turns into 50mg/ml solution, plus Enter 800 μ l triethylamines (TEA), then weigh 10g pNP-PEG-pNP and be dissolved in 50ml chloroforms, be added into above-mentioned preparing In PE-TEA- chloroform mixtures.Nitrogen will be filled with reaction flask, then room temperature magnetic stirrer over night uses rotary evaporator (volume 3L, rotating speed 80-180rpm) evaporates chloroform, adds 120ml 0.01M HCl, carries out water bath sonicator, room temperature reaction overnight, Nitrogen is protected, ultrasonically treated formation emulsion.Using CL-4B gel filtration chromatography reactant mixtures, chromatography solution is 0.01M HCl, 0.15M NaCl.PNP-PEG-PE components add chloroform purifying pNP-PEG-PE, end-product to be dissolved in chloroform after lyophilized In, -80 DEG C store for future use.

5. the synthesis of targeted polypeptide-PEG-PE/ penetrating peptides-PEG-PE：Take 200mg pNP-PEG-PE and be dissolved in 20ml chlorine It is imitative, to put in 100ml flasks, rotary evaporation removes chloroform.Targeted polypeptide/penetrating peptide is dissolved in 0.01M HCl/water solution, adds 10 Targeted polypeptide/the penetrating peptide of times pNP-PEG-PE molal quantitys, shakes 30min.40ml 50mM Tris (pH 8.7) are added, is filled Enter nitrogen protection, 4 DEG C are reacted 24 hours.Product crosses Sepharose CL-4B column chromatographies, and -20 DEG C of product freezes standby.

The product obtained by the step is had：aCD4-PEG-PE、aCCR5-PEG-PE、aCXCR4-PEG-PE、aCD4_ CCR5-PEG-PE、aCCR5_CD4-PEG-PE、aCD4_CXCR4-PEG-PE、aCXCR4_CD4-PEG-PE、aCCR5_CXCR4- PEG-PE, aCXCR4_CCR5-PEG-PE, aCCR5_CD4scFV-PEG-PE, penetrating peptide aCCR5_CD4scFV-PEG-PE.

(2) synthesis of hyaluronic acid-PEG-PE：Hyaluronic acid is in EDC (1- (3- dimethylamino-propyls) -3- ethyls first Carbodiimide hydrochloride)/NHS (N-hydroxy-succinamide) effect under carry out sulfhydrylation, take 100mg hyaluronic acids, be dissolved in In 10ml EDC/NHS containing 200mM solution, pH5.0 is suspended half an hour, adds 200mg Cys, and room temperature is suspended anti- Answer 4 hours.The hyaluronic acid of sulfhydrylation is dialysed by the pellicle of MWCO 20kDa, and dialyzate is the hydrochloric acid of pH5.0 The aqueous solution, dialysis 3 times after, be stored in the aqueous solution of pH5.0, -80 DEG C freeze it is standby.Maleimide-PEG-PE and sulfhydrylation Hyaluronic acid is with mol ratio 1:1 mixing (Hepes pH of buffer 7.4), room temperature reaction 12-16 hours under inflated with nitrogen, product is used Sepharose CL-4B are purified.

(3) preparation of endotoxin-free plasmid：Plasmid transfection STBL3 Host Strains, positive bacterium colony is seeded in the LB containing penicillin In culture medium, amplify culture (containing 200 μ g/ml penicillin) overnight, second day centrifugation thalline, using the big upgrading grain examination of endotoxin-free Agent box cracks thalline, crosses post purifying and wash-out plasmid, by after concentration mensuration and agarose electrophoresis identification, -20 DEG C save backup, Plasmid concentration is 200ng/ μ l-8000ng/ μ l.The plasmid for treating loading form is pure plasmid, can also add polyethyleneimine, fish , used as assistant carrier, polyethyleneimine molecular weight is in 600-75000, preferably band for protamine or 5-12 positive charges amino acid polypeptide The polyethyleneimine (molecular weight 25000) of side chain.

(4) preparation of the liposome of embedding plasmid：By DSPC：Cholesterol：Polypeptide-PEG2000-PE, in mass ratio 50: 15:1 is made uniform mixed liquor, is dissolved in 10 times of chloroforms of volume：Methyl alcohol (2:1, V/V) solution, then in a rotary evaporator Evaporation solvent, forms thin layer.Plasmid and its auxiliary ingredients are added in reconstitution process, the wherein buffer solution used by plasmid is 5-50mM HEPES (pH7.5-8.2), the concentration of plasmid is in 200ng/ μ l.50 DEG C of heating are vortexed and process 5 minutes, are repeated 3 times, and are embedded The liposome film bubble of plasmid.Liposome film bubble is extruded, it is equal with the aperture for reducing liposome and the particle diameter for improving liposome Evenness, liposome squeezes through the cellulose acetate sheets or polycarbonate membrane in 200nm apertures at 40-65 DEG C, and 5- is repeated 20 times, the unilamellar liposome film bubble for being obtained is purified through semi-permeable membrane dialysed overnight, or by SephadexG-50 posts or class Like gel column purifying, to remove free non-integral protein and impurity, or purified through semi-permeable membrane dialysed overnight.

The liposome molecule of obtained loading plasmid is determined by laser particle analyzer.Table 1 several represents property for selected The liposome average size of grain.The liposomal particle size uniformity made using the method is higher, and particle diameter concentrates on 120- substantially In the range of 180nm, design requirement is basically reached.

Table 1 loads the liposome average size of different plasmids

The plasmid of loading	p1	p4	p6-1	p7	p13	p15
							Particle diameter (nm)	163.9	168.4	158.2	166.7	160.8	163.3

Conveying effect analysis of liposome of the embodiment 12 with different targeting elements to plasmid

Homemade liposome with different targeting elements loads different plasmids, and transfection carries different expressed receptors Hela cells, not the Hela cells of isoacceptor be used to simulate lymphocyte of the human body with corresponding acceptor and the endocytosis of liposome made With.Wherein all cells have surely turned p16 plasmids, to the cellular environment of simulated infection HIV-1, phase are integrated to provide HIV-1 The enzyme of pass TAT protein related to expression control etc..

(A) 4 kinds of liposomes are transfected, empty plasmid (empty), the control liposome of p3-p5 plasmids is respectively loaded, is loaded The CD4 target liposomes (containing anti-CD4-PEG-PE) of p3-p5 plasmids and the CCR5 target liposomes of loading p3-p5 plasmids are (containing anti- CCR5-PEG-PE).Transfect 2 kinds of cells respectively, one is that p16 surely turns Hela cell lines, one kind be p16, pLVX-CD4 and The Hela cell lines of triple steady turn of pLVX-CCR5, transfect 2 times, are spaced 12 hours, and last time transfection determines fluorescence after 36 hours The activity of plain enzyme.The plasmid p3 collocation plasmid P5 of Figure 23 A display TAT regulation types can shear genome, make the table of luciferase Up to decline, after the PEG-PE of the small peptide coupling of anti-CD4 and anti-CCR5 loads liposome, it is possible to increase the cell of target practice plasmid is defeated Enter ability.

(B) 5 kinds of liposomes are transfected, empty plasmid (empty), the control liposome of p3-p5 plasmids is respectively loaded, is loaded CD4 the and CCR5 target liposomes (aCCR5_CD4scFV-PEG-PE) of p3-p5 plasmids, the CD4 and CCR5 that load p3-p5 plasmids CD4 the and CCR5 target liposomes (aCCR5_CD4- of target liposomes (aCCR5_CD4-PEG-PE) and loading p3-p5 plasmids PEG-PE and penetrating peptide-PEG-PE).Transfect 2 kinds of cells respectively, one is that p16 surely turns Hela cell lines, one kind be p16, The Hela cells of triple steady turn of pLVX-CD4 and pLVX-CCR5, transfect 2 times, are spaced 12 hours, after last time is transfected 36 hours Determine the activity of luciferase.Figure 23 B display aCCR5_CD4scFV-PEG-PE modified liposomes can improve plasmid p3-p5 groups The positive Hela cells of input CD4, CCR5 are closed, aCCR5_CD4-PEG-PE modified liposomes can also improve plasmid pair CD4-CCR5 The transfection abilities of positive Hela cells；The liposome effect that aCCR5_CD4-PEG-PE and penetrating peptide-PEG-PE is modified jointly The transfection of the relatively simple liposome modified using aCCR5_CD4-PEG-PE will get well (p<0.05).

(C) 3 kinds of liposomes are transfected, plasmid p6-1 is respectively loaded, is loaded plasmid p6-1 (coating CXCR4-PEG-PE), dress Carry the liposome (coating CXCR4-PEG-PE) of p7-p4 plasmid combinations.P6 is the abbreviation of plasmid p6-1 in figure.2 kinds are transfected respectively Cell, one is that p16 surely turns Hela cell lines, and a kind of is the Hela cells of double steady turn of p16, pLVX-CXCR4.With loading p7- The cell that the liposome (coating CXCR4-PEG-PE) of p4 plasmid combinations is processed makees 2 kinds for the treatment of, one kind plus Dox treatment, and one kind is not Plus Dox treatment, transfect 2 times, it is spaced 12 hours, last time transfection determines the activity of luciferase after 36 hours.Figure 23 C show The liposome of aCXCR4-PEG-PE modifications can also improve the positive cell inputs of target practice plasmid pair CXCR4, also show p6-1 mono- PUC pUC has genomic modification ability as double-mass model system.

(D) 4 kinds of liposomes are transfected, p6-1 plasmids is loaded respectively, p6-1 plasmids (aCCR5_CD4-PEG-PE) is loaded, is loaded P7-p4 plasmid combinations, loading p7-p4 plasmid combinations (aCCR5_CD4-PEG-PE, aCXCR4 and penetrating peptide).2 kinds are transfected respectively Cell, one is that p16 surely turns Hela cell lines, and a kind of is the Hela cells of triple steady turn of p16, pLVX-CD4 and pLVX-CCR5. Wherein with the cell of the liposome-treated for loading p7-p4 plasmid combinations (containing anti-CD4-CCR5-PEG-PE), 36 are processed with DOX small When.Transfection 2 times, is spaced 12 hours, and last time transfection determines the activity of luciferase after 36 hours.Figure 23 D show aCCR5_ The liposome of CD4-PEG-PE modifications has the liposome intake ability for improving the positive Hela cells of CD4, CCR5.P6 is in figure The abbreviation of plasmid p6-1.

In above-mentioned four groups of experiments, the molecular number ratio of plasmid is 1 in all many plasmid combinations:1, * in figure：p<0.05 (T inspections Test).

The Targeting Effect that embodiment 13 loads the liposome of the target spots of CCR5-CD4-CXCR4 tri- and penetrating peptide is determined：

5 kinds of liposomes of transfection, load empty plasmid, p1-p4 combinations plasmid, p1-p4 combination plasmids (aCCR5_CD4 respectively Combined peptide-PEG-PE), p1-p4 combine plasmid (aCCR5_CD4 combined peptide-PEG-PE and aCXCR4-PEG-PE) and p1-p4 groups Conjugative plasmid (aCCR5_CD4 combined peptides-PEG-PE, aCXCR4-PEG-PE and penetrating peptide-PEG-PE).Transfection p16 surely turns respectively The Hela cells that Hela cells or p16, pLVX-CD4, pLVX-CCR5 and pLVX-CXCR4 quadruple surely turn, transfect 2 times, between transfection Every 12 hours, the molecular number ratio of plasmid was 1 in many plasmid combinations:1, last time transfection determines the work of luciferase after 36 hours Property.

As shown in figure 24, naked liposome has certain transfection abilities to cell.ACCR5_CD4- is added in liposome After PEG-PE, the ability of Hela cell of the transfection without expression CD4, CCR5 and CXCR4 declines on the contrary.But when transfection expression CD4, After the Hela cells of CCR5 and CXCR4, the transfection abilities of the liposome of aCCR5_CD4-PEG-PE and aCXCR4-PEG-PE are added Increase.When in liposome simultaneously add penetrating peptide-PEG-PE after, to express CD4, CCR5 and CXCR4 Hela cells turn Dye ability is most strong, while the transfection abilities of the Hela cells to not expressing CD4-CCR5-CXCR4 are also improved, therefore penetrating peptide- The addition of PEG-PE is improved the ability of liposome transfection cell.

The genome conformity efficiency of embodiment 14

Representative plasmid p1, p4 and p7 are chosen, above-mentioned plasmid, transfection two is transfected with lipofactamine 2000 respectively It is secondary, it is spaced 12 hours.Last time transfect after 24 hours after, vitellophag does ten thousand times of 5000-2 dilution, allows cell monoclonal Grow into macroscopic big clone cell colony (maintaining 0.5-1 μ g/ml puromycins in growth course all the time).After 2-4 weeks, The cell of clonal growth is drawn with sample-adding pipette tips, 6 orifice plates of inoculation, 12 orifice plates or 24 orifice plates, each clone are inoculated with a hole, often Individual transfection treatment sample chooses 15 clones, amplifies growth.The cell of last each clone enters performing PCR mirror by extracting STb gene It is fixed, or a little cell is taken, directly cell is added using the kit of trace P CR templates enters performing PCR amplification in reaction tube.p1 With p7 identifications (sense primer is expanded with one section of primer for cas9：5’-GCCAGCCAGGAAGAGTTCTACA-3’；Draw in downstream Thing：5 '-TCAGCTCGTTATACACGGTGAAGTA-3 '), p4 with a pair of identification wPRE and ins sequences (sense primer 5 '- AATAGGGAGGGGGAAAGCGA-3’；- GGAGGGACGTAATTACATC the CCT-3 ' of anti-sense primer 5 ') special primer amplification. Stable transfection rate is the percentage that positive cell clone number clones number divided by total cell.

The cell clone PCR results of Figure 25 show, after liposome transfection p1, p4 and p7 plasmid, are found by after PCR identifications In p16 surely turns cell, this 3 representative plasmids there occurs the integration of genome, and it is normal it is non-it is steady turn Hela cells, Almost there is no integrator cell phenomenon, the intracellular of p16 simulation HIV-1 infection is illustrated, with these plasmid integration genomes of help Ability.

The virus-mediated genome conformity efficiency of the integrase Inactivating mutations packaging system of embodiment 15 packaging

Plasmid p1, p4 and p7 are packed by second generation slow virus packaging plasmid, and wherein integrase is by transformation, three Point mutation D64K, D116Q and E152Q.Collect within 48 hours and 72 hours after transfection 293T cells virus, purifying, after determining potency Hela cells and common Hela cells that infection p16 surely turns, 3 days rear clone cultured cells, specific steps with embodiment 14, carefully Born of the same parents extract genomic DNA or directly carry out cell PCR measure, with the genome conformity assay method of embodiment 4.

The cell clone PCR results of Figure 26 show that the integrase saltant type slow virus of p1, p4 and p7 plasmid packaging is surely turning In the Hela cells of p16, there occurs and effectively surely turn phenomenon, imply that there is obvious genome conformity phenomenon, and do not turn Then there is no visible plasmid and surely turn phenomenon in the Hela cells of dye p16 plasmids, imply do not exist obvious genome conformity phenomenon.

Seq ID No.1：G1,5 '-GGAATCGAAGAAGGAGG-3 '

Seq ID No.2：G2,5 '-AAACTACACACCAGGGCC-3 '

Seq ID No.3：G3,5 '-GATAGACCCATAAATCA-3 '

Seq ID No.4：G4,5 '-GCCAAAGAGTGATTTGA-3 '

Seq ID No.5：G5,5 '-GAATTGATACTGACTGTA-3 '

Seq ID No.6

1 ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT

61 TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG

121 CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC

181 TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC

241 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC

301 AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC

361 TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT

421 AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT

481 AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC

541 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT

601 TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG

661 ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC

721 ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA

781 TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG

841 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG

901 TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA

961 GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG

1021 CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA

1081 GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC

1141 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA

1201 AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG

1261 ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT

1321 AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC

1381 AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG

1441 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG

1501 GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT

1561 GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA

1621 GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA

1681 CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC

1741 ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTT AATACGACTC ACTATAGGGC

1801 GAGGAATTCT CACTCGAGAA GCTTGGATCC TGAACCGGTT GAGGTACCGA TATCTGATCA

1861 TGATCTAGAT CAGCGGCCGC ATACACTAGT TGATTAATTA A

Seq ID No.7：The sequence of plasmid p1

1 ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT

61 TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG

121 CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC

181 TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC

241 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC

301 AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC

361 TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT

421 AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT

481 AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC

541 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT

601 TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG

661 ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC

721 ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA

781 TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG

841 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG

901 TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA

961 GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG

1021 CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA

1081 GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC

1141 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA

1201 AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG

1261 ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT

1321 AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC

1381 AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG

1441 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG

1501 GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT

1561 GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA

1621 GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA

1681 CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC

1741 ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTT AATACGACTC ACTATAGGGC

1801 GAGGAATTCT GGAAGGGCTA ATTTGGTCCC AAAAAAGACA AGAGAATGCT TATGGACTAG

1861 GGACTGTTAC AAACAAATGA TGAATGGAAA CAGCTGAGCA TGACTCATAG CTGAAGCGCT

1921 AGCAGCTGCT TAACCGCAAA ACCACATCCT ATGTAAAGCT TGCTAATGAC GTATAAGTTG

1981 CTCCACTGTA AAAGTATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA

2041 CCAGATCTGA GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA

2101 AAGCTTGCCT TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA

2161 GAGATCCCTC AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG

2221 GACTTGAAAG CGAAAGTAAA GCCAGAGGAG ATCTCTCGAC GCAGGACTCG GCTTGCTGAA

2281 GCGCGCACGG CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC

2341 GGAGGCTAGA AGGAGAGAGA TGGGTGCGAG AGCGTCAGTA TTAAGCGGGG GAGGATCCGG

2401 ATCCTGAACC GGTATGCCCA AGAAGAAACG CAAAGTCGCT TCAGACAAGA AGTACAGCAT

2461 CGGCCTGGAC ATCGGCACCA ACTCTGTGGG CTGGGCCGTG ATCACCGACG AGTACAAGGT

2521 GCCCAGCAAG AAATTCAAGG TGCTGGGCAA CACCGACCGG CACAGCATCA AGAAGAACCT

2581 GATCGGAGCC CTGCTGTTCG ACAGCGGCGA AACAGCCGAG GCCACCCGGC TGAAGAGAAC

2641 CGCCAGAAGA AGATACACCA GACGGAAGAA CCGGATCTGC TATCTGCAAG AGATCTTCAG

2701 CAACGAGATG GCCAAGGTGG ACGACAGCTT CTTCCACAGA CTGGAAGAGT CCTTCCTGGT

2761 GGAAGAGGAT AAGAAGCACG AGCGGCACCC CATCTTCGGC AACATCGTGG ACGAGGTGGC

2821 CTACCACGAG AAGTACCCCA CCATCTACCA CCTGAGAAAG AAACTGGTGG ACAGCACCGA

2881 CAAGGCCGAC CTGCGGCTGA TCTATCTGGC CCTGGCCCAC ATGATCAAGT TCCGGGGCCA

2941 CTTCCTGATC GAGGGCGACC TGAACCCCGA CAACAGCGAC GTGGACAAGC TGTTCATCCA

3001 GCTGGTGCAG ACCTACAACC AGCTGTTCGA GGAAAACCCC ATCAACGCCA GCGGCGTGGA

3061 CGCCAAGGCC ATCCTGTCTG CCAGACTGAG CAAGAGCAGA CGGCTGGAAA ATCTGATCGC

3121 CCAGCTGCCC GGCGAGAAGA AGAATGGCCT GTTCGGAAAC CTGATTGCCC TGAGCCTGGG

3181 CCTGACCCCC AACTTCAAGA GCAACTTCGA CCTGGCCGAG GATGCCAAAC TGCAGCTGAG

3241 CAAGGACACC TACGACGACG ACCTGGACAA CCTGCTGGCC CAGATCGGCG ACCAGTACGC

3301 CGACCTGTTT CTGGCCGCCA AGAACCTGTC CGACGCCATC CTGCTGAGCG ACATCCTGAG

3361 AGTGAACACC GAGATCACCA AGGCCCCCCT GAGCGCCTCT ATGATCAAGA GATACGACGA

3421 GCACCACCAG GACCTGACCC TGCTGAAAGC TCTCGTGCGG CAGCAGCTGC CTGAGAAGTA

3481 CAAAGAGATT TTCTTCGACC AGAGCAAGAA CGGCTACGCC GGCTACATTG ACGGCGGAGC

3541 CAGCCAGGAA GAGTTCTACA AGTTCATCAA GCCCATCCTG GAAAAGATGG ACGGCACCGA

3601 GGAACTGCTC GTGAAGCTGA ACAGAGAGGA CCTGCTGCGG AAGCAGCGGA CCTTCGACAA

3661 CGGCAGCATC CCCCACCAGA TCCACCTGGG AGAGCTGCAC GCCATTCTGC GGCGGCAGGA

3721 AGATTTTTAC CCATTCCTGA AGGACAACCG GGAAAAGATC GAGAAGATCC TGACCTTCCG

3781 CATCCCCTAC TACGTGGGCC CTCTGGCCAG GGGAAACAGC AGATTCGCCT GGATGACCAG

3841 AAAGAGCGAG GAAACCATCA CCCCCTGGAA CTTCGAGGAA GTGGTGGACA AGGGCGCTTC

3901 CGCCCAGAGC TTCATCGAGC GGATGACCGC TTTCGATAAG AACCTGCCCA ACGAGAAGGT

3961 GCTGCCCAAG CACAGCCTGC TGTACGAGTA CTTCACCGTG TATAACGAGC TGACCAAAGT

4021 GAAATACGTG ACCGAGGGAA TGAGAAAGCC CGCCTTCCTG AGCGGCGAGC AGAAAAAGGC

4081 CATCGTGGAC CTGCTGTTCA AGACCAACCG GAAAGTGACC GTGAAGCAGC TGAAAGAGGA

4141 CTACTTCAAG AAAATCGAGT GCTTCGACTC CGTGGAAATC TCCGGCGTGG AAGATCGGTT

4201 CAACGCCTCC CTGGGCACAT ACCACGATCT GCTGAAAATT ATCAAGGACA AGGACTTCCT

4261 GGACAATGAG GAAAACGAGG ACATTCTGGA AGATATCGTG CTGACCCTGA CACTGTTTGA

4321 GGACAGAGAG ATGATCGAGG AACGGCTGAA AACCTATGCC CACCTGTTCG ACGACAAAGT

4381 GATGAAGCAG CTGAAGCGGC GGAGATACAC CGGCTGGGGA GCACTGAGCC GGAAGCTGAT

4441 CAACGGCATC CGGGACAAGC AGTCCGGCAA GACAATCCTG GATTTCCTGA AGTCCGACGG

4501 CTTCGCCAAC AGAAACTTCA TGGCTCTGAT CCACGACGAC AGCCTGACCT TTAAAGAGGA

4561 CATCCAGAAA GCCCAGGTGT CCGGCCAGGG CGATAGCCTG CACGAGCACA TTGCCAATCT

4621 GGCCGGCAGC CCCGCCATTA AGAAGGGCAT CCTGCAGACA GTGAAGGTGG TGGACGAGCT

4681 CGTGAAAGTG ATGGGCCGGC ACAAGCCCGA GAACATCGTG ATCGAAATGG CCAGAGAGAA

4741 CCAGACCACC CAGAAGGGAC AGAAGAACAG CCGCGAGAGA ATGAAGCGGA TCGAAGAGGG

4801 CATCAAAGAG CTGGGCAGCC AGATCCTGAA AGAACACCCC GTGGAAAACA CCCAGCTGCA

4861 GAACGAGAAG CTGTACCTGT ACTACCTGCA GAATGGGCGG GATATGTACG TGGACCAGGA

4921 ACTGGACATC AACCGGCTGT CCGACTACGA TGTGGACCAT ATCGTGCCTC AGAGCTTTCT

4981 GAAGGACGAC TCCATCGACA ACAAGGTGCT GACCAGAAGC GACAAGAACC GGGGCAAGAG

5041 CGACAACGTG CCCTCCGAAG AGGTCGTGAA GAAGATGAAG AACTACTGGC GGCAGCTGCT

5101 GAACGCCAAG CTGATTACCC AGAGAAAGTT CGACAATCTG ACCAAGGCCG AGAGAGGCGG

5161 CCTGAGCGAA CTGGATAAGG CCGGCTTCAT CAAGAGACAG CTGGTGGAAA CCAGGGCAAT

5221 CACAAAGCAC GTGGCACAGA TCCTGGACTC CCGGATGAAC ACTAAGTACG ACGAGAATGA

5281 CAAGCTGATC CGGGAAGTGA AAGTGATCAC CCTGAAGTCC AAGCTGGTGT CCGATTTCCG

5341 GAAGGATTTC CAGTTTTACA AAGTGCGCGA GATCAACAAC TACCACCACG CCCACGACGC

5401 CTACCTGAAC GCCGTCGTGG GAACCGCCCT GATCAAAAAG TACCCTAAGC TGGAAAGCGA

5461 GTTCGTGTAC GGCGACTACA AGGTGTACGA CGTGCGGAAG ATGATCGCCA AGAGCGAGCA

5521 GGAAATCGGC AAGGCTACCG CCAAGTACTT CTTCTACAGC AACATCATGA ACTTTTTCAA

5581 GACCGAGATT ACCCTGGCCA ACGGCGAGAT CCGGAAGCGG CCTCTGATCG AGACAAACGG

5641 CGAAACCGGG GAGATCGTGT GGGATAAGGG CCGGGATTTT GCCACCGTGC GGAAAGTGCT

5701 GAGCATGCCC CAAGTGAATA TCGTGAAAAA GACCGAGGTG CAGACAGGCG GCTTCAGCAA

5761 AGAGTCTATC CTGCCCAAGA GGAACAGCGA TAAGCTGATC GCCAGAAAGA AGGACTGGGA

5821 CCCTAAGAAG TACGGCGGCT TCGACAGCCC CACCGTGGCC TATTCTGTGC TGGTGGTGGC

5881 CAAAGTGGAA AAGGGCAAGT CCAAGAAACT GAAGAGTGTG AAAGAGCTGC TGGGGATCAC

5941 CATCATGGAA AGAAGCAGCT TCGAGAAGAA TCCCATCGAC TTTCTGGAAG CCAAGGGCTA

6001 CAAAGAAGTG AAAAAGGACC TGATCATCAA GCTGCCTAAG TACTCCCTGT TCGAGCTGGA

6061 AAACGGCCGG AAGAGAATGC TGGCCTCTGC CGGCGAACTG CAGAAGGGAA ACGAACTGGC

6121 CCTGCCCTCC AAATATGTGA ACTTCCTGTA CCTGGCCAGC CACTATGAGA AGCTGAAGGG

6181 CTCCCCCGAG GATAATGAGC AGAAACAGCT GTTTGTGGAA CAGCACAAGC ACTACCTGGA

6241 CGAGATCATC GAGCAGATCA GCGAGTTCTC CAAGAGAGTG ATCCTGGCCG ACGCTAATCT

6301 GGACAAAGTG CTGTCCGCCT ACAACAAGCA CCGGGATAAG CCCATCAGAG AGCAGGCCGA

6361 GAATATCATC CACCTGTTTA CCCTGACCAA TCTGGGAGCC CCTGCCGCCT TCAAGTACTT

6421 TGACACCACC ATCGACCGGA AGAGGTACAC CAGCACCAAA GAGGTGCTGG ACGCCACCCT

6481 GATCCACCAG AGCATCACCG GCCTGTACGA GACACGGATC GACCTGTCTC AGCTGGGAGG

6541 CGACTCTAAA AGACCAGCAG CCACTAAAAA GGCCGGACAG GCTAAAAAGA AAAAGTAAGG

6601 TACCAGGAGC TTTGTTCCTT GGGTTCTTGG GAGCAGCAGG AAGCACTATG GGCGCAGCGT

6661 CAATGACGCT GACGGTACAG GCCAGACAAT TATTGTCTGG TATAGTGCAG CAGCAGAACA

6721 ATTTGCTGAG GGCTATTGAG GCGCAACAGC ATCTGTTGCA ACTCACAGTC TGGGGCATCA

6781 AGCAGCTCCA GGCAAGAATC CTGGCTGTGG AAAGATACCT AAAGGATCAA CAGCTCCTTT

6841 TAAAAGAAAA GGGGGGATTG GGGGGTACAG TGCAGGGGAA AGAATAGTAG ACATAATAGC

6901 AACAGACATA CAAACTAAAG AATTACAAAA ACAAATTACA AAAATTCAAA ATTTTCGGGT

6961 TTTCTAGATG GAAGGGCTAA TTTGGTCCCA AAAAAGACAA GAGAATGCTT ATGGACTAGG

7021 GACTGTTACA AACAAATGAT GAATGGAAAC AGCTGAGCAT GACTCATAGC TGAAGCGCTA

7081 GCAGCTGCTT AACCGCAAAA CCACATCCTA TGTAAAGCTT GCTAATGACG TATAAGTTGC

7141 TCCACTGTAA AAGTATATAA GCAGCTGCTT TTTGCCTGTA CTGGGTCTCT CTGGTTAGAC

7201 CAGATCTGAG CCTGGGAGCT CTCTGGCTAA CTAGGGAACC CACTGCTTAA GCCTCAATAA

7261 AGCTTGCCTT GAGTGCTTCA AGTAGTGTGT GCCCGTCTGT TGTGTGACTC TGGTAACTAG

7321 AGATCCCTCA GACCCTTTTA GTCAGTGTGG AAAATCTCTA GCATTAATTA A

Seq ID No.8：The sequence of plasmid p6-1

1 ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT

61 TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG

121 CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC

181 TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC

241 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC

301 AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC

361 TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT

421 AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT

481 AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC

541 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT

601 TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG

661 ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC

721 ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA

781 TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG

841 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG

901 TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA

961 GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG

1021 CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA

1081 GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC

1141 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA

1201 AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG

1261 ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT

1321 AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC

1381 AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG

1441 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG

1501 GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT

1561 GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA

1621 GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA

1681 CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC

1741 ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTT AATACGACTC ACTATAGGGC

1801 GAGGAATTCT GGAAGGGCTA ATTTGGTCCC AAAAAAGACA AGAGAATGCT TATGGACTAG

1861 GGACTGTTAC AAACAAATGA TGAATGGAAA CAGCTGAGCA TGACTCATAG CTGAAGCGCT

1921 AGCAGCTGCT TAACCGCAAA ACCACATCCT ATGTAAAGCT TGCTAATGAC GTATAAGTTG

1981 CTCCACTGTA AAAGTATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA

2041 CCAGATCTGA GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA

2101 AAGCTTGCCT TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA

2161 GAGATCCCTC AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG

2221 GACTTGAAAG CGAAAGGGAA ACCAGAGGAG CTCTCTCGAC GCAGGACTCG GCTTGCTGAA

2281 GCGCGCACGG CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC

2341 GGAGGCTAGA AGGAGAGAGA TGGGTGCGAG AGCGTCAGTA TTAAGCGGGG GAGAATTAGA

2401 TCGCGATGGG AAAAAATTCG GTTAAGGCCA GGGGGAAAGA AAAAATATAA ATTAAAACAT

2461 ATAGTATGGG CAAGCAGGGA GCTAGAACGA TTCGCAGTTA ATCCTGGCCT GTTAGAAACA

2521 TCAGAAGGCT GTAGACAAAT ACTGGGACAG CTACAACCAT CCCTTCAGAC AGGATCAGAA

2581 GAACTTAGAT CATTATATAA TACAGTAGCA ACCCTCTATT GTGTGCATCA AAGGATAGAG

2641 ATAAAAGACA CCAAGGAAGC TTTAGACAAG ATAGAGGAAG AGCAAAACAA AAGTAAGACC

2701 ACCGCACAGC AAGCGGCCGG CCGCTGATCT TCAGACCTGG AGGAGGAGAT ATGAGGGACA

2761 ATTGGAGAAG TGAATTATAT AAATATAAAG TAGTAAAAAT TGAACCATTA GGAGTAGCAC

2821 CCACCAAGGC AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AGCAGTGGGA ATAGGAGCTT

2881 TGTTCCTTGG GTTCTTGGGA GCAGCAGGAA GCACTATGGG CGCAGCGTCA ATGACGCTGA

2941 CGGTACAGGC CAGACAATTA TTGTCTGGTA TAGTGCAGCA GCAGAACAAT TTGCTGAGGG

3001 CTATTGAGGC GCAACAGCAT CTGTTGCAAC TCACAGTCTG GGGCATCAAG CAGCTCCAGG

3061 CAAGAATCCT GGCTGTGGAA AGATACCTAA AGGATCAACA GCTCCTTTTA AAAGAAAAGG

3121 GGGGATTGGG GGGTACAGTG CAGGGGAAAG AATAGTAGAC ATAATAGCAA CAGACATACA

3181 AACTAAAGAA TTACAAAAAC AAATTACAAA AATTCAAAAT TTTCGGGTTT CTCGAGACGG

3241 GGACAGCCCC CCCCCAAAGC CCCCAGGGAT GTAATTACGT CCCTCCCCCG CTAGGGGGCA

3301 GCAGCGAGCC GCCCGGGGCT CCGCTCCGGT CCGGCGCTCC CCCCGCATCC CCGAGCCGGC

3361 AGCGTGCGGG GACAGCCCGG GCACGGGGAA GGTGGCACGG GATCGCTTTC CTCTGAACGC

3421 TTCTCGCTGC TCTTTGAGCC TGCAGACACC TGGGGGGATA CGGGGAAAAA GGATCCGTGG

3481 AGAAGAGCAT GCTTGAGGGC TGAGTGCCCC TCAGTGGGCA GAGAGCACAT GGCCCACAGT

3541 CCCTGAGAAG TTGGGGGGAG GGGTGGGCAA TTGAACTGGT GCCTAGAGAA GGTGGGGCTT

3601 GGGTAAACTG GGAAAGTGAT GTGGTGTACT GGCTCCACCT TTTTCCCCAG GGTGGGGGAG

3661 AACCATATAT AAGTGCAGTA GTCTCTGTGA ACATTCGGTC TCTCTGGTTA GACCAGATCT

3721 GAGCCTGGGA GCTCTCTGGC TAACTAGGGA ACCCACTGCT TAAGCCTCAA TAAAGCTTGC

3781 CTTGAGTGCT TCACCGGTAT GCCCAAGAAG AAACGCAAAG TCGCTTCAGA CAAGAAGTAC

3841 AGCATCGGCC TGGACATCGG CACCAACTCT GTGGGCTGGG CCGTGATCAC CGACGAGTAC

3901 AAGGTGCCCA GCAAGAAATT CAAGGTGCTG GGCAACACCG ACCGGCACAG CATCAAGAAG

3961 AACCTGATCG GAGCCCTGCT GTTCGACAGC GGCGAAACAG CCGAGGCCAC CCGGCTGAAG

4021 AGAACCGCCA GAAGAAGATA CACCAGACGG AAGAACCGGA TCTGCTATCT GCAAGAGATC

4081 TTCAGCAACG AGATGGCCAA GGTGGACGAC AGCTTCTTCC ACAGACTGGA AGAGTCCTTC

4141 CTGGTGGAAG AGGATAAGAA GCACGAGCGG CACCCCATCT TCGGCAACAT CGTGGACGAG

4201 GTGGCCTACC ACGAGAAGTA CCCCACCATC TACCACCTGA GAAAGAAACT GGTGGACAGC

4261 ACCGACAAGG CCGACCTGCG GCTGATCTAT CTGGCCCTGG CCCACATGAT CAAGTTCCGG

4321 GGCCACTTCC TGATCGAGGG CGACCTGAAC CCCGACAACA GCGACGTGGA CAAGCTGTTC

4381 ATCCAGCTGG TGCAGACCTA CAACCAGCTG TTCGAGGAAA ACCCCATCAA CGCCAGCGGC

4441 GTGGACGCCA AGGCCATCCT GTCTGCCAGA CTGAGCAAGA GCAGACGGCT GGAAAATCTG

4501 ATCGCCCAGC TGCCCGGCGA GAAGAAGAAT GGCCTGTTCG GAAACCTGAT TGCCCTGAGC

4561 CTGGGCCTGA CCCCCAACTT CAAGAGCAAC TTCGACCTGG CCGAGGATGC CAAACTGCAG

4621 CTGAGCAAGG ACACCTACGA CGACGACCTG GACAACCTGC TGGCCCAGAT CGGCGACCAG

4681 TACGCCGACC TGTTTCTGGC CGCCAAGAAC CTGTCCGACG CCATCCTGCT GAGCGACATC

4741 CTGAGAGTGA ACACCGAGAT CACCAAGGCC CCCCTGAGCG CCTCTATGAT CAAGAGATAC

4801 GACGAGCACC ACCAGGACCT GACCCTGCTG AAAGCTCTCG TGCGGCAGCA GCTGCCTGAG

4861 AAGTACAAAG AGATTTTCTT CGACCAGAGC AAGAACGGCT ACGCCGGCTA CATTGACGGC

4921 GGAGCCAGCC AGGAAGAGTT CTACAAGTTC ATCAAGCCCA TCCTGGAAAA GATGGACGGC

4981 ACCGAGGAAC TGCTCGTGAA GCTGAACAGA GAGGACCTGC TGCGGAAGCA GCGGACCTTC

5041 GACAACGGCA GCATCCCCCA CCAGATCCAC CTGGGAGAGC TGCACGCCAT TCTGCGGCGG

5101 CAGGAAGATT TTTACCCATT CCTGAAGGAC AACCGGGAAA AGATCGAGAA GATCCTGACC

5161 TTCCGCATCC CCTACTACGT GGGCCCTCTG GCCAGGGGAA ACAGCAGATT CGCCTGGATG

5221 ACCAGAAAGA GCGAGGAAAC CATCACCCCC TGGAACTTCG AGGAAGTGGT GGACAAGGGC

5281 GCTTCCGCCC AGAGCTTCAT CGAGCGGATG ACCGCTTTCG ATAAGAACCT GCCCAACGAG

5341 AAGGTGCTGC CCAAGCACAG CCTGCTGTAC GAGTACTTCA CCGTGTATAA CGAGCTGACC

5401 AAAGTGAAAT ACGTGACCGA GGGAATGAGA AAGCCCGCCT TCCTGAGCGG CGAGCAGAAA

5461 AAGGCCATCG TGGACCTGCT GTTCAAGACC AACCGGAAAG TGACCGTGAA GCAGCTGAAA

5521 GAGGACTACT TCAAGAAAAT CGAGTGCTTC GACTCCGTGG AAATCTCCGG CGTGGAAGAT

5581 CGGTTCAACG CCTCCCTGGG CACATACCAC GATCTGCTGA AAATTATCAA GGACAAGGAC

5641 TTCCTGGACA ATGAGGAAAA CGAGGACATT CTGGAAGATA TCGTGCTGAC CCTGACACTG

5701 TTTGAGGACA GAGAGATGAT CGAGGAACGG CTGAAAACCT ATGCCCACCT GTTCGACGAC

5761 AAAGTGATGA AGCAGCTGAA GCGGCGGAGA TACACCGGCT GGGGAGCACT GAGCCGGAAG

5821 CTGATCAACG GCATCCGGGA CAAGCAGTCC GGCAAGACAA TCCTGGATTT CCTGAAGTCC

5881 GACGGCTTCG CCAACAGAAA CTTCATGGCT CTGATCCACG ACGACAGCCT GACCTTTAAA

5941 GAGGACATCC AGAAAGCCCA GGTGTCCGGC CAGGGCGATA GCCTGCACGA GCACATTGCC

6001 AATCTGGCCG GCAGCCCCGC CATTAAGAAG GGCATCCTGC AGACAGTGAA GGTGGTGGAC

6061 GAGCTCGTGA AAGTGATGGG CCGGCACAAG CCCGAGAACA TCGTGATCGA AATGGCCAGA

6121 GAGAACCAGA CCACCCAGAA GGGACAGAAG AACAGCCGCG AGAGAATGAA GCGGATCGAA

6181 GAGGGCATCA AAGAGCTGGG CAGCCAGATC CTGAAAGAAC ACCCCGTGGA AAACACCCAG

6241 CTGCAGAACG AGAAGCTGTA CCTGTACTAC CTGCAGAATG GGCGGGATAT GTACGTGGAC

6301 CAGGAACTGG ACATCAACCG GCTGTCCGAC TACGATGTGG ACCATATCGT GCCTCAGAGC

6361 TTTCTGAAGG ACGACTCCAT CGACAACAAG GTGCTGACCA GAAGCGACAA GAACCGGGGC

6421 AAGAGCGACA ACGTGCCCTC CGAAGAGGTC GTGAAGAAGA TGAAGAACTA CTGGCGGCAG

6481 CTGCTGAACG CCAAGCTGAT TACCCAGAGA AAGTTCGACA ATCTGACCAA GGCCGAGAGA

6541 GGCGGCCTGA GCGAACTGGA TAAGGCCGGC TTCATCAAGA GACAGCTGGT GGAAACCAGG

6601 GCAATCACAA AGCACGTGGC ACAGATCCTG GACTCCCGGA TGAACACTAA GTACGACGAG

6661 AATGACAAGC TGATCCGGGA AGTGAAAGTG ATCACCCTGA AGTCCAAGCT GGTGTCCGAT

6721 TTCCGGAAGG ATTTCCAGTT TTACAAAGTG CGCGAGATCA ACAACTACCA CCACGCCCAC

6781 GACGCCTACC TGAACGCCGT CGTGGGAACC GCCCTGATCA AAAAGTACCC TAAGCTGGAA

6841 AGCGAGTTCG TGTACGGCGA CTACAAGGTG TACGACGTGC GGAAGATGAT CGCCAAGAGC

6901 GAGCAGGAAA TCGGCAAGGC TACCGCCAAG TACTTCTTCT ACAGCAACAT CATGAACTTT

6961 TTCAAGACCG AGATTACCCT GGCCAACGGC GAGATCCGGA AGCGGCCTCT GATCGAGACA

7021 AACGGCGAAA CCGGGGAGAT CGTGTGGGAT AAGGGCCGGG ATTTTGCCAC CGTGCGGAAA

7081 GTGCTGAGCA TGCCCCAAGT GAATATCGTG AAAAAGACCG AGGTGCAGAC AGGCGGCTTC

7141 AGCAAAGAGT CTATCCTGCC CAAGAGGAAC AGCGATAAGC TGATCGCCAG AAAGAAGGAC

7201 TGGGACCCTA AGAAGTACGG CGGCTTCGAC AGCCCCACCG TGGCCTATTC TGTGCTGGTG

7261 GTGGCCAAAG TGGAAAAGGG CAAGTCCAAG AAACTGAAGA GTGTGAAAGA GCTGCTGGGG

7321 ATCACCATCA TGGAAAGAAG CAGCTTCGAG AAGAATCCCA TCGACTTTCT GGAAGCCAAG

7381 GGCTACAAAG AAGTGAAAAA GGACCTGATC ATCAAGCTGC CTAAGTACTC CCTGTTCGAG

7441 CTGGAAAACG GCCGGAAGAG AATGCTGGCC TCTGCCGGCG AACTGCAGAA GGGAAACGAA

7501 CTGGCCCTGC CCTCCAAATA TGTGAACTTC CTGTACCTGG CCAGCCACTA TGAGAAGCTG

7561 AAGGGCTCCC CCGAGGATAA TGAGCAGAAA CAGCTGTTTG TGGAACAGCA CAAGCACTAC

7621 CTGGACGAGA TCATCGAGCA GATCAGCGAG TTCTCCAAGA GAGTGATCCT GGCCGACGCT

7681 AATCTGGACA AAGTGCTGTC CGCCTACAAC AAGCACCGGG ATAAGCCCAT CAGAGAGCAG

7741 GCCGAGAATA TCATCCACCT GTTTACCCTG ACCAATCTGG GAGCCCCTGC CGCCTTCAAG

7801 TACTTTGACA CCACCATCGA CCGGAAGAGG TACACCAGCA CCAAAGAGGT GCTGGACGCC

7861 ACCCTGATCC ACCAGAGCAT CACCGGCCTG TACGAGACAC GGATCGACCT GTCTCAGCTG

7921 GGAGGCGACT CTAAAAGACC AGCAGCCACT AAAAAGGCCG GACAGGCTAA AAAGAAAAAG

7981 TAAGGTACCC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC

8041 CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG

8101 CATCGCATTG TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA

8161 AGGGGGAGGA TTGGGAAGAC AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGGGT

8221 ACCAAGGTCG GGCAGGAAGA GGGCCTATTT CCCATGATTC CTTCATATTT GCATATACGA

8281 TACAAGGCTG TTAGAGAGAT AATTGGAATT AATTTGACTG TAAACACAAA GATATTAGTA

8341 CAAAATACGT GACGTAGAAA GTAATAATTT CTTGGGTAGT TTGCAGTTTT AAAATTATGT

8401 TTTAAAATGG ACTATCATAT GCTTACCGTA ACTTGAAAGT ATTTCGATTT CTTGGCTTTA

8461 TATATCTTGT GGAAAGGACG AAACACCGGA ATCGAAGAAG GAGGGTTTTA GAGCTAGAAA

8521 TAGCAAGTTA AAATAAGGCT AGTCCGTTAT CAACTTGAAA AAGTGGCACC GAGTCGGTGC

8581 TTTTTTAAAA AAGCACCGAC TCGGTGCCAC TTTTTCAAGT TGATAACGGA CTAGCCTTAT

8641 TTTAACTTGC TATTTCTAGC TCTAAAACGG CCCTGGTGTG TAGTTTGGGA AAGAGTGGTC

8701 TCATACAGAA CTTATAAGAT TCCCAAATCC AAAGACATTT CACGTTTATG GTGATTTCCC

8761 AGAACACATA GCGACATGCA AATATTGCAG GGCGCCACTC CCCTGTCCCT CACAGCCATC

8821 TTCCTGCCAG GGCGCACGCG CGCTGGGTGT TCCCGCCTAG TGACACTGGG CCCGCGATTC

8881 CTTGGAGCGG GTTGATGACG TCAGCGTTCG AATTCCATGG CGGCGCGGCG GCGACGGAGC

8941 ACCGGCGGCG GCAGGGCGAG AGGTTCGGAG CTCAATATCG CGGGACGGCA TGCGGGGGGC

9001 GGGCAGTCAG AAAGGAACGA TGCCACCTAC TGTGACCCCC TTCCCTTCAG CTCCCTATAA

9061 TCTAGAGATC CGACGCCGCC ATCTCTAGGC CCGCGCCGGC CCCCTCGCAC AGACTTGTGG

9121 GAGAAGCTCG GCTACTCCCC TGCCCCGGTT AATTTGCATA TAATATTTCC TAGTAACTAT

9181 AGAGGCTTAA TGTGCGATAA AAGACAGATA ATCTGTTCTT TTTAATACTA GCTACATTTT

9241 ACATGATAGG CTTGGATTTC TATAAGAGAT ACAAATACTA AATTATTATT TTAAAAAACA

9301 GCACAAAAGG AAACTCACCC TAACTGTAAA GTAATTGTGT GTTTTGAGAC TATAAATATC

9361 CCTTGGAGAA AAGCCTTGTT TGATAGACCC ATAAATCAGT TTTAGAGCTA GAAATAGCAA

9421 GTTAAAATAA GGCTAGTCCG TTATCAACTT GAAAAAGTGG CACCGAGTCG GTGCTTTTTT

9481 AAAAAAGCAC CGACTCGGTG CCACTTTTTC AAGTTGATAA CGGACTAGCC TTATTTTAAC

9541 TTGCTATTTC TAGCTCTAAA ACTCAAATCA CTCTTTGGCG AGGTACCCAG GCGGCGCACA

9601 AGCTATATAA ACCTGAAGGA AATCTCAACT TTACACTTAG GTCAAGTTAC TTATCGTACT

9661 AGAGCTTCAG CAGGAAATTT AACTAAAATC TAATTTAACC AGCATAGCAA ATATCATTTA

9721 TTCCCAAAAT GCTAAAGTTT GAGATAAACG GACTTGATTT CCGGCTGTTT TGACACTATC

9781 CAGAATGCCT TGCAGATGGG TGGGGCATGC TAAATACTGC AGACTAGTTG CGGGGAGGCG

9841 GCCCAAAGGG AGATCCGACT CGTCTGAGGG CGAAGGCGAA GACGCGGAAG AGGCCGCAGA

9901 GCCGGCAGCA GGCCGCGGGA AGGAAGGTCC GCTGGATTGA GGGCCGAAGG GACGTAGCAG

9961 AAGGACGTCC CGCGCAGAAT CCAGGTGGCA ACACAGGCGA GCAGCCAAGG AAAGGACGAT

10021 GATTTCCCCG ACAACACCAC GGAATTGTCA GTGCCCAACA GCCGAGCCCC TGTCCAGCAG

10081 CGGGCAAGGC AGGCGGCGAT GAGTTCCGCC GTGGCAATAG GGAGGGGGAA AGCGAAAGTC

10141 CCGGAAAGGA GCTGACAGGT GGTGGCAATG CCCCAACCAG TGGGGGTTGC GTCAGCAAAC

10201 ACAGTGCACA CCACGCCACG TTGCCTGACA ACGGGCCACA ACTCCTCATA AAGAGACAGC

10261 AACCAGGATT TATACAAGGA GGAGAAAATG AAAGCCATAC GGGAAGCAAT AGCATGATAC

10321 AAAGGCATTA AAGCAGCGTA TCCACATAGC GTAAAAGGAG CAACATAGTT AAGAATACCA

10381 GTCAATCTTT CACAAATTTT GTAATCCAGA GGTTGAACGG GGACAGCCCC CCCCCAAAGC

10441 CCCCAGGGAT GTAATTACGT CCCTCCCCCG CTAGGGGGCA GCAGCGAGCC GCCCGGGGCT

10501 CCGCTCCGGT CCGGCGCTCC CCCCGCATCC CCGAGCCGGC AGCGTGCGGG GACAGCCCGG

10561 GCACGGGGAA GGTGGCACGG GATCGCTTTC CTCTGAACGC TTCTCGCTGC TCTTTGAGCC

10621 TGCAGACACC TGGGGGGATA CGGGGAAAAA TGGAAGGGCT AATTTGGTCC CAAAAAAGAC

10681 AAGAGGTATA TAAGCAGCTG CTTTTTGCCT GTACTGGGTC TCTCTGGTTA GACCAGATCT

10741 GAGCCTGGGA GCTCTCTGGC TAACTAGGGA ACCCACTGCT TAAGCCTCAA TAAAGCTTGC

10801 CTTGAGTGCT TCAAGTAGTG TGTGCCCGTC TGTTGTGTGA CTCTGGTAAC TAGAGATCCC

10861 TCAGACCCTT TTAGTCAGTG TGGAAAATCT CTAGCATTAA TTAA

Seq ID No.9：The sequence of plasmid p7

1 ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT

61 TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG

121 CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC

181 TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC

241 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC

301 AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC

361 TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT

421 AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT

481 AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC

541 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT

601 TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG

661 ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC

721 ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA

781 TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG

841 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG

901 TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA

961 GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG

1021 CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA

1081 GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC

1141 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA

1201 AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG

1261 ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT

1321 AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC

1381 AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG

1441 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG

1501 GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT

1561 GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA

1621 GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA

1681 CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC

1741 ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTT AATACGACTC ACTATAGGGC

1801 GAGGAATTCT GGAAGGGCTA ATTTGGTCCC AAAAAAGACA AGAGAATGCT TATGGACTAG

1861 GGACTGTTAC AAACAAATGA TGAATGGAAA CAGCTGAGCA TGACTCATAG CTGAAGCGCT

1921 AGCAGCTGCT TAACCGCAAA ACCACATCCT ATGTAAAGCT TGCTAATGAC GTATAAGTTG

1981 CTCCACTGTA AAAGTATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA

2041 CCAGATCTGA GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA

2101 AAGCTTGCCT TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA

2161 GAGATCCCTC AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG

2221 GACTTGAAAG CGAAAGGGAA ACCAGAGGAG CTCTCTCGAC GCAGGACTCG GCTTGCTGAA

2281 GCGCGCACGG CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC

2341 GGAGGCTAGA AGGAGAGAGA TGGGTGCGAG AGCGTCAGTA TTAAGCGGGG GAGAATTAGA

2401 TCGCGATGGG AAAAAATTCG GTTAAGGCCA GGGGGAAAGA AAAAATATAA ATTAAAACAT

2461 ATAGTATGGG CAAGCAGGGA GCTAGAACGA TTCGCAGTTA ATCCTGGCCT GTTAGAAACA

2521 TCAGAAGGCT GTAGACAAAT ACTGGGACAG CTACAACCAT CCCTTCAGAC AGGATCAGAA

2581 GAACTTAGAT CATTATATAA TACAGTAGCA ACCCTCTATT GTGTGCATCA AAGGATAGAG

2641 ATAAAAGACA CCAAGGAAGC TTTAGACAAG ATAGAGGAAG AGCAAAACAA AAGTAAGACC

2701 ACCGCACAGC AAGCGGCCGG CCGCTGATCT TCAGACCTGG AGGAGGAGAT ATGAGGGACA

2761 ATTGGAGAAG TGAATTATAT AAATATAAAG TAGTAAAAAT TGAACCATTA GGAGTAGCAC

2821 CCACCAAGGC AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AGCAGTGGGA ATAGGAGCTT

2881 TGTTCCTTGG GTTCTTGGGA GCAGCAGGAA GCACTATGGG CGCAGCGTCA ATGACGCTGA

2941 CGGTACAGGC CAGACAATTA TTGTCTGGTA TAGTGCAGCA GCAGAACAAT TTGCTGAGGG

3001 CTATTGAGGC GCAACAGCAT CTGTTGCAAC TCACAGTCTG GGGCATCAAG CAGCTCCAGG

3061 CAAGAATCCT GGCTGTGGAA AGATACCTAA AGGATCAACA GCTCCTTTTA AAAGAAAAGG

3121 GGGGATTGGG GGGTACAGTG CAGGGGAAAG AATAGTAGAC ATAATAGCAA CAGACATACA

3181 AACTAAAGAA TTACAAAAAC AAATTACAAA AATTCAAAAT TTTCGGGTTT CTCGAGGTGG

3241 AGAAGAGCAT GCTTGAGGGC TGAGTGCCCC TCAGTGGGCA GAGAGCACAT GGCCCACAGT

3301 CCCTGAGAAG TTGGGGGGAG GGGTGGGCAA TTGAACTGGT GCCTAGAGAA GGTGGGGCTT

3361 GGGTAAACTG GGAAAGTGAT GTGGTGTACT GGCTCCACCT TTTTCCCCAG GGTGGGGGAG

3421 AACCATATAT AAGTGCAGTA GTCTCTGTGA ACATTCTCCC TATCAGTGAT AGAGAACTCC

3481 CTATCAGTGA TAGAGAGGAT CCTGAACCGG TATGCCCAAG AAGAAACGCA AAGTCGCTTC

3541 AGACAAGAAG TACAGCATCG GCCTGGACAT CGGCACCAAC TCTGTGGGCT GGGCCGTGAT

3601 CACCGACGAG TACAAGGTGC CCAGCAAGAA ATTCAAGGTG CTGGGCAACA CCGACCGGCA

3661 CAGCATCAAG AAGAACCTGA TCGGAGCCCT GCTGTTCGAC AGCGGCGAAA CAGCCGAGGC

3721 CACCCGGCTG AAGAGAACCG CCAGAAGAAG ATACACCAGA CGGAAGAACC GGATCTGCTA

3781 TCTGCAAGAG ATCTTCAGCA ACGAGATGGC CAAGGTGGAC GACAGCTTCT TCCACAGACT

3841 GGAAGAGTCC TTCCTGGTGG AAGAGGATAA GAAGCACGAG CGGCACCCCA TCTTCGGCAA

3901 CATCGTGGAC GAGGTGGCCT ACCACGAGAA GTACCCCACC ATCTACCACC TGAGAAAGAA

3961 ACTGGTGGAC AGCACCGACA AGGCCGACCT GCGGCTGATC TATCTGGCCC TGGCCCACAT

4021 GATCAAGTTC CGGGGCCACT TCCTGATCGA GGGCGACCTG AACCCCGACA ACAGCGACGT

4081 GGACAAGCTG TTCATCCAGC TGGTGCAGAC CTACAACCAG CTGTTCGAGG AAAACCCCAT

4141 CAACGCCAGC GGCGTGGACG CCAAGGCCAT CCTGTCTGCC AGACTGAGCA AGAGCAGACG

4201 GCTGGAAAAT CTGATCGCCC AGCTGCCCGG CGAGAAGAAG AATGGCCTGT TCGGAAACCT

4261 GATTGCCCTG AGCCTGGGCC TGACCCCCAA CTTCAAGAGC AACTTCGACC TGGCCGAGGA

4321 TGCCAAACTG CAGCTGAGCA AGGACACCTA CGACGACGAC CTGGACAACC TGCTGGCCCA

4381 GATCGGCGAC CAGTACGCCG ACCTGTTTCT GGCCGCCAAG AACCTGTCCG ACGCCATCCT

4441 GCTGAGCGAC ATCCTGAGAG TGAACACCGA GATCACCAAG GCCCCCCTGA GCGCCTCTAT

4501 GATCAAGAGA TACGACGAGC ACCACCAGGA CCTGACCCTG CTGAAAGCTC TCGTGCGGCA

4561 GCAGCTGCCT GAGAAGTACA AAGAGATTTT CTTCGACCAG AGCAAGAACG GCTACGCCGG

4621 CTACATTGAC GGCGGAGCCA GCCAGGAAGA GTTCTACAAG TTCATCAAGC CCATCCTGGA

4681 AAAGATGGAC GGCACCGAGG AACTGCTCGT GAAGCTGAAC AGAGAGGACC TGCTGCGGAA

4741 GCAGCGGACC TTCGACAACG GCAGCATCCC CCACCAGATC CACCTGGGAG AGCTGCACGC

4801 CATTCTGCGG CGGCAGGAAG ATTTTTACCC ATTCCTGAAG GACAACCGGG AAAAGATCGA

4861 GAAGATCCTG ACCTTCCGCA TCCCCTACTA CGTGGGCCCT CTGGCCAGGG GAAACAGCAG

4921 ATTCGCCTGG ATGACCAGAA AGAGCGAGGA AACCATCACC CCCTGGAACT TCGAGGAAGT

4981 GGTGGACAAG GGCGCTTCCG CCCAGAGCTT CATCGAGCGG ATGACCGCTT TCGATAAGAA

5041 CCTGCCCAAC GAGAAGGTGC TGCCCAAGCA CAGCCTGCTG TACGAGTACT TCACCGTGTA

5101 TAACGAGCTG ACCAAAGTGA AATACGTGAC CGAGGGAATG AGAAAGCCCG CCTTCCTGAG

5161 CGGCGAGCAG AAAAAGGCCA TCGTGGACCT GCTGTTCAAG ACCAACCGGA AAGTGACCGT

5221 GAAGCAGCTG AAAGAGGACT ACTTCAAGAA AATCGAGTGC TTCGACTCCG TGGAAATCTC

5281 CGGCGTGGAA GATCGGTTCA ACGCCTCCCT GGGCACATAC CACGATCTGC TGAAAATTAT

5341 CAAGGACAAG GACTTCCTGG ACAATGAGGA AAACGAGGAC ATTCTGGAAG ATATCGTGCT

5401 GACCCTGACA CTGTTTGAGG ACAGAGAGAT GATCGAGGAA CGGCTGAAAA CCTATGCCCA

5461 CCTGTTCGAC GACAAAGTGA TGAAGCAGCT GAAGCGGCGG AGATACACCG GCTGGGGAGC

5521 ACTGAGCCGG AAGCTGATCA ACGGCATCCG GGACAAGCAG TCCGGCAAGA CAATCCTGGA

5581 TTTCCTGAAG TCCGACGGCT TCGCCAACAG AAACTTCATG GCTCTGATCC ACGACGACAG

5641 CCTGACCTTT AAAGAGGACA TCCAGAAAGC CCAGGTGTCC GGCCAGGGCG ATAGCCTGCA

5701 CGAGCACATT GCCAATCTGG CCGGCAGCCC CGCCATTAAG AAGGGCATCC TGCAGACAGT

5761 GAAGGTGGTG GACGAGCTCG TGAAAGTGAT GGGCCGGCAC AAGCCCGAGA ACATCGTGAT

5821 CGAAATGGCC AGAGAGAACC AGACCACCCA GAAGGGACAG AAGAACAGCC GCGAGAGAAT

5881 GAAGCGGATC GAAGAGGGCA TCAAAGAGCT GGGCAGCCAG ATCCTGAAAG AACACCCCGT

5941 GGAAAACACC CAGCTGCAGA ACGAGAAGCT GTACCTGTAC TACCTGCAGA ATGGGCGGGA

6001 TATGTACGTG GACCAGGAAC TGGACATCAA CCGGCTGTCC GACTACGATG TGGACCATAT

6061 CGTGCCTCAG AGCTTTCTGA AGGACGACTC CATCGACAAC AAGGTGCTGA CCAGAAGCGA

6121 CAAGAACCGG GGCAAGAGCG ACAACGTGCC CTCCGAAGAG GTCGTGAAGA AGATGAAGAA

6181 CTACTGGCGG CAGCTGCTGA ACGCCAAGCT GATTACCCAG AGAAAGTTCG ACAATCTGAC

6241 CAAGGCCGAG AGAGGCGGCC TGAGCGAACT GGATAAGGCC GGCTTCATCA AGAGACAGCT

6301 GGTGGAAACC AGGGCAATCA CAAAGCACGT GGCACAGATC CTGGACTCCC GGATGAACAC

6361 TAAGTACGAC GAGAATGACA AGCTGATCCG GGAAGTGAAA GTGATCACCC TGAAGTCCAA

6421 GCTGGTGTCC GATTTCCGGA AGGATTTCCA GTTTTACAAA GTGCGCGAGA TCAACAACTA

6481 CCACCACGCC CACGACGCCT ACCTGAACGC CGTCGTGGGA ACCGCCCTGA TCAAAAAGTA

6541 CCCTAAGCTG GAAAGCGAGT TCGTGTACGG CGACTACAAG GTGTACGACG TGCGGAAGAT

6601 GATCGCCAAG AGCGAGCAGG AAATCGGCAA GGCTACCGCC AAGTACTTCT TCTACAGCAA

6661 CATCATGAAC TTTTTCAAGA CCGAGATTAC CCTGGCCAAC GGCGAGATCC GGAAGCGGCC

6721 TCTGATCGAG ACAAACGGCG AAACCGGGGA GATCGTGTGG GATAAGGGCC GGGATTTTGC

6781 CACCGTGCGG AAAGTGCTGA GCATGCCCCA AGTGAATATC GTGAAAAAGA CCGAGGTGCA

6841 GACAGGCGGC TTCAGCAAAG AGTCTATCCT GCCCAAGAGG AACAGCGATA AGCTGATCGC

6901 CAGAAAGAAG GACTGGGACC CTAAGAAGTA CGGCGGCTTC GACAGCCCCA CCGTGGCCTA

6961 TTCTGTGCTG GTGGTGGCCA AAGTGGAAAA GGGCAAGTCC AAGAAACTGA AGAGTGTGAA

7021 AGAGCTGCTG GGGATCACCA TCATGGAAAG AAGCAGCTTC GAGAAGAATC CCATCGACTT

7081 TCTGGAAGCC AAGGGCTACA AAGAAGTGAA AAAGGACCTG ATCATCAAGC TGCCTAAGTA

7141 CTCCCTGTTC GAGCTGGAAA ACGGCCGGAA GAGAATGCTG GCCTCTGCCG GCGAACTGCA

7201 GAAGGGAAAC GAACTGGCCC TGCCCTCCAA ATATGTGAAC TTCCTGTACC TGGCCAGCCA

7261 CTATGAGAAG CTGAAGGGCT CCCCCGAGGA TAATGAGCAG AAACAGCTGT TTGTGGAACA

7321 GCACAAGCAC TACCTGGACG AGATCATCGA GCAGATCAGC GAGTTCTCCA AGAGAGTGAT

7381 CCTGGCCGAC GCTAATCTGG ACAAAGTGCT GTCCGCCTAC AACAAGCACC GGGATAAGCC

7441 CATCAGAGAG CAGGCCGAGA ATATCATCCA CCTGTTTACC CTGACCAATC TGGGAGCCCC

7501 TGCCGCCTTC AAGTACTTTG ACACCACCAT CGACCGGAAG AGGTACACCA GCACCAAAGA

7561 GGTGCTGGAC GCCACCCTGA TCCACCAGAG CATCACCGGC CTGTACGAGA CACGGATCGA

7621 CCTGTCTCAG CTGGGAGGCG ACTCTAAAAG ACCAGCAGCC ACTAAAAAGG CCGGACAGGC

7681 TAAAAAGAAA AAGTAAGGTA CCCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC

7741 CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA

7801 AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT CTATTCTGGG GGGTGGGGTG

7861 GGGCAGGACA GCAAGGGGGA GGATTGGGAA GACAATAGCA GGCATGCTGG GGATGCGGTG

7921 GGCTCTATGG GGTACCGATT CCGGGTCACT GTGAGTGGGG GAGGCAGGGA AGAAGGGCTC

7981 ACAGGACAGT CAAACCATGC CCCCTGTTTT TCCTTCTTCA AGTAGACCTC TATAAGACAA

8041 CAGAGACAAC TAAGGCTGAG TGGCCAGGCG AGGAGAAACC ATCTCGCCGT AAAACATGGA

8101 AGGAACACTT CAGGGGAAAG GTGGTATCTC TAAGCAAGAG AACTGAGTGG AGTCAAGGCT

8161 GAGAGATGCA GGATAAGCAA ATGGGTAGTG AAAAGACATT CATGAGGACA GCTAAAACAA

8221 TAAGTAATGT AAAATACAGC ATAGCAAAAC TTTAACCTCC AAATCAAGCC TCTACTTGAA

8281 TCCTTTTCTG AGGGATGAAT AAGGCATAGG CATCAGGGGC TGTTGCCAAT GTGCATTAGC

8341 TGTTTGCAGC CTCACCTTCT TTCATGGAGT TTAAGATATA GTGTATTTTC CCAAGGTTTG

8401 AACTAGCTCT TCATTTCTTT ATGTTTTAAA TGCACTGACC TCCCACATTC CCTTTTTAGT

8461 AAAATATTCA GAAATAATTT AAATACATCA TTGCAATGAA AATAAATGTT TTTTATTAGG

8521 CAGAATCCAG ATGCTCAAGG CCCTTCATAA TATCCCCCAG TTTAGTAGTT GGACTTAGGG

8581 AACAAAGGAA CCTTTAATAG AAATTGGACA GCAAGAAAGC GAGCTTAGTG ATACTTGTGG

8641 GCCAGGGCAT TAGCCACACC AGCCACCACT TTCTGATAGG CAGCCTGCAC TGGTGGGGTG

8701 AATTAATAAG ATCTGAATTC CCGGGATCCG CTGTACGCGG ACCCACTTTC ACATTTAAGT

8761 TGTTTTTCTA ATCCGCATAT GATCAATTCA AGGCCGAATA AGAAGGCTGG CTCTGCACCT

8821 TGGTGATCAA ATAATTCGAT AGCTTGTCGT AATAATGGCG GCATACTATC AGTAGTAGGT

8881 GTTTCCCTTT CTTCTTTAGC GACTTGATGC TCTTGATCTT CCAATACGCA ACCTAAAGTA

8941 AAATGCCCCA CAGCGCTGAG TGCATATAAT GCATTCTCTA GTGAAAAACC TTGTTGGCAT

9001 AAAAAGGCTA ATTGATTTTC GAGAGTTTCA TACTGTTTTT CTGTAGGCCG TGTACCTAAA

9061 TGTACTTTTG CTCCATCGCG ATGACTTAGT AAAGCACATC TAAAACTTTT AGCGTTATTA

9121 CGTAAAAAAT CTTGCCAGCT TTCCCCTTCT AAAGGGCAAA AGTGAGTATG GTGCCTATCT

9181 AACATCTCAA TGGCTAAGGC GTCGAGCAAA GCCCGCTTAT TTTTTACATG CCAATACAAT

9241 GTAGGCTGCT CTACACCTAG CTTCTGGGCG AGTTTACGGG TTGTTAAACC TTCGATTCCG

9301 ACCTCATTAA GCAGCTCTAA TGCGCTGTTA ATCACTTTAC TTTTATCTAA TCTAGACATG

9361 GTTTAGTTCC TCACCTTGTC GTATTATACT ATGCCGATAT ACTATGCCGA TGATTAATTG

9421 TCAACACGTG CTGCTGCAGG TCGAAAGGCC CGGAGATGAG GAAGAGGAGA ACAGCGCGGC

9481 AGACGTGCGC TTTTGAAGCG TGCAGAATGC CGGGCCTCCG GAGGACCTTC GGGCGCCCGC

9541 CCCGCCCCTG AGCCCGCCCC TGAGCCCGCC CCCGGACCCA CCCCTTCCCA GCCTCTGAGC

9601 CCAGAAAGCG AAGGAGTAAA GCTGCTATTG GCCGCTGCCC CAAAGGCCTA CCCGCTTCCA

9661 TTGCTCAGCG GTGCTGTCCA TCTGCACGAG ACTAGTGAGA CGTGCTACTT CCATTTGTCA

9721 CGTCCTGCAC GACGCGAGCT GCGGGGCGGG GGGGAACTTC CTGACTAGGG GAGGAGTAGA

9781 AGGTGGCGCG AAGGGGCCAC CAAAGAACGG AGCCGGTTGG CGCCTACCGG TGGATGTGGA

9841 ATGTGTGCGA GGCCAGAGGC CACTTGTGTA GCGCCAAGTG CCCAGCGGGG CTGCTAAAGC

9901 GCATGCTCCA GACTGCCTTG GGAAAAGCGC CTCCCCTACC CGGTAGGCGG CCGCATACAC

9961 TAGTTGGAAG GGCTAATTTG GTCCCAAAAA AGACAAGAGG TATATAAGCA GCTGCTTTTT

10021 GCCTGTACTG GGTCTCTCTG GTTAGACCAG ATCTGAGCCT GGGAGCTCTC TGGCTAACTA

10081 GGGAACCCAC TGCTTAAGCC TCAATAAAGC TTGCCTTGAG TGCTTCAAGT AGTGTGTGCC

10141 CGTCTGTTGT GTGACTCTGG TAACTAGAGA TCCCTCAGAC CCTTTTAGTC AGTGTGGAAA

10201 ATCTCTAGCA TTAATTAA

Seq ID No.10：ARRPKFYRAPYVKNHPNVWGPWVAYGP

Seq ID No.11：H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg- NH2

Seq ID No.12：H-ARRPKFYRAPYVKNHPNVWGPWVAYGPSASTTTNYT-NH2

Seq ID No.13：H-ASTTTNYTSARRPKFYRAPYVKNHPNVWGPWVAYGP-NH2

Seq ID No.14：H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg- Ser-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH2

Seq ID No.15：Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Ser-Arg-Arg-Nal-Cys-Tyr-Cit- Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2

Seq ID No.16：Ala-Arg-Arg-Pro-Lys-Phe-Tyr-Arg-Ala-Pro-Tyr-Val-Lys-Asn-His- Pro-Asn-Val-Trp-Gly-Pro-Trp-Val-Ala-Tyr-Gly-Pro-Arg-Arg-Nal-Cys-Tyr-Cit-Lys- D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2

Seq ID No.17：H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-D-Lys-Pro-Tyr-Arg-Cit-Cys-Arg- Ala-Arg-Arg-Pro-Lys-Phe-Tyr-Arg-Ala-Pro-Tyr-Val-Lys-Asn-His-Pro-Asn-Val-Trp- Gly-Pro-Trp-Val-Ala-Tyr-Gly-Pro-NH2

Seq ID No.18：

1 ATGAAATACCTGCTGCCGACGGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCG

1 M K Y L L P T A A A G L L L L A A Q P A

61 ATGGCCAGTCAGGTGCAGTTACAGCAATGGGGCGCAGGTCTGTTGAAACCTTCGGAAACC

21 M A S Q V Q L Q Q W G A G L L K P S E T

121 CTGTCCCTTACCTGCGCTGTCTATGGTGGCTCCTTTAGTGGTTACTACTGGAGCTGGATT

41 L S L T C A V Y G G S F S G Y Y W S W I

181 CGTCAGCCGCCAGGTAAAGGGCTGGAATGGATTGGGGAAATCAATCATAGTGGAAGCACC

61 R Q P P G K G L E W I G E I N H S G S T

241 AACTACAACCCGTCCCTGAAGAGTCGTGTCACCATTTCAGTCGATACGTCCAAGAACCAG

81 N Y N P S L K S R V T I S V D T S K N Q

301 TTTTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGATACGGCTGTGTATTACTGTGCGAGA

101 F S L K L S S V T A A D T A V Y Y C A R

361 GTAATTAATTGGTTCGATCCCTGGGGCCAAGGTACCCTGGTCACCGTCTCCTCAGGTGGC

121 V I N W F D P W G Q G T L V T V S S G G

421 GGTGGCTCTGGCGGTGGAGGGAGTGGTGGCGGTGGCTCTGATATTCAGATGACCCAGTCT

141 G G S G G G G S G G G G S D I Q M T Q S

481 CCATCTTCCGTGTCTGCGTCTGTAGGAGACCGCGTCACCATCACTTGTCGGGCGAGTCAG

161 P S S V S A S V G D R V T I T C R A S Q

541 GATATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACCAGGCAAAGCCCCTAAGTTACTG

181 D I S S W L A W Y Q H K P G K A P K L L

601 ATCTATGCTGCGTCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCT

201 I Y A A S S L Q S G V P S R F S G S G S

661 GGGACAGATTTCACTCTCACCATTAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTAT

221 G T D F T L T I S S L Q P E D F A T Y Y

721 TGTCAACAGGCTAATAGTTTCCCGTACACTTTTGGCCAGGGTACCAAACTGGAGATCAAA

241 C Q Q A N S F P Y T F G Q G T K L E I K

781 TAA

261 *

Seq ID No. 19：

1 ATGAAATACCTGCTGCCGACGGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCG

1 M K Y L L P T A A A G L L L L A A Q P A

61 ATGGCCGCAAGCACCACGACTAATTATACCAGTCAGGTGCAGTTACAGCAATGGGGCGCA

21 M A A S T T T N Y T S Q V Q L Q Q W G A

121 GGTCTGTTGAAACCTTCGGAAACCCTGTCCCTTACCTGCGCTGTCTATGGTGGCTCCTTT

41 G L L K P S E T L S L T C A V Y G G S F

181 AGTGGTTACTACTGGAGCTGGATTCGTCAGCCGCCAGGTAAAGGGCTGGAATGGATTGGG

61 S G Y Y W S W I R Q P P G K G L E W I G

241 GAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTGAAGAGTCGTGTCACCATT

81 E I N H S G S T N Y N P S L K S R V T I

301 TCAGTCGATACGTCCAAGAACCAGTTTTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGAT

101 S V D T S K N Q F S L K L S S V T A A D

361 ACGGCTGTGTATTACTGTGCGAGAGTAATTAATTGGTTCGATCCCTGGGGCCAAGGTACC

121 T A V Y Y C A R V I N W F D P W G Q G T

421 CTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCTGGCGGTGGAGGGAGTGGTGGCGGTGGC

141 L V T V S S G G G G S G G G G S G G G G

481 TCTGATATTCAGATGACCCAGTCTCCATCTTCCGTGTCTGCGTCTGTAGGAGACCGCGTC

161 S D I Q M T Q S P S S V S A S V G D R V

541 ACCATCACTTGTCGGGCGAGTCAGGATATTAGCAGCTGGTTAGCCTGGTATCAGCATAAA

181 T I T C R A S Q D I S S W L A W Y Q H K

601 CCAGGCAAAGCCCCTAAGTTACTGATCTATGCTGCGTCCAGTTTGCAAAGTGGGGTCCCA

201 P G K A P K L L I Y A A S S L Q S G V P

661 TCACGTTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAGCCTGCAG

221 S R F S G S G S G T D F T L T I S S L Q

721 CCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGTACACTTTTGGC

241 P E D F A T Y Y C Q Q A N S F P Y T F G

781 CAGGGTACCAAACTGGAGATCAAATAA

261 Q G T K L E I K *

Seq ID No.20, the sequence of plasmid p5

1 ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT

61 TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG

121 CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC

181 TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC

241 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC

301 AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC

361 TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT

421 AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT

481 AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC

541 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT

601 TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG

661 ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC

721 ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA

781 TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG

841 GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG

901 TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA

961 GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG

1021 CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA

1081 GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC

1141 ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA

1201 AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG

1261 ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT

1321 AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC

1381 AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG

1441 GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG

1501 GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT

1561 GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA

1621 GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA

1681 CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC

1741 ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTT AATACGACTC ACTATAGGGC

1801 GAGGAATTCT GGAAGGGCTA ATTTGGTCCC AAAAAAGACA AGAGAATGCT TATGGACTAG

1861 GGACTGTTAC AAACAAATGA TGAATGGAAA CAGCTGAGCA TGACTCATAG CTGAAGCGCT

1921 AGCAGCTGCT TAACCGCAAA ACCACATCCT ATGTAAAGCT TGCTAATGAC GTATAAGTTG

1981 CTCCACTGTA AAAGTATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA

2041 CCAGATCTGA GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA

2101 AAGCTTGCCT TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA

2161 GAGATCCCTC AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG

2221 GACTTGAAAG CGAAAGGGAA ACCAGAGGAG CTCTCTCGAC GCAGGACTCG GCTTGCTGAA

2281 GCGCGCACGG CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC

2341 GGAGGCTAGA AGGAGAGAGA TGGGTGCGAG AGCGTCAGTA TTAAGCGGGG GAGAATTAGA

2401 TCGCGATGGG AAAAAATTCG GTTAAGGCCA GGGGGAAAGA AAAAATATAA ATTAAAACAT

2461 ATAGTATGGG CAAGCAGGGA GCTAGAACGA TTCGCAGTTA ATCCTGGCCT GTTAGAAACA

2521 TCAGAAGGCT GTAGACAAAT ACTGGGACAG CTACAACCAT CCCTTCAGAC AGGATCAGAA

2581 GAACTTAGAT CATTATATAA TACAGTAGCA ACCCTCTATT GTGTGCATCA AAGGATAGAG

2641 ATAAAAGACA CCAAGGAAGC TTTAGACAAG ATAGAGGAAG AGCAAAACAA AAGTAAGACC

2701 ACCGCACAGC AAGCGGCCGG CCGCTGATCT TCAGACCTGG AGGAGGAGAT ATGAGGGACA

2761 ATTGGAGAAG TGAATTATAT AAATATAAAG TAGTAAAAAT TGAACCATTA GGAGTAGCAC

2821 CCACCAAGGC AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AGCAGTGGGA ATAGGAGCTT

2881 TGTTCCTTGG GTTCTTGGGA GCAGCAGGAA GCACTATGGG CGCAGCGTCA ATGACGCTGA

2941 CGGTACAGGC CAGACAATTA TTGTCTGGTA TAGTGCAGCA GCAGAACAAT TTGCTGAGGG

3001 CTATTGAGGC GCAACAGCAT CTGTTGCAAC TCACAGTCTG GGGCATCAAG CAGCTCCAGG

3061 CAAGAATCCT GGCTGTGGAA AGATACCTAA AGGATCAACA GCTCCTGGGG ATTTGGGGTT

3121 GCTCTGGAAA ACTCATTTGC ACCACTGCTG TGCCTTGGAA TGCTAGTTGG AGTAATAAAT

3181 CTCTGGAACA GATTTGGAAT CACACGACCT GGATGGAGTG GGACAGAGAA ATTAACAATT

3241 ACACAAGCTT AATACACTCC TTAATTGAAG AATCGCAAAA CCAGCAAGAA AAGAATGAAC

3301 AAGAATTATT GGAATTAGAT AAATGGGCAA GTTTGTGGAA TTGGTTTAAC ATAACAAATT

3361 GGCTGTGGTA TATAAAATTA TTCATAATGA TAGTAGGAGG CTTGGTAGGT TTAAGAATAG

3421 TTTTTGCTGT ACTTTCTATA GTGAATAGAG TTAGGCAGGG ATATTCACCA TTATCGTTTC

3481 AGACCCACCT CCCAACCCCG AGGGGACCCG ACAGGCCCGA AGGAATAGAA GAAGAAGGTG

3541 GAGAGAGAGA CAGAGACAGA TCCATTCGAT TAGTGAACGG ATCTCGACGG TATCGCCTTT

3601 AAAAGAAAAG GGGGGATTGG GGGGTACAGT GCAGGGGAAA GAATAGTAGA CATAATAGCA

3661 ACAGACATAC AAACTAAAGA ATTACAAAAA CAAATTACAA AAATTCAAAA TTTTCGGGTT

3721 TCTCGAGACG GGGACAGCCC CCCCCCAAAG CCCCCAGGGA TGTAATTACG TCCCTCCCCC

3781 GCTAGGGGGC AGCAGCGAGC CGCCCGGGGC TCCGCTCCGG TCCGGCGCTC CCCCCGCATC

3841 CCCGAGCCGG CAGCGTGCGG GGACAGCCCG GGCACGGGGA AGGTGGCACG GGATCGCTTT

3901 CCTCTGAACG CTTCTCGCTG CTCTTTGAGC CTGCAGACAC CTGGGGGGAT ACGGGGAAAA

3961 AGGATCCAAG GTCGGGCAGG AAGAGGGCCT ATTTCCCATG ATTCCTTCAT ATTTGCATAT

4021 ACGATACAAG GCTGTTAGAG AGATAATTGG AATTAATTTG ACTGTAAACA CAAAGATATT

4081 AGTACAAAAT ACGTGACGTA GAAAGTAATA ATTTCTTGGG TAGTTTGCAG TTTTAAAATT

4141 ATGTTTTAAA ATGGACTATC ATATGCTTAC CGTAACTTGA AAGTATTTCG ATTTCTTGGC

4201 TTTATATATC TTGTGGAAAG GACGAAACAC CGGAATCGAA GAAGGAGGGT TTTAGAGCTA

4261 GAAATAGCAA GTTAAAATAA GGCTAGTCCG TTATCAACTT GAAAAAGTGG CACCGAGTCG

4321 GTGCTTTTTT AAAAAAGCAC CGACTCGGTG CCACTTTTTC AAGTTGATAA CGGACTAGCC

4381 TTATTTTAAC TTGCTATTTC TAGCTCTAAA ACGGCCCTGG TGTGTAGTTT GGGAAAGAGT

4441 GGTCTCATAC AGAACTTATA AGATTCCCAA ATCCAAAGAC ATTTCACGTT TATGGTGATT

4501 TCCCAGAACA CATAGCGACA TGCAAATATT GCAGGGCGCC ACTCCCCTGT CCCTCACAGC

4561 CATCTTCCTG CCAGGGCGCA CGCGCGCTGG GTGTTCCCGC CTAGTGACAC TGGGCCCGCG

4621 ATTCCTTGGA GCGGGTTGAT GACGTCAGCG TTCGAATTCC ATGGCGGCGC GGCGGCGACG

4681 GAGCACCGGC GGCGGCAGGG CGAGAGGTTC GGAGCTCAAT ATCGCGGGAC GGCATGCGGG

4741 GGGCGGGCAG TCAGAAAGGA ACGATGCCAC CTACTGTGAC CCCCTTCCCT TCAGCTCCCT

4801 ATAAACCGGT GATCCGACGC CGCCATCTCT AGGCCCGCGC CGGCCCCCTC GCACAGACTT

4861 GTGGGAGAAG CTCGGCTACT CCCCTGCCCC GGTTAATTTG CATATAATAT TTCCTAGTAA

4921 CTATAGAGGC TTAATGTGCG ATAAAAGACA GATAATCTGT TCTTTTTAAT ACTAGCTACA

4981 TTTTACATGA TAGGCTTGGA TTTCTATAAG AGATACAAAT ACTAAATTAT TATTTTAAAA

5041 AACAGCACAA AAGGAAACTC ACCCTAACTG TAAAGTAATT GTGTGTTTTG AGACTATAAA

5101 TATCCCTTGG AGAAAAGCCT TGTTTGATAG ACCCATAAAT CAGTTTTAGA GCTAGAAATA

5161 GCAAGTTAAA ATAAGGCTAG TCCGTTATCA ACTTGAAAAA GTGGCACCGA GTCGGTGCTT

5221 TTTTAAAAAA GCACCGACTC GGTGCCACTT TTTCAAGTTG ATAACGGACT AGCCTTATTT

5281 TAACTTGCTA TTTCTAGCTC TAAAACTCAA ATCACTCTTT GGCGAGGTAC CCAGGCGGCG

5341 CACAAGCTAT ATAAACCTGA AGGAAATCTC AACTTTACAC TTAGGTCAAG TTACTTATCG

5401 TACTAGAGCT TCAGCAGGAA ATTTAACTAA AATCTAATTT AACCAGCATA GCAAATATCA

5461 TTTATTCCCA AAATGCTAAA GTTTGAGATA AACGGACTTG ATTTCCGGCT GTTTTGACAC

5521 TATCCAGAAT GCCTTGCAGA TGGGTGGGGC ATGCTAAATA CTGCAGGGTA CCTGCGGGGA

5581 GGCGGCCCAA AGGGAGATCC GACTCGTCTG AGGGCGAAGG CGAAGACGCG GAAGAGGCCG

5641 CAGAGCCGGC AGCAGGCCGC GGGAAGGAAG GTCCGCTGGA TTGAGGGCCG AAGGGACGTA

5701 GCAGAAGGAC GTCCCGCGCA GAATCCAGGT GGCAACACAG GCGAGCAGCC AAGGAAAGGA

5761 CGATGATTTC CCCGACAACA CCACGGAATT GTCAGTGCCC AACAGCCGAG CCCCTGTCCA

5821 GCAGCGGGCA AGGCAGGCGG CGATGAGTTC CGCCGTGGCA ATAGGGAGGG GGAAAGCGAA

5881 AGTCCCGGAA AGGAGCTGAC AGGTGGTGGC AATGCCCCAA CCAGTGGGGG TTGCGTCAGC

5941 AAACACAGTG CACACCACGC CACGTTGCCT GACAACGGGC CACAACTCCT CATAAAGAGA

6001 CAGCAACCAG GATTTATACA AGGAGGAGAA AATGAAAGCC ATACGGGAAG CAATAGCATG

6061 ATACAAAGGC ATTAAAGCAG CGTATCCACA TAGCGTAAAA GGAGCAACAT AGTTAAGAAT

6121 ACCAGTCAAT CTTTCACAAA TTTTGTAATC CAGAGGTTGA ACGGGGACAG CCCCCCCCCA

6181 AAGCCCCCAG GGATGTAATT ACGTCCCTCC CCCGCTAGGG GGCAGCAGCG AGCCGCCCGG

6241 GGCTCCGCTC CGGTCCGGCG CTCCCCCCGC ATCCCCGAGC CGGCAGCGTG CGGGGACAGC

6301 CCGGGCACGG GGAAGGTGGC ACGGGATCGC TTTCCTCTGA ACGCTTCTCG CTGCTCTTTG

6361 AGCCTGCAGA CACCTGGGGG GATACGGGGA AAAATCTAGA TGGAAGGGCT AATTTGGTCC

6421 CAAAAAAGAC AAGAGGTATA TAAGCAGCTG CTTTTTGCCT GTACTGGGTC TCTCTGGTTA

6481 GACCAGATCT GAGCCTGGGA GCTCTCTGGC TAACTAGGGA ACCCACTGCT TAAGCCTCAA

6541 TAAAGCTTGC CTTGAGTGCT TCAAGTAGTG TGTGCCCGTC TGTTGTGTGA CTCTGGTAAC

6601 TAGAGATCCC TCAGACCCTT TTAGTCAGTG TGGAAAATCT CTAGCAACTA GTAGCAAGGT

6661 CGCCACGCAC AAGATCAATA TTAACAATCA GTCATCTCTC TTTAGCAATA AAAAGGTGAA

6721 AAATTACATT TTAAAAATGA CACCATAGAC GATGTATGAA AATAATCTAC TTGGAAATAA

6781 ATCTAGGCAA AGAAGTGCAA GACTGTTACC CAGAAAACTT ACAAATTGTA AATGAGAGGT

6841 TAGTGAAGAT TTAAATGAAT GAAGATCTAA ATAAACTTAT AAATTGTGAG AGAAATTAAT

6901 GAATGTCTAA GTTAATGCAG AAACGGAGAG ACATACTATA TTCATGAACT AAAAGACTTA

6961 ATATTGTGAA GGTATACTTT CTTTTCACAT AAATTTGTAG TCAATATGTT CACCCCAAAA

7021 AAGCTGTTTG TTAACTTGTC AACCTCATTT CAAAATGTAT ATAGAAAGCC CAAAGACAAT

7081 AACAAAAATA TTCTTGTAGA ACAAAATGGG AAAGAATGTT CCACTAAATA TCAAGATTTA

7141 GAGCAAAGCA TGAGATGTGT GGGGATAGAC AGTGAGGCTG ATAAAATAGA GTAGAGCTCA

7201 GAAACAGACC CATTGATATA TGTAAGTGAC CTATGAAAAA AATATGGCAT TTTACAATGG

7261 GAAAATGATG ATCTTTTTCT TTTTTAGAAA AACAGGGAAA TATATTTATA TGTAAAAAAT

7321 AAAAGGGAAC CCATATGTCA TACCATACAC ACAAAAAAAT TCCAGTGAAT TATAAGTCTA

7381 AATGGAGAAG GCAAAACTTT AAATCTTTTA GAAAATAATA TAGAAGCATG CCATCATGAC

7441 TTCAGTGTAG AGAAAAATTT CTTATGACTC AAAGTCCTAA CCACAAAGAA AAGATTGTTA

7501 ATTAGATTGC ATGAATATTA AGACTTATTT TTAAAATTAA AAAACCATTA AGAAAAGTCA

7561 GGCCATAGAA TGACAGAAAA TATTTGCAAC ACCCCAGTAA AGAGAATTGT AATATGCAGA

7621 TTATAAAAAG AAGTCTTACA AATCAGTAAA AAATAAAACT AGACAAAAAT TTGAACAGAT

7681 GAAAGAGAAA CTCTAAATAA TCATTACACA TGAGAAACTC AATCTCAGAA ATCAGAGAAC

7741 TATCATTGCA TATACACTAA ATTAGAGAAA TATTAAAAGG CTAAGTAACA TCTGTGGCAA

7801 TATTGATGGT ATATAACCTT GATATGATGT GATGAGAACA GTACTTTACC CCATGGGCTT

7861 CCTCCCCAAA CCCTTACCCC AGTATAAATC ATGACAAATA TACTTTAAAA ACCATTACCC

7921 TATATCTAAC CAGTACTCCT CAAAACTGTC AAGGTCATCA AAAATAAGAA AAGTCTGAGG

7981 AACTGTCAAA ACTAAGAGGA ACCCAAGGAG ACATGAGAAT TATATGTAAT GTGGCATTCT

8041 GAATGAGATC CCAGAACAGA AAAAGAACAG TAGCTAAAAA ACTAATGAAA TATAAATAAA

8101 GTTTGAACTT TAGTTTTTTT TAAAAAAGAG TAGCATTAAC ACGGCAAAGT CATTTTCATA

8161 TTTTTCTTGA ACATTAAGTA CAAGTCTATA ATTAAAAATT TTTTAAATGT AGTCTGGAAC

8221 ATTGCCAGAA ACAGAAGTAC AGCAGCTATC TGTGCTGTCG CCTAACTATC CATAGCTGAT

8281 TGGTCTAAAA TGAGATACAT CAACGCTCCT CCATGTTTTT TGTTTTCTTT TTAAATGAAA

8341 AACTTTATTT TTTAAGAGGA GTTTCAGGTT CATAGCAAAA TTGAGAGGAA GGTACATTCA

8401 AGCTGAGGAA GTTTTCCTCT ATTCCTAGTT TACTGAGAGA TTGCATCATG AATGGGTGTT

8461 AAATTTTGTC AAATGCTTTT TCTGTGTCTA TCAATATGAC CATGTGATTT TCTTCTTTAA

8521 CCTGTTGATG GGACAAATTA CGTTAATTGA TTTTCAAACG TTGAACCACC CTTACATATC

8581 TGGAATAAAT TCTACTTGGT TGTGGTGTAT ATTTTTTGAT ACATTCTTGG ATTCTTTTTG

8641 CTAATATTTT GTTGAAAATG TTTGTATCTT TGTTCATGAG AGATATTGGT CTGTTGTTTT

8701 CTTTTCTTGT AATGTCATTT TCTAGTTCCG GTATTAAGGT AATGCTGGCC TAGTTGAATG

8761 ATTTAGGAAG TATTCCCTCT GCTTCTGTCT TCTGAAAGAG ATTGTAGAAA GTTGATACAA

8821 TTTTTTTTTC TTTAAATATC TTGATATTAA TTAA

Seq ID No.21,5 heavy gRNA expressed sequences

1 AAGGTCGGGC AGGAAGAGGG CCTATTTCCC ATGATTCCTT CATATTTGCA TATACGATAC

61 AAGGCTGTTA GAGAGATAAT TGGAATTAAT TTGACTGTAA ACACAAAGAT ATTAGTACAA

121 AATACGTGAC GTAGAAAGTA ATAATTTCTT GGGTAGTTTG CAGTTTTAAA ATTATGTTTT

181 AAAATGGACT ATCATATGCT TACCGTAACT TGAAAGTATT TCGATTTCTT GGCTTTATAT

241 ATCTTGTGGA AAGGACGAAA CACCGGAATC GAAGAAGGAG GGTTTTAGAG CTAGAAATAG

301 CAAGTTAAAA TAAGGCTAGT CCGTTATCAA CTTGAAAAAG TGGCACCGAG TCGGTGCTTT

361 TTTAAAAAAG CACCGACTCG GTGCCACTTT TTCAAGTTGA TAACGGACTA GCCTTATTTT

421 AACTTGCTAT TTCTAGCTCT AAAACGGCCC TGGTGTGTAG TTTGGGAAAG AGTGGTCTCA

481 TACAGAACTT ATAAGATTCC CAAATCCAAA GACATTTCAC GTTTATGGTG ATTTCCCAGA

541 ACACATAGCG ACATGCAAAT ATTGCAGGGC GCCACTCCCC TGTCCCTCAC AGCCATCTTC

601 CTGCCAGGGC GCACGCGCGC TGGGTGTTCC CGCCTAGTGA CACTGGGCCC GCGATTCCTT

661 GGAGCGGGTT GATGACGTCA GCGTTCGAAT TCCATGGCGG CGCGGCGGCG ACGGAGCACC

721 GGCGGCGGCA GGGCGAGAGG TTCGGAGCTC AATATCGCGG GACGGCATGC GGGGGGCGGG

781 CAGTCAGAAA GGAACGATGC CACCTACTGT GACCCCCTTC CCTTCAGCTC CCTATAAACC

841 GGTGATCCGA CGCCGCCATC TCTAGGCCCG CGCCGGCCCC CTCGCACAGA CTTGTGGGAG

901 AAGCTCGGCT ACTCCCCTGC CCCGGTTAAT TTGCATATAA TATTTCCTAG TAACTATAGA

961 GGCTTAATGT GCGATAAAAG ACAGATAATC TGTTCTTTTT AATACTAGCT ACATTTTACA

1021 TGATAGGCTT GGATTTCTAT AAGAGATACA AATACTAAAT TATTATTTTA AAAAACAGCA

1081 CAAAAGGAAA CTCACCCTAA CTGTAAAGTA ATTGTGTGTT TTGAGACTAT AAATATCCCT

1141 TGGAGAAAAG CCTTGTTTGA TAGACCCATA AATCAGTTTT AGAGCTAGAA ATAGCAAGTT

1201 AAAATAAGGC TAGTCCGTTA TCAACTTGAA AAAGTGGCAC CGAGTCGGTG CTTTTTTAAA

1261 AAAGCACCGA CTCGGTGCCA CTTTTTCAAG TTGATAACGG ACTAGCCTTA TTTTAACTTG

1321 CTATTTCTAG CTCTAAAACT CAAATCACTC TTTGGCGAGG TACCCAGGCG GCGCACAAGC

1381 TATATAAACC TGAAGGAAAT CTCAACTTTA CACTTAGGTC AAGTTACTTA TCGTACTAGA

1441 GCTTCAGCAG GAAATTTAAC TAAAATCTAA TTTAACCAGC ATAGCAAATA TCATTTATTC

1501 CCAAAATGCT AAAGTTTGAG ATAAACGGAC TTGATTTCCG GCTGTTTTGA CACTATCCAG

1561 AATGCCTTGC AGATGGGTGG GGCATGCTAA ATACTGCAGG TCGACATGTC ACCAATAGAG

1621 ACGGCAATAT TTAGCATGCT CCACCCATCT GCAAGGCATG CTGGATAATA CGCAAACAAC

1681 TCCAAATCAA GCCCTTTTTA ATAGCAAGTT TTGCAATTTA AGGGAATAAT TTTTAAGTGT

1741 TAGTTAGGTT TGAACCTTAG TTTAAGATAT AACTTAGAGA TCCAGCATTT AGAAAGCAAG

1801 GAGAAAGTCG ACATATGCGT AAAGATCGAA ATTTCTTGTA GCTTATATAC TGCGTGCACT

1861 AGCTCAGTCT TTCGAATTGA TACTGACTGT AGTTTTAGAG CTAGAAATAG CAAGTTAAAA

1921 TAAGGCTAGT CCGTTATCAA CTTGAAAAAG TGGCACCGAG TCGGTGCTTT TTT

Claims (8)

1. a kind of controllability HIV-1 genomes target editing system, it is characterised in that including expressing controllable editing enzymes, collocation Multiple guiding RNA (gRNA)；

Specifically, expression of the pUC pUC to editing enzymes with the addition of control element, makes the expression of editing enzymes or is opened by LTR Mover and TAT protein regulating and expressing, or strong promoter and the R element of HIV-1 by built-in mammal constitute it is chimeric Promoter controls it to express, or is reached by the chimeric promoters control table that built-in strong mammalian promoters and TetO are constituted；Its It is characterised by, the editing enzymes expression plasmid includes following plasmid：P1, p2, p3, p7, p8, p10, p11 and p13；The gRNA Expression plasmid includes following plasmid：P4, p5, p9, p12, p14 and p15.

The multiple gRNA refers to the encoder block genome sequence design for HIV-1 genomes multiple site and/or CCR5 Targeting sequence.Characterized in that, in sequence containing Seq ID No.1-5 in ordered list in the multiple gRNA expression vectors At least 2 kinds.The gRNA expression plasmids include following plasmid：P4, p5, p9, p12, p14 and p15.

The editing enzymes, gRNA co-expression plasmids include following plasmid：P6-1, p6-2 and p6-3；

The sequence of the p1 as shown in Seq ID No.7 in sequence table, the sequence of p5 as shown in Seq ID No.20 in sequence table,

The sequence of p6-1 as shown in Seq ID No.8 in sequence table, the sequence of p7 as shown in Seq ID No.9 in sequence table, p6- The five heavy gRNA expressed sequences of 3 and p15 are as shown in Seq ID No.21.

2. controllability HIV-1 genome editing systems according to claim 1, the plasmid for carrying 5 '-and 3 '-LTR System can integrator gene group, its integration to genome depends on intracellular infected HIV-1 live viruses.

3. a kind of targeting carrier system for conveying any editing systems of claim 1-2, it is characterised in that the fat Plastid is mainly prepared from the following components：

DSPC DSPC：Cholesterol：Polypeptide-PEG- phosphatide mass ratio=20-70:10-30:0.0001-2；

Or, DPPC DPPC：DSPC DSPC：1,2- dioleyl phosphatidyl cholines DOPC：Cholesterol：Mol ratio=5 of polypeptide-PEG- phosphatide:1:0.5-3:0.5-2:0.0001-0.5.

4. the carrier system according to claim 1-3, it is characterised in that the carrier system contains the targeting fat of PEGylation Plastid, PEG is bi-functional, in addition to being covalently attached phospholipid composition, goes back the special target polypeptide of covalent coupling；The target Include any one or several combinations in single target spot polypeptide and Mutiple Targets polypeptide to polypeptide；Single target spot polypeptide includes anti-CD4 Polypeptide aCD4 or aCD4scFV, anti-CCR5 polypeptides aCCR5, anti-CXCR4 polypeptides aCXCR4；The Mutiple Targets polypeptide includes aCD4_ CCR5、aCCR5_CD4、aCD4_CXCR4、aCXCR4_CD4、aCCR5_CXCR4、aCXCR4_CCR5；The PEG of the coupling points Son amount scope is 200-10000, and the phosphatide of the coupling is one or more in phosphatidyl-ethanolamine or its derivative；

The coded sequence of aCD4scFV is Seq ID No.18 in sequence table；

The sequence of aCD4_CCR5 is Seq ID No.12 in sequence table, and wherein its amino can be put into left-hand end or right-hand end；

The sequence of aCCR5_CD4 is Seq ID No.13 in sequence table；

The coded sequence of aCCR5_CD4scFV is Seq ID No.19 in sequence table；

The sequence of aCD4_CXCR4 is Seq ID No.16 in sequence table；

The sequence of aCXCR4_CD4 is Seq ID No.17 in sequence table；

The sequence of aCCR5_CXCR4 is Seq ID No.14 in sequence table；

The sequence of aCXCR4_CCR5 is Seq ID No.15 in sequence table.

Above-mentioned functions polypeptide, can also directly with the lipid molecular covalent coupling such as PE, can directly add and be integrated into liposome membrane In molecular layer.

5. the carrier system according to claim 3 and 4, it is characterised in that penetrating peptide can be also coupled on PEG- phospholipid molecules, Formed " penetrating peptide-PEG- phosphatide ", the molecule is used with the collocation of target polypeptide-PEG- phosphatide；Penetrating peptide-PEG- phosphatide and other The mol ratio of target polypeptide-PEG- phosphatide is 0.0001-2:1.

6. the carrier system according to claim 3-5, it is characterised in that hyaluronic acid can be also added in liposome components Phosphatide, with target spot polypeptide-PEG- phosphatide and penetrating peptide-PEG- phosphatide collocation use, the phosphatide of the hyaluronic acid and its The mol ratio of his polypeptide-PEG- phosphatide is 0.00001-1：1.

7. the plasmid and liposome according to claim 1-6, load can also add in plasmid cationic polymer or Other polypeptide auxiliary ingredients, the auxiliary ingredients are polyethyleneimine, protamine sulfate, positively charged amino acid polypeptide or thin Born of the same parents promote one or more compositions in fusogenic peptide.

8. a kind of targeting carrier system for conveying any editing systems of claim 1-2, it is characterised in that the load Fortune system can also be slow virus；

The slow virus is that the targeting editing system is arranged in pairs or groups with the second generation or third generation slow virus packaging system, is carried out Virus packaging；Described packaging virus capsid protein is VSVG, or is the capsid in measles virus (Measles Virus) source Albumen, or be sindbis alphavirus (Sindbis Virus) source, and other viral sources capsid protein；It is described slow The integrase inactivation of virus system is lacked, and its integration to genome depends on the HIV-1 live viruses that cell is infected.