CN102337251A - Chimaera for HK97 bacteriophage virus-like particle and application of chimaera - Google Patents
Chimaera for HK97 bacteriophage virus-like particle and application of chimaera Download PDFInfo
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- CN102337251A CN102337251A CN2011101717304A CN201110171730A CN102337251A CN 102337251 A CN102337251 A CN 102337251A CN 2011101717304 A CN2011101717304 A CN 2011101717304A CN 201110171730 A CN201110171730 A CN 201110171730A CN 102337251 A CN102337251 A CN 102337251A
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Abstract
The invention provides a chimaera for an HK97 bacteriophage virus-like particle and application of the chimaera, and application of the chimaera to bio-immune targeted therapy. A base sequence for expressing AFHYESQ short peptide is added at the C end of a GP5 gene of the HK97 bacteriophage virus-like particle, the GP5 gene and a GP4 gene for coding protein at the head end of the HK97 bacteriophage virus-like particle are inserted into pETDuet-1 plasmid, a chimaera expression plasmid of the HK97 bacteriophage virus-like particle is constructed, and a precursor after the expression plasmid is subjected to induction expression is purified and acidified to be induced into a mature chimaera for the HK97 bacteriophage virus-like particle. The chimaera for the HK97 bacteriophage virus-like particle serves as a carrier for the bio-immune targeted therapy and is prepared into a vaccine to achieve the bio-immune targeted therapeutic effect.
Description
Invention field
The invention belongs to molecular biology genetically engineered field, be specifically related to a kind of chimeric structure and purifying thereof of HK97 phage virus-like particle protein, and the effect in the biological immune targeted therapy.
Background of invention
Therapeutic vaccine to chronic disease is one of focus of present vaccine research, and these diseases comprise: chronic viral infection, anaphylactic disease, tumour, mellitus, hypertension and alzheimer's disease etc.Different with common preventative vaccine is: what therapeutic vaccine was directed against is autoantigen or ectophylaxination tolerance antigen; Therefore; Therapeutic vaccine will be brought into play therapeutic action must at first break immunological tolerance, produces autoantibody or id reaction T cell to specific antigen.Because target antigen that therapeutic vaccine was directed against generally is a small peptide, antigenicity a little less than, must be with after suitable carriers combines, competence exertion effect under the help of immunological adjuvant, therefore, selection suitable carriers and adjuvant be the successful key of therapeutic vaccine often.
Carrier such as KLH, Toxoid,tetanus etc. commonly used at present; Render a service generally not by force, must use adjuvant, though and adjuvant such as freund's adjuvant usefulness commonly used are stronger; But can not be applied to human body; Though the aluminum adjuvant can be applicable to human body, immunizing potency very a little less than, and exist like untoward reactions such as cerebral arteriosclerosis, aluminium salt local accumulation.Therefore, ideal therapeutic vaccine carrier should under the situation of not using immunological adjuvant, just can effectively be broken the immunological tolerance of body with after target antigen combines, and produces high titre antibody, the performance therapeutic action.
The virus-like particle one type of ideal vaccine carrier that comes to this.Virus-like particle (VLP) is the hollow shell structure that does not contain viral nucleic acid that is assembled into by viral capsid proteins; Profile and immunogenicity with virus; Can induce body to produce with the humoral immunization is main reaction; Be directed against bonded antibody with it thereby produce,, therefore avoided the risk of virus replication propagation owing to do not have the nucleic acid component of virus.Virus-like particle is formed by the capsid protein monomer polymerization of a large amount of repeating structures; Can combine and activate the B cell with the B-cell receptor on specific b cells surface after getting in the body; Simultaneously, VLP also can provide exogenous t cell epitope and activate helper T cell, thereby breaks through the autoimmunization tolerance.Each capsid protein monomer generally has the basic polypeptide territory in addition; As Methionin-NH2; These basic polypeptide territories are towards the particulate outside surface, as long as small molecules such as small peptide have suitable amino acid group, as-SH; Just can combine and be showed in the VLPs surface through the isodigeranyl functional cross-link agent, stimulate body to produce antibody.
Prepared multiple phage virus-like particle at present both at home and abroad, like Q phagus beta virus-like particle, human papillomavirus virus-like particles (HPV), MS
2Virus-like particle etc.Switzerland Cytos company is that carrier has been developed a series of therapeutic vaccines with Q phagus beta virus-like particle; Comprise alzheimer's disease AD vaccine (CAD106), Nicotine vaccine (NIC002), allergic asthma vaccine (CYT003-QbG10) etc.; Wherein its development to the step-down vaccine (CYT006-AngQb) of Angiotensin II; Accomplished IIa phase clinical experiment, accomplished IIb phase clinical experiment to the vaccine (CYT003-QbG10) of allergia nose's conjunctivitis development.And the main vaccine composition of the vaccine of cervical cancer Cervarix (TM) that has gone on the market of Belgian GlaxoSmithKline PLC company (GSK) development is exactly HPV-16 and HPV-18.These virus-like particles have shown that powerful immunogenicity and people use security afterwards.Up to now, though the research of HK97 phage virus-like particle has bibliographical information, but rare, and do not appear in the newspapers as the purposes of biological immune targeted therapeutic carrier to its chimeric research.
Summary of the invention
First purpose of the present invention provides a kind of mosaic of HK97 phage virus-like particle.
The effect of mosaic in the biological immune targeted therapy that second purpose of the present invention is the HK97 phage virus-like particle.
The present invention realizes through following technical scheme:
The preparation of HK97 phage virus-like particle protein mosaic HII-HK97-P:
(1) will the encode Protein G P4 gene of HK97 phage virus-like particle head end inserts on the MCS of pETDuet-1 plasmid (available from Novagen company);
(2) the C end at the Protein G P5 gene of coding HK97 phage virus-like particle head end adds the base sequence of expressing the AFHYESQ small peptide, this GP5 gene is inserted on the MCS of pETDuet-1 plasmid, makes up mosaic expression plasmid pETDuet-HK97-P;
(3) plasmid pETDuet-HK97-P is converted into BL21 (DE3) competence bacterium, IPTG abduction delivering virus-like particle mosaic precursor P II-HK97-P;
(4) 6%PEG80004 ℃ of sedimentation virus-like particle mosaic precursor PII-HK97-P;
(5) the said virus-like particle mosaic of reinforcing yin essence ion exchange chromatography purifying precursor PII-HK97-P;
(6) acidifying induces said virus-like particle mosaic precursor PII-HK97-P maturation to be expanded to ripe virus-like particle mosaic HII-HK97-P;
(7) the ripe virus-like particle mosaic of gel chromatography HII-HK97-P.
Preservation culture called after Escherichia coli BL21 of the present invention (DE3)/pETDuet-HK97-P is deposited in Chinese typical culture collection center on June 21st, 2011, and its deposit number is CCTCC NO:M2011206.
Advantage of the present invention:
1, HK97 phage virus-like particle GP5 protein carboxyl groups end can add oneself's assembling of merging 1-12 amino acid and not influencing virus-like particle, thereby with the mosaic form small peptide epi-position directly is illustrated in the virus-like particle surface.
2, the HK97 phage virus-like particle protein mosaic HII-HK97-P of the present invention's preparation can be used for the biological immune targeted therapy.
Description of drawings
Sequence table SEQ ID NO:1 is the nucleotide sequence that the present invention makes up the used GP4 gene of HK97 phage virus-like particle mosaic.
Sequence table SEQ ID NO:2 is the nucleotide sequence that the present invention makes up the used GP5 gene of HK97 phage virus-like particle mosaic.
Sequence table SEQ ID NO:3 is the aminoacid sequence that the present invention makes up the used GP4 genes encoding of HK97 phage virus-like particle mosaic.
Sequence table SEQ ID NO:4 is the aminoacid sequence that the present invention makes up the used GP5 genes encoding of HK97 phage virus-like particle mosaic.
Fig. 1: agarose gel electrophoresis is identified mosaic expression plasmid pETDuet-HK97-P.
Fig. 2: the maturation of HII-HK97-P virus-like particle and purge process.Get sample (0min) after acidifying is induced, neutralization back sample (5min, 10min, 30min, 60min 2hrs) carries out the SDS-PAGE electrophoresis and identifies and purity check, along with in the prolongation of time, pentamer and six aggressiveness proportions are increasing.
Fig. 3: HK97-P virus-like particle mosaic precursor PII-HK97-P and ripe body HII-HK97-P Electronic Speculum figure.Left side figure is PII-HK97-P Electronic Speculum figure, and right figure is the HII-HK97-P Electronic Speculum, and the latter compares precursor and has thinner shell on morphological structure, and diameter is extended to about 66nm by 54nm.
Fig. 4: anti-P1 small peptide antibody titers behind the HII-HK97-P vaccine immunity normal SD male rat.7th, the mensuration of the anti-P1 small peptide antibody titers of 21,35,45 days immune rats (serum is all with dilution in 1: 1000).
Embodiment
Following case study on implementation is to further specify the present invention, but does not limit its scope.
The structure of case study on implementation 1 mosaic expression plasmid pETDuet-HK97-P
With primer 5 '-CCATGGCTGAAATCGTAAAAACGCTGT-3 '; 5 '-CCGGAATTCTTATTTACCTAAGTTAGAAGGG-3 ', through pcr amplification obtain the encoding Protein G P4 gene of HK97 phage virus-like particle head end, size is 678bp.With primer 5 '-CCATATGTCTGAACTCGCTCTCATTC-3 '; 5 '-CTCGAGTCAACGAGATTCGTAGTGG-3 ', through pcr amplification obtain the encoding Protein G P5 gene of HK97 phage virus-like particle head end, size is 1158bp.
With two MCSs of above-mentioned GP5 gene fragment that obtains and GP4 gene fragment insertion pETDuet-1 plasmid (available from Novagen company), make up mosaic expression plasmid pETDuet-HK97-P, enzyme is cut and is identified its molecular weight size (Fig. 1).
The maturation and the purifying thereof of case study on implementation 2 HII-HK97-P virus-like particles
(1) plasmid pETDuet-HK97-P is converted into BL21 (DE3) competence bacterium, the 37 ℃ of amplifications 16 hours in the 20ml liquid nutrient medium of the single bacterium colony of picking are got this bacterium liquid 1ml and are added in the 1L liquid nutrient medium, several hours OD to substratum of 37 ℃ of amplifications
600nmBe about 0.6, add IPTG (20ul, 0.2g/m1) abduction delivering virus-like particle mosaic precursor PII-HK97-P.
Cracking bacterium centrifuging and taking supernatant spends the night in 6%PEG80004 ℃ of sedimentation virus-like particle mosaic precursor PII-HK97-P after (2) 4 hours, and PII-HK97-P is through UnoQ reinforcing yin essence ion exchange chromatography (20mM Tris-HCl, 1M NaCl) purifying.
(3) behind the anionresin purifying PII-HK97-P is induced maturation: 1.5ml is diluted in 150ml acidifying damping fluid (pH3.8 with 30mg/ml PII-HK97-P virus-like particle mosaic precursor; 250mM KCl; 50mM Citrate) in 1 hour; The pH of the neutralization buffer (pH8.3,1M Tris-HCl) that adds 1/6 volume subsequently to total system is more than 7.0, and room temperature was placed 2 hours or the longer time.SDS-PAGE electrophoresis identifying virus appearance particle mosaic precursor, midbody and ripe body (are seen case study on implementation 3, Fig. 2).
(4) acidifying induce sophisticated virus-like particle mosaic HII-HK97-P through the gel permeation chromatography purifying to reach higher purity.
The evaluation of case study on implementation 3 HII-HK97-P virus-like particles
Get HK97-P virus-like particle mosaic precursor, midbody and each 20ug of ripe body and carry out 6%SDS-PAGE electrophoresis (Fig. 2); And get HK97-P virus-like particle mosaic precursor PII-HK97-P and ripe body HII-HK97-P and carry out form and size observation (Fig. 3) under the transmission electron microscope (nickel screen, 4% uranyl acetate negative staining).Electron microscopy observation is found; HK97-P virus-like particle mosaic precursor PII-HK97-P is the prismatic virus-like particle structure of assembling voluntarily, the about 54nm of diameter, and ripe body HII-HK97-P compares precursor and has thinner shell on morphological structure; Diameter enlarges a little, is about 66nm.
Anti-P small peptide production of antibodies behind the case study on implementation 4 HII-HK97-P mosaic vaccine immunity rats
10 of cleaning level SD rats are divided into two groups at random, and 5 every group, raise in cleaning level Animal House, adopt 12h/12h illumination and non-limiting full diet.Get HII-HK97-P vaccine immunity rat: adopt the scheme of back subcutaneous 3-4 point vaccinate, 400ul/, the about 300ug/ of vaccine dose, two weeks were strengthened 2 times at interval behind initial immunity.
0.3% glutaraldehyde cross-linking agent coupling joins bovine serum albumin (BSA) and small peptide P, encapsulates 96 hole elisa plates with this coupling matter (10ug/ hole).Getting rat caudal vein blood in 7,21,35,45 days uses mensuration (serum is all with dilution in 1: 1000) that ELISA carries out anti-P small peptide antibody titers (Fig. 4).
Claims (6)
1. the mosaic of a HK97 phage virus-like particle is characterized in that: the mosaic of this virus-like particle is obtained through abduction delivering, purifying and acidifying are ripe by the mosaic expression plasmid pETDuet-HK97-P that makes up.
2. the mosaic of HK97 phage virus-like particle as claimed in claim 1 contains the Protein G P4 gene of HK97 phage virus-like particle head end among the wherein said mosaic expression plasmid pETDuet-HK97-P.
3. the mosaic of HK97 phage virus-like particle as claimed in claim 1; The Protein G P5 gene that contains HK97 phage virus-like particle head end among the wherein said mosaic expression plasmid pETDuet-HK97-P, the C end of this GP5 gene has added the base sequence of expressing the AFHYESQ small peptide.
4. the mosaic of HK97 phage virus-like particle as claimed in claim 3, the base sequence of wherein said AFHYESQ small peptide is 5 '-GCGTTCCACTACGAATCTCGT-3 '.
5. the mosaic of HK97 phage virus-like particle as claimed in claim 1, it is as the purposes of biological targeting treatment carrier.
6. the mosaic of HK97 phage virus-like particle as claimed in claim 1; Wherein said mosaic expression plasmid pETDuet-HK97-P is deposited in Chinese typical culture collection center; Its preserving number is CCTCC NO:M2011206, the preservation called after: Escherichia coli BL21 (DE3)/pETDuet-HK97-P.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019047921A1 (en) * | 2017-09-08 | 2019-03-14 | 武汉华纪元生物技术开发有限公司 | Short peptide atr001, monoclonal antibody prepared by short peptide and having function of biased regulation of at1r, and application thereof |
| CN109679981A (en) * | 2019-02-21 | 2019-04-26 | 武汉大学 | A kind of formyl peptides directed evolution bacteriophage and the preparation method and application thereof |
| CN114369610A (en) * | 2021-12-01 | 2022-04-19 | 四川大学 | T7 phage virus-like particle self-assembly method based on single plasmid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896094B (en) * | 2006-06-08 | 2010-08-04 | 华中科技大学同济医学院附属协和医院 | Use of angiotensin II receptors I immunogenic peptide section |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896094B (en) * | 2006-06-08 | 2010-08-04 | 华中科技大学同济医学院附属协和医院 | Use of angiotensin II receptors I immunogenic peptide section |
Non-Patent Citations (3)
| Title |
|---|
| GERTSMAN ET AL.: "Structural characterization of viral capsid maturation in bacteriophage HK97", 《ESCHOLARSHI UNIVERSITY OF OF CALIFORNIA》, 31 December 2009 (2009-12-31) * |
| 朱峰等: "血管紧张素1型受体胞外第二环肽抗体对大鼠血管平滑肌细胞质游离钙水平的影响", 《中华老年心脑血管病杂志》, vol. 8, no. 11, 30 November 2006 (2006-11-30) * |
| 赵朴等: "病毒样颗粒疫苗及载体的研究进展", 《中国生物制品学杂志》, vol. 22, no. 5, 31 May 2009 (2009-05-31) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019047921A1 (en) * | 2017-09-08 | 2019-03-14 | 武汉华纪元生物技术开发有限公司 | Short peptide atr001, monoclonal antibody prepared by short peptide and having function of biased regulation of at1r, and application thereof |
| CN109467599A (en) * | 2017-09-08 | 2019-03-15 | 武汉华纪元生物技术开发有限公司 | Small peptide ATR001 and the monoclonal antibody and application that by small peptide preparation there is skewed popularity to adjust AT1R function |
| CN109467599B (en) * | 2017-09-08 | 2021-06-04 | 武汉华纪元生物技术开发有限公司 | Short peptide ATR001, monoclonal antibody prepared from short peptide and having function of preferentially regulating AT1R, and application of monoclonal antibody |
| CN109679981A (en) * | 2019-02-21 | 2019-04-26 | 武汉大学 | A kind of formyl peptides directed evolution bacteriophage and the preparation method and application thereof |
| CN114369610A (en) * | 2021-12-01 | 2022-04-19 | 四川大学 | T7 phage virus-like particle self-assembly method based on single plasmid |
| CN114369610B (en) * | 2021-12-01 | 2023-01-31 | 四川大学 | T7 bacteriophage virus-like particle self-assembly method based on single plasmid |
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