CN103923943A - Expression vector based on adenovirus AdC7 and its construction method - Google Patents
Expression vector based on adenovirus AdC7 and its construction method Download PDFInfo
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Abstract
The invention relates to an expression vector based on adenovirus AdC7 and its construction method. An effective strategy is designed for successfully construct the new expression vector based on chimpanzee adenovirus AdC7. The vaccine vector can be applied to the preparation of high expression viral vaccines with good immunogenicity.
Description
Technical field
The invention belongs to biotechnology and field of virology; More specifically, the present invention relates to a kind of expression vector and construction process thereof based on adenovirus AdC7.
Background technology
Recombinant adenoviral vector has high transduction efficiency, expression level is high, the time length is long, be easy to the features such as purifying, and can the specific cellular immunization of induction exogenous gene and humoral immune reaction after immunity, is therefore a kind of desirable vaccine carrier.Based on adenovirus human serum type 2 types (AdHu2) and the expression vector of 5 types (AdHu5), in vaccine research and gene therapy, be once widely used, but because the individuality of 40-60% in crowd has infected corresponding adenovirus, there is the neutralizing antibody that prestores accordingly, thus the clinical application of restriction AdHu2 and AdHu5 carrier.For overcoming the impact of the neutralizing antibody that prestores, a kind of method is the capsid protein of transformation AdHu2 and AdHu5 carrier.Some studies show that, can realize this idea by exchange fiber protein gene.But this embedded virus still can be neutralized by the antibody for hexon epi-position, and some hexon albumen are unstable through improved embedded virus; Another kind method is that the adenovirus selecting the rare serotype of people or derive from other animal host is as expression vector.Owing to generally can not containing the neutralizing antibody of anti-chimpanzee type adenovirus in crowd, so the vaccine carrier based on chimpanzee type adenovirus, its immune effect is significantly better than the vaccine carrier with human serum type adenovirus construction.At present, the novel vaccine carrier based on chimpanzee type adenovirus becomes important selection in vaccine research and development.In May, 2013, Bill is covered 2,900 ten thousand dollars of the research units (company) that the U.S., Britain and Switzerland are subsidized in hereby foundation (Bill & Melinda Gates Foundation), for the research and development of Novel black orangutan adenovirus carrier.
The method that builds recombinant adenoviral vector has three kinds.Traditional method is in package cell line, to carry out the homologous recombination of foreign gene and wild-type virus, thereby obtains recombinant adenovirus, but this method complicated operation wastes time and energy, and is not easy to obtain single recombinant adenovirus; Second method is based on homologous recombination in intestinal bacteria, and its shortcoming is relatively time-consuming, easily undergos mutation.The third method be by adenoviral gene group Direct Cloning to plasmid vector, then linearizing transfection package cell line, saves out adenovirus.Because adenoviral gene group is relatively large, size is about 36kb, its genome is carried out to Direct Cloning and have certain difficulty.
Summary of the invention
The object of the present invention is to provide a kind of expression vector and construction process thereof based on adenovirus AdC7.
In a first aspect of the present invention, a kind of method of preparing adenovirus expression carrier is provided, described method comprises: with wild-type adenovirus AdC7 genome (preferably, sequence is as GenBank accession number AY530878.1) be basis, deletion E1 coding region and whole E3 coding region, at E1, delete district and increase I-Ceu I and PI-Sce I restriction enzyme site, at E3, delete district and increase Rsr II restriction enzyme site, obtain the recombinant adenoviral expressing vector of replication defect type.
In a preference, described deletion E1 coding region refers to the sequence of deleting 458-3026 position (the sequence sequence of calculation site based on GenBank accession number AY530878.1) in wild-type AdC7 genome.
In another preference, the whole E3 of described deletion coding region refers to the sequence of deleting 27094-31799 position (the sequence sequence of calculation site based on GenBank accession number AY530878.1) in wild-type AdC7 genome.
In another preference, described method comprises:
(1) by deriving from the origin sequence of pNEB193, the LITR fragment that derives from AdC7 adenoviral gene group, the Nde I-Age I fragment that derives from AdC7 adenoviral gene group, by merging PCR, be fused into fragment OLN, with Spe I and Age I endonuclease bamhi OLN; With Nde I, Age I and Spe I enzyme, cut AdC7 adenoviral gene group, the Segment A ge cutting (4028)-SpeI (10610) (the sequence sequence of calculation site based on GenBank accession number AY530878.1) is connected with enzyme was cut fragment OLN and forms plasmid pOIN;
(2) with Hind III and Avr II enzyme, cut AdC7 genome, object fragment Hind III (the 7153)-Avr II (23363) cutting is inserted in the Hind III and Avr II site of plasmid pOIN to the plasmid called after pOINH of acquisition (the sequence sequence of calculation site based on GenBank accession number AY530878.1);
(3) amplification fragment between 31800-36535 in AdC7 adenoviral gene from AdC7 adenoviral gene group, at 5 ' end, introduce Avr II and Rsr II restriction enzyme site, at 3 ' end, introduce Pac I and Asis I site, the fragment of amplification is inserted in the Avr II and Asis I site of pOINH to the plasmid called after pOINHR of acquisition;
(4) amplification fragment between 23165-27093 in AdC7 adenoviral gene from AdC7 adenoviral gene group, at 3 ' end, introduce Rsr II site, the fragment of amplification is inserted in the Avr II and Rsr II site of pOINHR, obtains the recombinant adenoviral expressing vector of replication defect type.
In another preference, described origin sequence is the sequence shown in 1-1882 position in SEQ ID NO:1 (being the nucleotide sequence of accompanying pAdC7 carrier).
In another preference, the described LITR fragment that derives from AdC7 adenoviral gene group is the sequence shown in 1883-2382 position in SEQID NO:1.
In another preference, the described NdeI-AgeI fragment that derives from AdC7 adenoviral gene group is the sequence shown in 2383-3422 position in SEQ ID NO:1.
In another preference, the fragment between described Rsr II-Asis I is the sequence shown in 26483-31241 position in SEQ ID NO:1.
In another preference, the fragment between described Avr II-Rsr II is the sequence shown in 22751-26482 position in SEQ ID NO:1.
In another aspect of this invention, a kind of adenovirus expression carrier is provided, described expression vector is deletion E1 coding region and whole E3 coding region on wild-type adenovirus AdC7 genome basis, and, at E1, delete district and increase I-Ceu I and PI-Sce I restriction enzyme site, at E3, delete district and increase Rsr II restriction enzyme site.
In a preference, between the restriction enzyme site PI-Sce I and I-Ceu I of described expression vector, also comprise the antigen encoding gene of external source.
In another preference, described exogenous antigen is the hemagglutinin antigen (as H5N1HA) of (but being not limited to) influenza virus.
In another aspect of this invention, provide a kind of method of preparing vaccine, described method comprises:
(1) provide described adenovirus expression carrier;
(2) antigen encoding gene of external source is inserted between the restriction enzyme site PI-Sce I and I-Ceu I of (1) expression vector;
(3) recombinant expression vector of (2) is infected to virus production cell, virus, in cell internal packing, has immunogenic vaccine thereby obtain.
In another aspect of this invention, provide a kind of test kit for the preparation of vaccine, described test kit comprises: arbitrary described adenovirus expression carrier above.
In a preference, described test kit also comprises: virus production cell; And/or antigen encoding gene.
In another preference, described virus production cell is HEK293 cell.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
The enzyme of Fig. 1, AdC7 genomic dna is cut evaluation.
1,1kb ladder (NEB); 2, Bgl II enzyme is cut.
The enzyme of Fig. 2, recombinant adenoviral vector pAdC7 (being pAdC7-Δ E1 Δ E3) is cut evaluation.
1、1kb?ladder(NEB)；2、Bgl?II；3.Xho?I；4.Mfe?I。
Fig. 3, structure recombinant adenovirus AdC7-eGFP.
A: the enzyme of recombinant adenoviral vector pAdC7-eGFP is cut evaluation;
B: the plaque formation of recombinant adenovirus AdC7-eGFP
Fig. 4, structure recombinant adenovirus AdC7-H5N1HA.
A:pAdC7-H5N1HA enzyme is cut evaluation;
B:AdC7-H5N1 enzyme is cut evaluation;
C: detect the expression of HA albumen.
The strategy of Fig. 5, structure replication-defective adenoviral vector pAdC7.
Embodiment
The inventor, through deep research, has designed a kind of effective strategy, successfully based on chimpanzee type adenovirus AdC7, is built into a kind of new expression vector.Described vaccine carrier can be applicable to preparation can high efficient expression and have good immunogenic virus vaccines.
The invention provides a kind of adenovirus expression carrier, described expression vector comprises: described expression vector is deletion E1 coding region and whole E3 coding region on wild-type adenovirus AdC7 genome basis, and, at E1, delete district and increase I-Ceu I and PI-Sce I restriction enzyme site, at E3, delete district and increase Rsr II restriction enzyme site.
For adenovirus AdC7 genome, the inventor, through careful sequence alignment, has finally determined that application restriction enzyme site I-Ceu I and PI-Sce I are as the insertion point of foreign gene, thereby can not cause causing shearing in other position of adenovirus expression carrier.
Chimpanzee type adenovirus AdC7, SAdV24, is named as again Pan7, and from chimpanzee lymphoglandula, separation obtains.The present invention be take wild-type AdC7 as basis, by genome Direct Cloning method, deletion E1 and whole E3 coding region, at E1, delete district and increase I-Ceu I and PI-Sce I restriction enzyme site, at E3, delete district and increase Rsr II enzyme site, finally obtained the AdC7 recombinant adenoviral expressing vector of replication defect type.
Described expression vector also contains replication orgin and/or marker gene etc.Method well-known to those having ordinary skill in the art can be for building expression vector required for the present invention.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.The suitable promotor (as CMV) that described DNA sequence dna can be effectively connected in expression vector is upper, to instruct mRNA synthetic.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.In addition, expression vector preferably comprises one or more selected markers, to be provided for the phenotypic character of the host cell of selection conversion.
The present invention also provides a kind of method of preparing adenovirus expression carrier, described method comprises: take wild-type adenovirus AdC7 genome as basis, deletion E1 coding region and whole E3 coding region, at E1, delete district and increase I-Ceu I and PI-Sce I restriction enzyme site, at E3, delete district and increase Rsr II restriction enzyme site, obtain the recombinant adenoviral expressing vector of replication defect type.
As optimal way of the present invention, clone's step comprises: the fragment that 1. Overlap PCR obtains, and after Age I and Spe I enzyme are cut, with NdeI, AgeI and SpeI enzyme are cut the fragment that AdC7 postgenome obtains, and connect into plasmid pOIN; 2. Hind IIII and Avr II digested plasmid pOIN and AdC7 genome, connect into plasmid pOINH; 3. Rsr II and Asis I enzyme are cut fragment and the plasmid pOINH that PCR obtains, and connect into plasmid pOINHR; 4. Avr II and Rsr II enzyme are cut fragment and the plasmid pOINHR that PCR obtains, and connect into plasmid pAdC7.
Obtain after described adenovirus expression carrier, by it transfection virus production cell, carry out viral breeding.After for some time after transfection, can gather in the crops virus.As optimal way of the present invention, the virus of results can repeated infection virus production cell, continues to go down to posterity.Virus titer (TCID
50) mensuration can carry out according to this area ordinary method.
Adenovirus expression carrier of the present invention, as an expression vector platform, is applicable to express plurality of antigens, thereby prepares virus vaccines.Described antigen has no particular limits.
As optimal way of the present invention, validity for checking AdC7, the inventor by the HA gene clone of influenza virus H 5 N 1 to AdC7 carrier, studies show that AdC7 carrier energy overexpression protection protogene (as HA gene), the proof inventor has successfully obtained a kind of novel adenovirus carrier, can be used for vaccine research and development and other biomedical fundamental research, for new generation vaccine research and development provide a kind of novel carrier platform.
The present invention is by the method for adenoviral gene group Direct Cloning, and utilize STBL2 intestinal bacteria stable conversion system, avoid traditional homologous recombination method of passing through and built adenovirus carrier, reduced largely the possibility that adenovirus is undergone mutation, the recombinant adenoviral vector genetic stability of acquisition is good.Therefore, utilize the adenovirus carrier expression alien gene that the present invention builds will be more stable, efficient.The present invention utilizes external direct enzyme cutting method of attachment to build adenovirus carrier, at bacteria levels screening positive clone more easily, fast, has greatly shortened the structure time of adenovirus carrier.In addition, the present invention is applicable to the structure of all adenovirus carriers, only need adenoviral gene group restriction enzyme site to analyze and rationally apply, all biomedical laboratory can be generally applied to, and to adenovirus internal sequence, can be optimized transformation, build each species specific adenovirus carrier.Therefore, the present invention not only provides a kind of new expression vector, and provide a kind of construction process of novel adenovirus carrier, more be conducive to promote adenovirus carrier bringing into play its advantageous feature aspect biomedical fundamental research field and clinical application exploitation, for guarantee human health, improving the quality of living lays the foundation.
Based on new improvement of the present invention, the present invention also provides a kind of test kit for the preparation of vaccine, and described test kit comprises described adenovirus expression carrier.
Described test kit also can comprise virus production cell, the encoding gene of the antigen that institute's wish is expressed.In addition, in described test kit, also can comprise the working instructions that vaccine preparation method is described.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.
1, material
1.1 virus strain and cell
Wild-type adenovirus AdC7 is purchased from ATCC (Manassas, VA); HEK293 cell strain is purchased from Chinese Academy of Sciences's Shanghai biological chemistry and cell biological institute, and substratum is the DMEM containing 10% foetal calf serum (FCS).
1.2 restriction enzymes, bacterial classification and plasmid
Restriction enzyme is purchased from New England Biolabs; DH5 α and Stbl2 are purchased from Invitrogen; PNEB193 is purchased from New England Biolabs.
Pshuttle-CMV carrier is purchased from CLONTECH Laboratories, Inc.
PUC57-EGFP plasmid: EGFP sequence (SEQ ID NO:2) is become and is cloned into pUC57 carrier by Nanjing Jin Sirui company, obtains pUC57-EGFP.
PUC57-H5N1HA plasmid: H5N1HA sequence (SEQ ID NO:3) is synthesized and is cloned into pUC57 carrier by Nanjing Jin Sirui company, obtains pUC57-H5N1HA.
2, method
The amplification of 2.1 adenovirus and purifying
HEK293 cell is cultivated in containing the DMEM of 10%FCS, and AdC7 adenovirus is cultivated amplification in HEK293 cell.Adenovirus infection HEK293 carries out at the DMEM without FCS, after two hours, adds FCS and makes FCS final concentration reach 5%.When cell all shows after viral plaque (CPE), collect infected cell, centrifugal.Use DMEM re-suspended cell, multigelation cell three times, makes lysis.Virus, through CsCl density gradient centrifugation purifying, then adds glycerine to put-80 ℃ of Refrigerator stores.
2.2 extract adenovirus genomic dna
After AdC7 adenovirus purifying, with standard method, extract genomic dna.With the evaluation that performs an analysis of BglII digested genomic dna.
2.3 build the adenovirus carrier of replication defect type
2.3.1 build plasmid pOIN
Take pNEB193 as template, with primer outside primer of the origin and antisense primer of the origin (table 1), by pcr amplification, obtain fragment the origin.
The AdC7 adenoviral gene group of take is template, with primer sense primer of LITR and antisense primer of LITR.I-Cue, by pcr amplification, obtains fragment LITR.
The AdC7 adenoviral gene group of take is template, with primer sense primer of NdeI-AgeI.PI-Sce and outside primer of NdeI-AgeI, by pcr amplification, obtains fragment NdeI-AgeI.
By merging PCR, by fragment the origin, fragment LITR and fragment NdeI-AgeI are fused into fragment OLN, while merging PCR, use primer inside primer of origin and inside primer of NdeI-AgeI.By PCR and fusion PCR, the 458-3026 bit slice section of E1 in AdC7 genome is deleted to (the sequence sequence of calculation site based on GenBank accession number AY530878.1), introduce some restriction enzyme sites simultaneously, 5 ' the end at fragment the origin is introduced spe I, Avr II and Asis I restriction enzyme site, between fragment the origin and LITR, introduce Pac I restriction enzyme site, between fragment LITR and NdeI-AgeI, introduce I-Ceu I and PI-Sce I restriction enzyme site.With Spe I and Age I endonuclease bamhi OLN, with DNA common products purification kit, reclaim; With Nde, Spe I and Age I, (with Spe I and Age I enzyme, cut AdC7, can obtain two fragments that size is almost identical, be Segment A ge I (4028)-Spe I (10601) and AgeI (25823)-Spe I (32770), inseparable in low melting-point agarose electrophoresis, therefore with three kinds of enzyme enzymes, cut AdC7 genome) enzyme cuts AdC7 genome, low melting-point agarose electrophoresis, can obtain 6.5kb object Segment A geI (4028)-Spe I (10610) (the sequence sequence of calculation site based on GenBank accession number AY530878.1).Then, in low melting-point agarose, connect reaction, transform, the selected clone evaluation that performs an analysis; Obtain pOIN plasmid.
Table 1, the primer
2.3.2 build plasmid pOINH
With Hind III and Avr II enzyme, cut pOIN, agarose electrophoresis reclaims object fragment (fragment of 6.5kb); With Hind III and Avr II enzyme, cut AdC7 genome, low melting-point agarose electrophoresis, obtains object fragment: the fragment from Hind III (7153) to Avr II (23363) (the sequence sequence of calculation site based on GenBank accession number AY530878.1).Then, in low melting-point agarose, connect reaction, transform, identify; Obtain pOINH.
2.3.3 build plasmid pOINHR
The AdC7 adenoviral gene group of take is template, with primer sense primer of RsrII.AsisI.PacI and antisense primer of RsrII.AsisI.PacI, by pcr amplification, obtain fragment RsrII-AsisI, purified pcr product, Rsr II and Asis I endonuclease bamhi RsrII-AsisI.With Rsr II and Asis I enzyme, cut pOINHR, low melting-point agarose electrophoresis, obtains object fragment.Then, in low melting-point agarose, connect reaction, transform, identify.
2.3.4 build plasmid pAdC7
Take AdC7 adenovirus genomic dna as template, with primer sense primer of AvrII-RsrII and antisense primer of AvrII-RsrII, by pcr amplification, obtain Segment A vr II-Rsr II, purified pcr product; With Avr II and Rsr II endonuclease bamhi AvrII-RsrII.With Avr II and Rsr II enzyme, cut pOINHR, low melting-point agarose electrophoresis, obtains object fragment.Then, in low melting-point agarose, connect reaction, transform, identify.Obtain plasmid pAdC7, by 2.3.2 and the similar step of 2.3.3,27094-31799 bit sequence in AdC7 genome is deleted to (deleting E3).
2.4 build recombinant adenovirus AdC7-eGFP
2.4.1 eGFP is cloned into pAdC7
With Nhe I and Not I enzyme, cut pUC57-EGFP plasmid, sepharose reclaims object fragment EGFP, is connected to the pShuttle-CMV carrier of cutting through same enzyme, and enzyme obtains plasmid pShuttle-CMV-EGFP after cutting and identifying.With PI-Sce I and I-Ceu I enzyme, cut pshuttle-eGFP, run glue and reclaim object fragment; With PI-SceI and I-Ceu I enzyme, cut pAdC7, low melting-point agarose electrophoresis, obtains object fragment.Then, in low melting-point agarose, connect reaction, transform, identify.Obtain pAdC7-eGFP.
2.4.2 rescue AdC7-eGFP recombinant adenovirus
Pac I enzyme is cut pAdC7-eGFP, makes its linearizing.With HEK293 cell, spread 6 orifice plates, when cell degree of collecting reaches 70-80%, with the linearizing pAdC7-eGFPDNA of X-tremeGENE transfection 1.5 μ g.Every day observation of cell fluorescence situation.
2.5 build recombinant adenovirus AdC7-H5N1HA
2.5.1 H5N1HA is cloned into pAdC7
With Apa I and Not I enzyme, cut pUC57-H5N1HA plasmid, sepharose reclaims object fragment HA, is connected to the pShuttle-CMV carrier of cutting through same enzyme, and enzyme is cut and identified and check order and obtain plasmid pShuttle-H5N1HA.With PI-Sce I and I-Ceu I enzyme, cut pshuttle-H5N1HA, run glue and reclaim object fragment; With PI-Sce I and I-Ceu I enzyme, cut pAdC7, low melting-point agarose electrophoresis, obtains object fragment.Then, in low melting-point agarose, connect reaction, transform, identify; Obtain pAdC7-H5N1HA.
2.5.2 rescue AdC7-H5N1HA recombinant adenovirus
Pac I enzyme is cut pAdC7-H5N1HA, makes its linearizing.With HEK293 cell, spread 6 orifice plates, when cell degree of collecting reaches 70-80%, with the linearizing pAdC7-H5N1HA of X-tremeGENE transfection 1.5 μ g.
2.5.3 amplification and purifying group adenovirus AdC7-H5N1HA
HEK293 cell is cultivated in containing the DMEM of 10%FCS, and AdC7-H5N1HA adenovirus is cultivated amplification in HEK293 cell.Adenovirus infection HEK293 carries out at the DMEM without FCS, after two hours, adds FCS and makes FCS final concentration reach 5%.When cell all shows after viral plaque (CPE), collect infected cell, centrifugal.Use DMEM re-suspended cell, multigelation cell three times, makes lysis.Virus, through CsCl density gradient centrifugation purifying, then adds glycerine to put-80 ℃ of Refrigerator stores.
2.5.4 enzyme is cut and is identified AdC7-H5N1HA
After AdC7-H5N1HA adenovirus purifying, with standard method, extract genomic dna.With the evaluation that performs an analysis of Bgl II and Xho I digested genomic dna.
2.5.5Western blot detects the expression of H5N1HA
With HEK293 cell, spread 6 orifice plates.When cell degree of collecting reaches 80-90%, 1 * 10
9vp, 3 * 10
9vp and 6 * 10
9vp AdC7-H5N1HA virus is removed cells infected, uses 6 * 10 simultaneously
9vp AdC7 virus is removed cells infected, after 24 hours, receives cell, and Western blot detects the expression of H5N1.
3, embodiment
The extraction of the amplification of embodiment 1, adenovirus, purifying and adenoviral gene group
Wild-type AdC7 obtains purifying through CsCl density gradient centrifugation after increasing in HEK293 cell, extracts afterwards virus genom DNA, with Bgl II enzyme, cuts genome, carries out agarose electrophoresis.Result shows, increase, the virus of purifying is AdC7 adenovirus.See Fig. 1.
The adenovirus carrier of embodiment 2, structure replication defect type
According to schema (Fig. 5), by PCR, obtain fragment the ori, LITR, NdeI-AgeI; By merging PCR, this three segment composition is become to fragment OLN; With Spe I and Age I enzyme, cut OLN, with Hind III, Age I, Spe I enzyme, cut AdC7 genome, be connected to form afterwards plasmid pLITR.
With Hind III and Avr II enzyme, cut AdC7 genome, then enzyme is cut and obtained large dna fragment cloning to pLITR, form plasmid pOINH.
To obtain fragment RsrII-AsisI by PCR, be cloned into pOINH, form pOINHR.
Finally, will obtain Segment A vrII-RsrII by PCR, be cloned into plasmid pOINHR, obtain pAdC7-Δ E1 Δ E3.
With Bgl II, Xho I, Mfe I enzyme, cut pAdC7-Δ E1 Δ E3, carry out agarose electrophoresis, result shows that pAdC7-Δ E1 Δ E3 is correct object carrier.See Fig. 2.
Embodiment 3, structure recombinant adenovirus AdC7-eGFP
EGFP is cloned into after pAdC7, and the enzyme of recombinant adenoviral vector pAdC7-eGFP is cut and is identified as Fig. 3 A.
, there is viral plaque on the tenth day in transfected HEK 293 after recombinant plasmid dna linearizing, plaque expands gradually subsequently.As Fig. 3 B.
Embodiment 4, structure recombinant adenovirus AdC7-H5N1HA
H5N1HA is cloned into after pAdC7, and transfected HEK 293 is packed out virus.After amplification, purified virus, carry genome enzyme and cut evaluation, as Fig. 4 A-B.
With the AdC7-H5N1HA of different titers, infect HEK293 cell.After 24 hours, collecting cell, Western blot detects the expression of HA.As Fig. 4 C.
Conclusion
By the method for above viral genome Direct Cloning, the inventor is built into wild-type adenovirus AdC7 in the carrier of energy expression alien gene.Therefore the inventor, for new generation vaccine research and development and various biomedical fundamental research provide a kind of new expression vector based on AdC7, provides a kind of method of novel structure adenovirus carrier simultaneously.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a method of preparing adenovirus expression carrier, is characterized in that, described method comprises:
Take wild-type adenovirus AdC7 genome as basis, deletion E1 coding region and whole E3 coding region, at E1, delete district and increase I-Ceu I and PI-Sce I restriction enzyme site, at E3, delete district and increase Rsr II restriction enzyme site, obtain the recombinant adenoviral expressing vector of replication defect type.
2. the method for claim 1, is characterized in that, described deletion E1 coding region refers to the sequence of deleting 458-3026 position in wild-type AdC7 genome.
3. the method for claim 1, is characterized in that, the whole E3 of described deletion coding region refers to the sequence of deleting 27094-31799 position in wild-type AdC7 genome.
4. the method for claim 1, is characterized in that, described method comprises:
(1) by deriving from the origin sequence of pNEB193, the LITR fragment that derives from AdC7 adenoviral gene group, the Nde I-Age I fragment that derives from AdC7 adenoviral gene group, by merging PCR, be fused into fragment OLN, with Spe I and Age I endonuclease bamhi OLN; With Nde I, Age I and Spe I enzyme, cut AdC7 adenoviral gene group, the Segment A ge cutting (4028)-SpeI (10610) is connected with enzyme was cut fragment OLN and forms plasmid pOIN;
(2) with Hind III and Avr II enzyme, cut AdC7 genome, object fragment Hind III (the 7153)-Avr II (23363) cutting is inserted in the Hind III and Avr II site of plasmid pOIN to the plasmid called after pOINH of acquisition;
(3) amplification fragment between 31800-36535 in AdC7 adenoviral gene from AdC7 adenoviral gene group, at 5 ' end, introduce Avr II and Rsr II restriction enzyme site, at 3 ' end, introduce Pac I and Asis I site, the fragment of amplification is inserted in the Avr II and Asis I site of pOINH to the plasmid called after pOINHR of acquisition; With
(4) amplification fragment between 23165-27093 in AdC7 adenoviral gene from AdC7 adenoviral gene group, at 3 ' end, introduce Rsr II site, the fragment of amplification is inserted in the Avr II and Rsr II site of pOINHR, obtains the recombinant adenoviral expressing vector of replication defect type.
5. an adenovirus expression carrier, it is characterized in that, described expression vector is deletion E1 coding region and whole E3 coding region on wild-type adenovirus AdC7 genome basis, and, at E1, delete district and increase I-Ceu I and PI-Sce I restriction enzyme site, at E3, delete district and increase Rsr II restriction enzyme site.
6. adenovirus expression carrier as claimed in claim 5, is characterized in that, between the restriction enzyme site PI-Sce I and I-Ceu I of described expression vector, also comprises the antigen encoding gene of external source.
7. adenovirus expression carrier as claimed in claim 6, is characterized in that, described exogenous antigen is the hemagglutinin antigen of influenza virus.
8. a method of preparing vaccine, is characterized in that, described method comprises:
(1) provide adenovirus expression carrier claimed in claim 5;
(2) antigen encoding gene of external source is inserted between the restriction enzyme site PI-Sce I and I-Ceu I of (1) expression vector;
(3) recombinant expression vector of (2) is infected to virus production cell, virus, in cell internal packing, has immunogenic vaccine thereby obtain.
9. for the preparation of a test kit for vaccine, it is characterized in that, described test kit comprises:
The arbitrary described adenovirus expression carrier of claim 5-7.
10. test kit as claimed in claim 9, is characterized in that, described test kit also comprises:
Virus production cell; And/or
Antigen encoding gene.
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| CN107574175A (en) * | 2017-09-11 | 2018-01-12 | 南方医科大学 | A kind of expression vector and its construction method based on recombined adhenovirus |
| CN107686843A (en) * | 2017-09-11 | 2018-02-13 | 南方医科大学 | A kind of expression vector and its construction method based on adenovirus AdC6 |
| CN107988258A (en) * | 2017-12-12 | 2018-05-04 | 中国科学院微生物研究所 | A kind of zika virus vaccine based on chimpanzee adenoviral vector and preparation method thereof |
| CN112760341A (en) * | 2020-07-13 | 2021-05-07 | 中国科学院微生物研究所 | Recombinant vector and application of chimpanzee adenovirus packaged with recombinant vector in preparation of 2019 novel coronavirus vaccine |
| CN113930452A (en) * | 2020-07-13 | 2022-01-14 | 中国科学院微生物研究所 | Recombinant vector based on chimpanzee adenovirus vector, adenovirus and 2019 novel coronavirus vaccine and preparation method thereof |
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