CN106868047B - Recombinant adenovirus vector and construction method and application thereof - Google Patents
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Abstract
The invention provides a recombinant adenovirus vector and a construction method thereof, which are characterized in that the pre-existing immunity aiming at an Ad5 vector can be well avoided, namely the immunogenicity of the vector is hardly interfered by a neutralizing antibody of Ad5 widely existing in a human body. The invention was achieved based on the replacement of the corresponding part of Ad5 with the antigenic determinant gene of the human rare serotype adenovirus Ad 35. The choice of rare serotype adenovirus for replacement and replacement genes is based on considerations that facilitate packaging of the engineered viral vector and do not affect the level of immunity of the original viral vector.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant adenovirus vector for gene therapy and vaccine vectors and a construction method thereof.
Background
The development of adenovirus (Ad) as a gene expression vector began in the early 60's of the 20 th century, when virologists observed that the adenovirus genome could hybridize to the simian virus 40 (SV 40) genome, suggesting that the adenovirus genome could carry heterologous genes. Thereafter, adenovirus has evolved into an important vector system.
At present, 57 different adenovirus serotypes have been identified, and recombinant adenovirus vectors have been used in gene therapy and vaccine vectors, especially human serum type 5 adenovirus, which is also the main viral vector in the current research of tumor gene therapy and Human Immunodeficiency Virus (HIV) vaccines. In addition, the development of vaccines against pathogens such as hepatitis B virus, malaria, tuberculosis, etc. has also placed considerable attention on the use of type 5 adenoviral vectors.
However, since the immune response against Ad5 adenovirus itself is ubiquitous in human, such pre-existing immune response includes anti-adenovirus neutralizing antibody and killer T cell against adenovirus, so as to inhibit its entry into body cells while the target gene carried by it is not expressed at the expected level or even not expressed at all, and furthermore, CTL response against adenovirus vector with cross property can kill target cells infected with adenovirus vector and block the continuous expression of foreign gene. In summary, the preexisting immune response can greatly reduce the effectiveness of adenoviral vector-based vaccine immunization or gene therapy.
To overcome the effects of preexisting immune responses on adenoviral vectors, researchers have tried a variety of approaches: (1) carrying recombinant Ad5 vector by using a new method; (2) the development of novel recombinant adenoviral vectors from other human adenoviral serotypes and even other animal species; (3) the Ad5 vector is genetically engineered, and the method is safer and more applicable compared with other methods. The method specifically replaces corresponding genes of an Ad5 vector with antigenic determinant genes derived from human rare serotype adenoviruses with better human safety, such as Ad35, Ad48, Ad49 and the like, so as to obtain the chimeric vector which not only retains the immunogenicity of the Ad5 vector, but also has the serological property of rare adenovirus subtype. Patent CN104419717A discloses a method for constructing recombinant adenovirus, which comprises replacing HVR5, 7 of RAd5 vector with HVR5, 7 of another human adenovirus serotype, wherein said another human adenovirus serotype is Ad43 or Ad37, but the method has the following defects: (1) it simply replaces the HVR and cannot escape the preexisting immunity completely; (2) the preparation method uses at least four carriers of different companies, and has high price and inconvenient operation.
Therefore, there is still a need in the art to further modify an adenovirus vector by genetic engineering technology to obtain a recombinant adenovirus vector which is easy and convenient to operate, easy to package, high in immune level and capable of well avoiding pre-existing.
Disclosure of Invention
In order to solve the technical problems, the invention provides a recombinant adenovirus vector and a construction method thereof, the recombinant adenovirus vector can well avoid the pre-existing immunity aiming at the Ad5 vector, simultaneously not only keeps the strong immunogenicity of the Ad5 vector, but also has the serological property of rare serotype adenovirus, and is simple and convenient to operate.
The technical problem to be solved by the invention is realized by the following technical scheme:
a recombinant adenovirus vector is prepared by replacing the whole Hexon (Hexon) in a backbone plasmid of an Ad5 vector with the Hexon of a human rare adenovirus serotype Ad35 by adopting a molecular cloning method to obtain the recombinant adenovirus vector RAd 5-35-Hexon-Adbone.
The invention also provides a construction method of the recombinant adenovirus vector, which comprises the following steps:
firstly, transforming an intermediate vector, introducing AscI and NruI between restriction enzyme sites XbaI and BamHI of a Puc19 vector to obtain an intermediate vector Puc 19-AscI-NruI;
secondly, performing single enzyme digestion on the original Ad5 skeleton plasmid by using AscI, recovering a 9620bp fragment, performing single enzyme digestion on the Puc19-AscI-NruI by using AscI, recovering a 2680bp fragment, and connecting the recovered two parts of fragments to construct a plasmid 9620-Puc19-AscI;
(III) singly digesting the plasmid 9620-Puc19-AscI with HindIII to recover a 7000bp fragment, simultaneously singly digesting the vector Puc19 with HindIII to recover a 2680bp fragment, and connecting the recovered two parts of fragments to construct a plasmid 7000-Puc 19-HindIII;
(IV) plasmid-based: 7000-Puc19-HindIII, inserting restriction enzyme site ClaI at the 5 'end of Hexon of Ad5 skeleton plasmid, PCR amplifying two sections of A and B at the 5' end of Hexon, inserting single restriction enzyme site ClaI at the junction of A and B, and designing primers as follows:
fragment A
Upstream: 5'-TCTAGACTGGTGACGCAAATAGACGA-3'
Downstream: 5'-TCTATCGATGGGGTAGCCATAAGCTT-3'
Fragment B
Upstream: 5'-TCTAGACCCATCGATGATGCCGCA-3'
Downstream: 5'-CTTGCTCGTCTACTTCGTCTAAGCTT-3'
(V) respectively connecting the fragments A and B to a Puc19 vector, performing double enzyme digestion on plasmids Puc19-A and Puc19-B by ClaI and HindIII, respectively recovering fragments of 3100bp and 500bp, and connecting the recovered two parts of fragments to construct a plasmid Puc19-A + B;
(VI) the plasmid Puc19-A + B was digested with XbaI and HindIII, and a 1000bp fragment was recovered while
Using DraIII-HF and RsrII to double-enzyme cut plasmid 7000-Puc19-HindIII, recycling 9800bp fragment, connecting the two recycled fragments by using an EsayGeno rapid recombinant cloning kit of Tiangen, namely constructing plasmid 7000-Puc 19-ClaI;
(VII) PCR amplifying Ad35hexon, wherein the primers are designed as follows:
upstream: 5'-ATGGCCACCCCATCGATGCTGCC-3'
Downstream: 5'-TACGTGGTAGCGTTGCCGGCCGAGA-3'
Recovering a 2800bp fragment of Ad35 hexon;
(eight) carrying out double enzyme digestion on the plasmid 7000-Puc19-ClaI by ClaI and NaeI, recovering a 7000bp fragment, simultaneously carrying out double enzyme digestion on Ad35hexon recovered by PCR by ClaI and NaeI, recovering a 2800bp fragment, and connecting the recovered two parts of fragments to construct a plasmid: 7000-Puc19-Ad 35;
(nine) HindIII single enzyme digestion 7000-Puc19-Ad35, recover 7000bp fragment, at the same time, HindIII single enzyme digestion plasmid 9620-Puc19-AscI, recover 5400bp fragment, will reclaim two fragments to link, construct plasmid: 9620-Puc19-Ad 35;
(ten) performing single enzyme digestion on the original Ad5 backbone plasmid pBHGlox (delta) E1,3Cre by AscI, recovering a 25000bp fragment, performing single enzyme digestion on the plasmid 9620-Puc19-Ad35 by AscI, recovering a 9620bp fragment, and connecting the two recovered fragments to construct a plasmid: RAd 5-35-Hexon-Adbone.
Further, the step of engineering the Puc19 vector in the step (one) comprises the following steps:
A. enzyme digestion: the enzyme digestion reaction system is as follows: 2 mu g of Puc19 vector, 1 mu L of XbaI, 1 mu L of BamHI, 5 mu L of Cusmartbuffer and a proper amount of sterilized water, wherein the total volume is 50 mu L; the reaction conditions are as follows: the 2680bp fragment was recovered by digestion at 37 ℃ for 3 h.
B. Preparation of upstream and downstream reaction fragments: the mixture is: 100mmol/L of upstream primer, 100mmol/L of downstream primer and 8 mu L of sterilized water, wherein the total volume is 10 mu L, the upstream primer: 5' -CTAGATAGGCGCGCCTACCTCGCGAGGAGG CGCGCCTTG-3'; a downstream primer: 5' -GATCC AAGGCGCGCCTCCTCGCGAGGTAGGCGCGCCTAT-3; the reaction procedure is as follows: the temperature is reduced from 95 ℃ to 4 ℃ in a gradient way, and the temperature is reduced by 5 ℃ every 5 min.
C. Construction of the plasmid Puc 19-AscI-NruI: the recovered 2680bp fragment was ligated with upstream and downstream reaction fragments by the ligation scheme: 10 XBuffer 1 mu L, T4DNA ligase 1 mu L, the 2680bp fragment recovered 1 mu L, the upstream and downstream reaction fragments 5 mu L and sterile water 2 mu L, the total volume is 10 mu L.
D. Transforming, selecting bacteria, cloning and extracting plasmid to obtain the vector Puc 19-AscI-NruI.
The invention also provides the application of the recombinant adenovirus vector in preparing vaccines or gene therapy medicaments.
The invention has the following beneficial effects
(1) The specific neutralizing antibody of Ad5 mainly aims at adenovirus hexon, the immunity of adenovirus is serotype specific, and the Ad5 is replaced by the rare serotype adenovirus hexon of human body, so that the pre-existing immunity of Ad5 can be avoided; and HVRs are only small fragments in the hexon, the replacement of the whole hexon by the method has higher probability of escaping from pre-existing immunity in vivo compared with replacement of the HVRs.
(2) The difficulty in replacing the hexon is: the framework plasmid pBHGlox (delta) E1,3Cre of the Ad5 vector is about 34kb, and basically comprises the existing enzyme cutting sites, so that the replacement of the whole hexon cannot be completed by simple enzyme cutting; according to the invention, the special enzyme cutting sites on the Ad5 vector skeleton plasmid are utilized to connect the segment containing the hexon to the Puc19 vector, so that the plasmid segments are reduced, more enzyme cutting sites are increased, and the operation is more convenient.
(3) The invention only uses the molecular cloning method to complete the construction of the whole vector through enzyme digestion and connection, only uses the exogenous classical cloning vector Puc19, does not need special reagents and other cloning vectors, does not need special competence, has simple method and strong feasibility, and avoids the uncertainty of other methods such as homologous recombination and the like to operate large-fragment plasmids.
(4) In order to ensure that the recombinant adenovirus can be packaged, the base outside the hexon is not mutated in the process of replacing the hexon; according to the invention, a section containing hexon is connected to a Puc19 vector, and although a single enzyme cutting site ClaI is inserted into the 5' end of hexon, the basic group is changed, but the coded amino acid is not changed, so that the adenovirus skeleton plasmid is beneficial to packaging of viruses.
(5) The vector of the invention not only retains the strong immunogenicity of the RAd5 vector, but also has the serological properties of rare serotype adenovirus.
Description of the drawings:
FIG. 1 is a schematic structural diagram of a recombinant adenovirus vector;
FIG. 2 is a flow chart of the construction of chimeric RAd5 backbone plasmids;
FIG. 3 is a schematic illustration of viral vector packaging;
FIG. 4 is an identifying electropherogram of the constructed plasmid 9620-puc19-AscI, where lane 1 is plasmid: 9620-puc19-AscI was not digested, lane 2 is Adbone, lane 3 is 9620-puc 19-the correct clone of AscI, lane 4 is puc19-AscI, lane 5 is Maker;
FIG. 5 is an electrophoretic image of the construction of the plasmid 7000-puc19-HindIII, in which lane 1 is Maker, lanes 2-5 are different 7000-puc19-HindIII clones, respectively, and lane 2 is the correct 7000-puc19-HindIII clone;
FIG. 6 is an identification electrophoretogram of the constructed plasmid 7000-puc19-Ad35, in which lanes 1-6 are different 7000-puc19-Ad35 clones, lanes 2, 4, 5 have correct bands, the sequencing is correct, and lane 7 is marker;
FIG. 7 shows the identification of the plasmid 9620-puc19-Ad35, in lane 1, plasmid: 9620-puc19-Ad35, lane 2 is Maker;
FIG. 8 shows the identification of the construction of the plasmid RAd5-35-Hexon-Adbone, in which lanes 1-10 represent different clones of the plasmid RAd5-35-Hexon-Adbone, respectively, and only lane 6 shows the correct band, lane M represents marker, lane + pBHGlox (delta) E1,3Cre plasmid;
FIG. 9 is a graph of the working performance of shuttle plasmid pDC315-GFP transfected cells;
FIG. 10 is a graph of the working efficacy of recombinant adenoviruses, in which A is Ad5-35-hexon-Adbone packaging virus on day seven; the sixteenth day of the Ad5-35-hexon-Adbone packaging virus; c: rAd5 recombinant adenovirus packaging virus eighth day;
fig. 11 is a graph of packaging recombinant adenovirus supernatant infected 293A cells, wherein a.rad 5 virus supernatant infected 293A cells on day three; B. infection of 293A cells with Ad5-35-hexon-Adbone virus supernatant on day ten;
FIG. 12 shows PCR amplification to identify hexon in rAd5-35-hexon-Adbone packaging virus, in which lane 1 shows PCR amplified Ad35hexon, and lane 2 shows marker.
Detailed Description
The present invention will be described in detail with reference to examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. Ad35hexon sequence: AB330116.1 GenBank
Example 1
RAd5 vector and a method for constructing a shuttle plasmid for expressing a Green Fluorescent Protein (GFP) gene, comprising
1. Construction of chimeric Ad5 backbone plasmid
The first step is as follows: modifying an intermediate carrier: AscI and NruI were introduced between restriction sites XbaI and BamHI of the Puc19 vector to obtain an intermediate vector Puc 19-AscI-NruI.
A. Enzyme digestion: the enzyme digestion reaction system is as follows: 2 mu g of Puc19 vector, 1 mu L of XbaI, 1 mu L of BamHI, 5 mu L of Cusmartbuffer and a proper amount of sterilized water, wherein the total volume is 50 mu L; the reaction conditions are as follows: the 2680bp fragment was recovered by digestion at 37 ℃ for 3 h.
B. Preparation of upstream and downstream reaction fragments: the mixture is: 100mmol/L of upstream primer, 100mmol/L of downstream primer and 8 mu L of sterilized water, wherein the total volume is 10 mu L, the upstream primer: 5' -CTAGATAGGCGCGCCTACCTCGCGAGGAGG CGCGCCTTG-3' (SEQ ID NO: 1); a downstream primer: 5' -GATCCAAGGCGCGCCTCCTCGCGAGGTAGGCGCGCCTAT-3' (SEQ ID NO: 2); the reaction procedure is as follows: the temperature is reduced from 95 ℃ to 4 ℃ in a gradient way, and the temperature is reduced by 5 ℃ every 5 min.
Note: underlined are restriction sites
C. Construction of the plasmid Puc 19-AscI-NruI: the recovered 2680bp fragment was ligated with upstream and downstream reaction fragments by the ligation scheme: 10 XBuffer 1 u L, T4DNA ligase 1 uL, the recovered 2680bp fragment 1 uL, the upstream and downstream reaction fragments 5 uL and sterile water 2 uL, the total volume is 10 uL.
D. Transforming, selecting bacteria, cloning and extracting plasmid to obtain the vector Puc 19-AscI-NruI.
The second step is that: construction of viral vectors: vector RAd5-35-Hexon-Adbone was constructed by a multi-step cloning, the Hexon of which was replaced by adenovirus type 35 Hexon.
A. An original RAd5 skeleton plasmid, namely pBHGlox (delta) E1,3Cre plasmid of an AdMax system, is subjected to single enzyme digestion by AscI, a 9620bp fragment is recovered, meanwhile, an AscI enzyme digestion vector Puc19-AscI-NruI is subjected to enzyme digestion, a 2680bp fragment is recovered, and the two parts of fragments are connected to construct a plasmid 9620-Puc19-AscI;
B. plasmid 9620-Puc19-AscI is subjected to single enzyme digestion by HindIII, a 7000bp fragment is recovered, meanwhile, a HindIII single enzyme digestion vector Puc19 is subjected to single enzyme digestion to recover a 2680bp fragment, and the recovered two parts of fragments are connected to construct plasmid 7000-Puc 19-HindIII;
C. based on the plasmid 7000-Puc19-HindIII, enzyme cutting sites ClaI are inserted into the 5 'end of the Hexon of RAd5 skeleton plasmid, two sections of A and B of the 5' end of the Hexon are amplified by PCR, a single enzyme cutting site ClaI is inserted into the joint of the sections A and B, and primers are designed as follows:
fragment A
Upstream: 5'-CTGGTGACGCAAATAGACGA-3' (SEQ ID NO: 3)
Downstream: 5' -TCTATCGATGGGGTAGCCAT-3’ （SEQ ID NO：4）
Fragment B
Upstream: 5' -CCCATCGATGATGCCGCA-3’（SEQ ID NO：5）
Downstream: 5'-CTTGCTCGTCTACTTCGTCT-3' (SEQ ID NO: 6);
note: underlined are restriction sites
D. Connecting the fragment A to a Puc19 vector, carrying out double digestion on the plasmid Puc19-A by ClaI and HindIII, and recovering a 3100bp fragment; connecting the fragment B to a Puc19 vector, performing double enzyme digestion on the plasmid Puc19-B by ClaI and HindIII, and recovering a 500bp fragment; connecting the two recovered fragments to construct a plasmid Puc19-A + B;
E. digesting the plasmid Puc19-A + B by XbaI and HindIII, recovering a 1000bp fragment, digesting the plasmid 7000-Puc19-HindIII by DraIII-HF and RsrII, recovering a 9800bp fragment, and connecting the recovered two parts of fragments by using an EsayGeno rapid recombinant cloning kit of Tiangen to construct a plasmid 7000-Puc 19-ClaI;
f, carrying out PCR amplification on Ad35hexon, introducing single enzyme cutting sites ClaI and NaeI, and designing primers as follows:
upstream: 5' -ATGGCCACCCCATCGATGCTGCC-3’ （SEQ ID NO：7）
Downstream: 5' -TACGTGGTAGCGTTGCCGGCCGAGA-3’ （SEQ ID NO：8）
Recovering a 2800bp fragment of Ad35 hexon;
note: underlined are restriction sites
G, digesting the plasmid 7000-Puc19-ClaI by ClaI and NaeI, recovering a 7000bp fragment, simultaneously digesting the Ad35hexon recovered by PCR by ClaI and NaeI, recovering about 2800bp, and connecting the recovered two parts of fragments to construct the plasmid 7000-Puc19-Ad 35;
HindIII enzyme digestion 7000-Puc19-Ad35, 7000bp fragments are recovered, simultaneously plasmid 9620-Puc19-AscI is digested with HindIII enzyme, 5400bp fragments are recovered, the recovered two parts of fragments are connected, and plasmid 9620-Puc19-Ad35 is constructed;
I. the plasmid pBHGlox (delta) E1,3Cre is digested by AscI, a 25000bp fragment is recovered, meanwhile, the plasmid 9620-Puc19-Ad35 is digested by AscI, a 9620bp fragment is recovered, and the recovered two fragments are connected to construct a plasmid RAd 5-35-Hexon-Adbone.
The third step: and (5) verifying the recombinant adenovirus vector. Because the backbone plasmid is not recovered and purified and is subjected to single enzyme digestion, the backbone plasmid can be self-connected or the target fragment is connected to generate a reverse result, and enzyme digestion electrophoresis is needed to identify whether the size of the fragment is correct or not and to perform sequencing identification.
A. Electrophoresis experiment: agarose gel electrophoresis was performed using the original Ad5 backbone vector as a positive control.
B. Sequencing and identifying: the constructed plasmid RAd5-35-Hexon-Adbone was sent for sequencing, and the sequencing primers were:
5'-GAGGAAGTACGGCGCCGCCA-3' (SEQ ID NO: 9) (identification of correct orientation of the NruI linkage)
5'-GAGGACATGAACGATCATGCCATTC-3' (SEQ ID NO: 10) (identification of the correct orientation of the ligation with AscI)
5'-ATCGTTGCGGCCCGTAGCCAGTG-3' (identify if Ad35hexon replacement is correct)
2. Construction of shuttle plasmid expressing Green Fluorescent Protein (GFP) Gene and photographing with fluorescent microscope to observe cell changes.
In the first step, the GFP gene obtained by PCR was cloned into the eukaryotic expression vector pDC 315.
A. PCR amplification of GFP gene was carried out using pHAGE-CMV-MCS-GFP (plasmid with GFP gene stored in laboratory, or other plasmid with GFP gene may be selected) as template, and upstream primer 5'-CCGGAATCCGATGTCTAAAGGTGAAGAATTATTCA-3' (SEQ ID NO: 11) and downstream primer 5'-CGCGGATCCGCGTTATTTGTACAATTCATCCATACC-3' (SEQ ID NO: 12);
the reaction system of PCR is: 10 XBuffer 5 mu L, DNTP 4 mu L, upstream primer 1 mu L, downstream primer 1 mu L, high fidelity enzyme 1 mu L, template 1 mu L and a proper amount of sterilized water, wherein the total volume is 50 mu L.
The PCR reaction program is: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 68 deg.C for 1min for 30 cycles; finally, the extension is carried out for 10min at 68 ℃.
B. After PCR obtains a target gene, carrying out double digestion on a PCR product and a pDC315 vector by EcoRI and BamHI, and recovering a target fragment; ligation was performed overnight at 16 ℃; then, the plasmid pDC315-GFP was transformed and extracted.
C. EcoRI and BamHI are used for double digestion of pDC315-GFP plasmid, two bands of 700bp and 3900bp are cut out, and the correct pDC315-GFP clone is identified through sequencing.
In the second step, human embryonic kidney (293A) cells were transiently transfected after successful cloning and GFP expression was observed by fluorescence microscopy. The steps, reagents and conditions are as follows:
A. 293A cells at 5X 105The mixture is spread in a 6-hole plate with a density of one hole and put in 5% CO2The cells were cultured in the cell culture chamber of (1) at 37 ℃ until the cell growth area was 80% -90% of the bottom area of the well plate, and 3ug of pDC315-GFP plasmid was transfected into the cells using X-tremagene (available from Roche) as a transfection reagent, and the transfection procedure was as described in the reagent manual.
B. The expression of Green Fluorescent Protein (GFP) was observed by fluorescence microscopy: after 72 hours of transfection, the green fluorescent protein expression was observed under a fluorescent microscope and photographed, and the result is shown in fig. 9.
Example 2
Packaging and infectivity validation of recombinant adenoviruses
The first step is as follows: the recombinant adenovirus vector rAd5-35-Hexon-Adbone and the original adenovirus vector RAd5 constructed in example 1 and the shuttle plasmid containing the Green Fluorescent Protein (GFP) gene constructed in example 1 are co-transfected to 293A cells through X-tremagene (purchased from Roche), as shown in FIG. 3, the shuttle plasmid and the adenovirus vector co-transfected to 293A cells are packaged into recombinant adenovirus under the action of recombinase, and the obtained recombinant virus is replication-defective chimeric adenovirus with E1/E3 deletion. The result shows that the constructed recombinant adenovirus vector and the original adenovirus vector (namely rAd 5) are packaged successfully to obtain the virus primarily. The experimental procedures, reagents, conditions and operating methods were as follows:
each hole is laid with 3 multiplied by 10 in a six-hole plate5293A cells, carrying green fluorescent protein gene of the adenovirus shuttle plasmid and adenovirus carrier skeleton plasmid each 1.5ug, with X-tremeGENE (purchased from Roche) 9 u L cotransfection 293A cells, every 2-3 days change liquid 1 time, about 16 days appeared in cytopathic effect, i.e. cells become round and grow, shed and become grape bead-like aggregation, the results are shown in figure 10A and 10B, positive rAd5 packaging virus about the eighth day appeared in cytopathic effect, the results are shown in figure 10C.
The second step is that: collecting and packaging the chimeric virus, packaging the virus for 14 days, collecting the virus cells packaged in a six-hole plate after the cells are diseased, repeatedly freezing and thawing for 3 times in a water bath at-80-37 ℃, centrifuging at 4000rpm for 2 minutes to remove cell debris, and sucking cell culture supernatant containing the virus to infect new 293A cells. Green fluorescence and cytopathic effects were seen approximately 4 days after infection of 293A cells with positive rAd5 adenovirus, the results are shown in FIG. 11A, but only after approximately 10 days with rAd 5-35-hexon-Adbone-packaged adenovirus 293A, the results are shown in FIG. 11B.
The third step: and (3) collecting 293A cells infected by the adenovirus packaged by rAd5-35-hexon-Adbone in the previous step, repeatedly freezing and thawing at-80 ℃ to-37 ℃ for 3 times, and centrifuging at 4000rpm for 2 minutes to absorb supernatant. Ad35hexon was PCR amplified using primers F: 5'-Atggccaccccatcgatgct-3' (SEQ ID NO: 13) and R: 5'-TTACGTGGTAGCGTTACCG-3' (SEQ ID NO: 14) using the virus supernatant as a template, and sequencing identified that the hexon of rAd5 was successfully replaced by Ad35, the results of which are shown in FIG. 12.
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
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cttgctcgtc tacttcgtct 20
<210>7
<211>23
<212>DNA
<213> Artificial sequence
<400>7
atggccaccc catcgatgct gcc 23
<210>8
<211>25
<212>DNA
<213> Artificial sequence
<400>8
tacgtggtag cgttgccggc cgaga 25
<210>9
<211>20
<212>DNA
<213> Artificial sequence
<400>9
gaggaagtac ggcgccgcca 20
<210>10
<211>25
<212>DNA
<213> Artificial sequence
<400>10
gaggacatga acgatcatgc cattc 25
<210>11
<211>35
<212>DNA
<213> Artificial sequence
<400>11
ccggaatccg atgtctaaag gtgaagaatt attca 35
<210>12
<211>36
<212>DNA
<213> Artificial sequence
<400>12
cgcggatccg cgttatttgt acaattcatc catacc 36
<210>13
<211>20
<212>DNA
<213> Artificial sequence
<400>13
atggccaccc catcgatgct 20
<210>14
<211>19
<212>DNA
<213> Artificial sequence
<400>14
ttacgtggta gcgttaccg 19
Claims (2)
1. A recombinant adenovirus vector is characterized in that a molecular cloning method is adopted, and Hexon in a backbone plasmid of a type 5 adenovirus rAd5 vector is replaced by Hexon of human rare adenovirus serotype Ad35 to obtain the recombinant adenovirus vector rAd 5-35-Hexon-Adbone; the molecular cloning method comprises the following steps:
the transformation of the Puc19 vector comprises the following steps: introducing AscI and NruI between restriction sites XbaI and BamHI of the Puc19 vector to obtain an intermediate vector Puc 19-AscI-NruI;
(II) performing single enzyme digestion on the original Ad5 backbone plasmid by using AscI, recovering a 9620bp fragment, performing single enzyme digestion on the intermediate vector Puc19-AscI-NruI by using AscI, recovering a 2680bp fragment, and connecting the recovered two parts of fragments to construct a plasmid 9620-Puc19-AscI;
(III) singly digesting the plasmid 9620-Puc19-AscI with HindIII to recover a 7000bp fragment, simultaneously singly digesting the vector Puc19 with HindIII to recover a 2680bp fragment, and connecting the recovered two parts of fragments to construct a plasmid 7000-Puc 19-HindIII;
(IV) based on the plasmid 7000-Puc19-HindIII, inserting a restriction site ClaI at the 5 'end of the hexon of the Ad5 framework plasmid, carrying out PCR amplification on two sections of A and B at the 5' end of the hexon, inserting a single restriction site ClaI at the joint of the sections A and B, and designing primers as follows:
fragment A
Upstream: 5'-TCTAGACTGGTGACGCAAATAGACGA-3'
Downstream: 5'-TCTATCGATGGGGTAGCCATAAGCTT-3'
Fragment B
Upstream: 5'-TCTAGACCCATCGATGATGCCGCA-3'
Downstream: 5'-CTTGCTCGTCTACTTCGTCTAAGCTT-3', respectively;
(V) connecting the fragment A to a Puc19 vector, and digesting the plasmid Puc19-A by ClaI and HindIII to recover a 3100bp fragment; connecting the fragment B to a Puc19 vector, performing double enzyme digestion on the plasmid Puc19-B by ClaI and HindIII, and recovering a 500bp fragment; connecting the two recovered fragments to construct a plasmid Puc19-A + B;
(VI) double restriction of plasmid Puc19-A + B by XbaI and HindIII, recycling 1000bp fragment, simultaneously double restriction of plasmid 7000-Puc19-HindIII by DraIII-HF and RsrII, recycling 9800bp fragment, connecting the two recycled fragments by a rapid recombinant cloning kit, namely constructing plasmid 7000-Puc 19-ClaI;
(VII) PCR amplifying Ad35hexon, wherein the primers are designed as follows:
upstream: 5'-ATGGCCACCCCATCGATGCTGCC-3'
Downstream: 5'-TACGTGGTAGCGTTGCCGGCCGAGA-3'
Recovering a 2800bp fragment of Ad35 hexon;
(eight) carrying out double enzyme digestion on the plasmid 7000-Puc19-ClaI by ClaI and NaeI, recovering a 7000bp fragment, simultaneously carrying out double enzyme digestion on Ad35hexon recovered by PCR by ClaI and NaeI, recovering a 2800bp fragment, and connecting the recovered two parts of fragments to construct a plasmid 7000-Puc19-Ad 35;
(nine) HindIII single enzyme digestion 7000-Puc19-Ad35, recovering 7000bp fragments, simultaneously using HindIII single enzyme digestion plasmid 9620-Puc19-AscI to recover 5400bp fragments, connecting the recovered two parts of fragments, and constructing plasmid 9620-Puc19-Ad 35;
tenthly, performing single enzyme digestion on an original Ad5 skeleton plasmid by AscI, recovering a 25000bp fragment, performing single enzyme digestion on the plasmid 9620-Puc19-Ad35 by the AscI, recovering a 9620bp fragment, and connecting the two recovered fragments to construct a plasmid Ad 5-35-Hexon-Adbone;
wherein, the original Ad5 skeleton plasmid is pBHGlox (delta) E1,3 Cre.
2. Use of the recombinant adenoviral vector according to claim 1 for the preparation of a vaccine or a gene therapy drug.
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