CN1276777C

CN1276777C - Adenoviral vector SARS vaccine and preparation method thereof, application of coronavirus S gene

Info

Publication number: CN1276777C
Application number: CN 200410044285
Authority: CN
Inventors: 黄文林; 曾益新; 汪健; 刘然义; 黄嘉凌; 黄必军; 赖坤; 吴立志; 梁志慧; 柯妙拉; 吴秀菊
Original assignee: TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
Current assignee: TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
Priority date: 2003-05-21
Filing date: 2004-05-18
Publication date: 2006-09-27
Anticipated expiration: 2024-05-18
Also published as: CN1562365A

Abstract

本发明属于生物基因工程领域，具体涉及腺病毒载体SARS疫苗，其制备方法以及相关冠状病毒S基因在制备预防非典型肺炎(SARS)疫苗方面的应用。通过生物工程手段，将相关冠状病毒S基因与缺陷型腺病毒重组结合，使保护性免疫原蛋白或多肽在其中表达，经过扩增培养、纯化、制剂而制成一种能引起粘膜免疫原性的基因疫苗，诱导呼吸道粘膜免疫反应，使机体产生相应抗体，防止病毒侵染。本发明与传统的灭活病毒颗粒疫苗比较，安全性高，使用方便，不受肌肉注射等特定条件限制，具有广泛的临床应用前景。The invention belongs to the field of biogenetic engineering, and in particular relates to an adenovirus vector SARS vaccine, a preparation method thereof and the application of related coronavirus S gene in preparation of SARS vaccine. By means of bioengineering, the related coronavirus S gene is recombined with the defective adenovirus, so that the protective immunogenic protein or polypeptide is expressed in it, and after amplified culture, purification, and preparation, a drug that can cause mucosal immunogenicity is produced. The genetic vaccine can induce respiratory mucosal immune response, make the body produce corresponding antibodies, and prevent virus infection. Compared with the traditional inactivated virus particle vaccine, the invention has high safety, is convenient to use, is not limited by specific conditions such as intramuscular injection, and has broad clinical application prospects.

Description

Adenovirus Carrier SARS Vaccines And Its Preparation Method, the application of coronavirus S gene

One, technical field

The invention belongs to the biological gene engineering field, be specifically related to adenovirus vector SARS vaccine, its preparation method and the application of announcement associated coronavirus S gene aspect preparation prevention of atypical pneumonia (SARS) vaccine.

Two, background technology

Atypical pneumonia (atypical pneumonia) is broken out in the Chinese Guangdong area since the end of last year, morbidity is anxious, and infectiousness is strong, and antibiotic therapy is invalid.At present, this disease has been transmitted to more than 30 countries and regions, WHO with the respiratory complication of it called after serious acute (severe acuterespiratory syndrome, SARS).

At present, the scientist of world many countries all from different patients serums independent separate to novel coronavirus (coronavirus), the virus gene sequence analysis result shows, they and known coronavirus have 50%～60% homology, The World Health Organization (WHO) announces that the pathogen that causes SARS is the variant of coronavirus.

Coronavirus is a kind of RNA viruses of non-segmental normal chain, and the nearly 30kb of its genome can propagate in humans and animals, the main respiratory system that infects humans and animals, the coronavirus granule is a kind ofly to have inner core and cyst membrane bag by ground virus, has four kinds of structural protein: and spike protein (spike, S), memebrane protein (membrance, M), and envelope protein (envelop, E), and nucleoprotein (nucleoprotein, N).Because coronavirus is a kind of RNA viruses, and is very unstable, undergo mutation easily to escape host's immunological surveillance and repulsion.Therefore, must search out good stability in the SARS associated coronavirus and have the antigen of immanoprotection action, with the development of the vaccine of being correlated with.

The virus particle vaccine of present less application deactivation, because whole virus particles has been carried viral full genome, safety concerns is bigger.Though coronavirus previously easily obtains at In vitro culture, this time the coronavirus toxicity that SARS is relevant greatly and genetic background imperfectly understand, so mass preparation coronavirus granule is infeasible.The exploitation that develops into subunit vaccine of technique for gene engineering provides a great convenience, and the operability of subunit vaccine and biological safety are good.Have immunogenic virus antigen determinant if can screen, in conjunction with technique for gene engineering, can transform antigenic determinant easily, to strengthen its stability, immunogenicity, biological safety.Obviously, by technique for gene engineering, can prepare effective subunit vaccine easily.

According to studies show that at present; in the known four kinds of structural protein of coronavirus; spike protein (spike S) has the structural protein of inducing protective immunological reaction; part scholar's research structure is verified, and coronavirus spike protein C-terminal is its antigen decision family place; simultaneously; the SARS associated coronavirus causes infection by respiratory tract, and the vaccine that can prevent SARS is not arranged at present yet.

In addition, data shows, adenovirus itself is easy to infect the respiratory mucosa epithelium, more easily induce the respiratory mucosa immunoreation, and adenovirus is made the immunogen of vector expression and is expressed in host cell, processes, folds, modifies, offers, basically kept immunogenic native conformation, the biological activity height.

Described condition provides solid theory for utilizing defective adenoviral as carrier development and production adenovirus SARS vaccine.

Three, summary of the invention

First purpose of the present invention is to provide a kind of adenovirus SARS vaccine that can prevent epidemic disease " atypical pneumonia ", better prevents the generation and the propagation of " atypical pneumonia "; Another object of the present invention is to provide a kind of defective adenoviral that utilizes as carrier, by means such as gene clone, reorganization, prepares the method for adenovirus vector SARS vaccine and discloses the application of SARS associated coronavirus S gene in the preparation vaccine.

The object of the present invention is achieved like this: by the biological engineering means, (coronavirus has four structural genes with SARS associated coronavirus S gene, the S gene is one of them, detailed description sees below) combine with the defective adenoviral reorganization, constitute a kind of gene vaccine that can cause mucosal immunity originality.

Used Spike gene fragment order is:

The S gene order of being announced among the use Gene bank (Gene bank query ID:, as follows according to sequential design PCR primer gbAY278554.2) as masterplate:

V1 GGTCTAGAGT?TGTGGTTTCA?AGTGAT

V2 TTTCTAGACC?ATGGGTTGTG?TCCTTGCT

V3 TTTCTAGACC?ATGGCATATA?GGTTCAATG

V4 TAGGTACCAA?TGCCAGTAGT?GGTG

V5 TTGGTACCTC?CGCCTCGACT?TT

V6 CCGGTACCAT?AAGTTCGTTT?ATGTGT

Wherein V1 and V4 are a pair of, amplification S gene N end fragment; V2 and V5 are a pair of primer, amplification S gene M district fragment; V3 and V6 are a pair of primer, amplification S gene C end fragment (as Fig. 1), and the structure after the amplification is seen Fig. 2.

The preparation of adenovirus SARS vaccine:

At first, collect to close and separate rehabilitation patient's serum, obtain the total RNA of coronavirus, by reverse transcription, step such as check order, select and obtain the S gene by separation and Extraction.Then, the S gene clone is obtained clone body (depositary institution: Chinese typical culture collection center to the pShuttle plasmid; Preservation date: on May 17th, 2003; Preserving number: CCTCC M 203036 E.coliDH5a/pShuttle-SN and CCTCC M 203037 E.coli DH5a/pShuttle-SC), again with itself and adenovirus skeleton plasmid (pAdeno-X ^TM) connect (wherein pShuttle, pAdeno-X be all available from CLONTECH Laboratory, Inc, USA).Connect the common rotaring redyeing 293 cell in back, after further purification is identified, increase in a large number with 293 cells, collect viral liquid, separation and purification is adenovirus SARS vaccine.It can make the medicament of spray or other form.The major technology route is seen Fig. 4.

The present invention is a kind of recombinant vaccine, promptly be to use the gene vaccine of defective adenoviral as carrier, the adenovirus that its utilization itself is easy to infect the respiratory mucosa epithelium expresses former albumen of protective immunity or polypeptide as carrier therein, induces the respiratory mucosa immunoreation; By the mucosa immunity-inducing reaction, make body produce corresponding antibodies, prevent virus infection.The present invention and traditional inactivated virus particle vaccine relatively, it is safe, and easy to use, not limited by specified conditions such as intramuscular injection.

At present, SARS propagates all over the world fast, as a kind of viral infectious, does not find the medicine that can effectively treat now as yet, and in such cases, prevention is best means.Confirmed that now SARS associated coronavirus spike protein C-terminal is its antigen decision family place.Therefore, the present invention is just according to this discovery, synthetic SARS associated coronavirus spike protein gene, it is cloned into adenovirus vector, form through amplification cultivation, purification, preparation, can effectively induce mucosa to produce antibody, produce humoral immunization and prevent the virus infection body, have potential applicability in clinical practice widely.

Four, description of drawings

Fig. 1 is a S gene fragment amplification sketch map.

Fig. 2 is the recombination deficient mutant adenovirus structure chart that has S gene (Spike-S).

Fig. 3 is pShuttle-Sc, pShuttle-S _M, pShuttle-S _NRecombinant enzyme action result.

Fig. 4 is pShuttle-Sc, pShuttle-S _M, pShuttle-S _NThe recombinant sequencing result.

Fig. 5 is that RT-PCR detects Ad-S _NAnd Ad-S _MThe expression result of the test

Fig. 6 is that Western blot detects Ad-S _NThe expression result of the test

Fig. 7 is Ad-S _NInjection or respiratory immunity all induce female rats to produce the expression in vivo result of the test of specific antibody (IgG)

Fig. 8 is Ad-S _NInjection or respiratory immunity all induce male rat to produce the expression in vivo result of the test of specific antibody (IgG)

Fig. 9 is the technology path figure of vaccine production of the present invention.

Five, the specific embodiment

Below will further specify the present invention by specific embodiment.

Embodiment 1

The preparation method of adenovirus vector SARS vaccine:

Two parts that the preparation of adenovirus vector SARS vaccine is divided into that make up early stage and the later stage increases:

Make up early stage:

Obtain SARS associated coronavirus spike (S _F, S _N, S _M, S _C) behind the gene, increase with PCR method, behind PCR,, use this enzyme enzyme action pShuttle simultaneously with 37 ℃ of enzyme action of Xbal+Kpnl, connect, transformed into escherichia coli DH5a utilizes kanamycin (Kan ^R) the resistance screening positive colony, obtain pS behind cultivation, the purification _F/ S _N/ S _M/ S _C-Shuttle uses the l-Ceul+Pl-Scel enzyme action, uses this enzyme enzyme action PAdeno-X simultaneously ^TM, connecting behind the enzyme action, transformed into escherichia coli DH5a utilizes ammonia benzyl (Amp ⁺) the resistance screening positive colony promptly gets pAd-S _F/ S _N/ S _M/ S _C

Amplification culture:

Obtain pAd-S _F/ S _N/ S _M/ S _CAfter, again through Pacl enzyme action, plasmid transfection incasing cells after linearize, incasing cells is for having integrated the cell line (strain) of C subclass 5 type adenovirus (Ad5) E1 district genes, for example 293 cells.Through plaque select, PCR is accredited as Ad-S _F/ S _N/ S _M/ S _CAfter, cultivate 293 cells in a large number, use Ad-S _F/ S _N/ S _M/ S _CInfect 293 cells, through the cesium chloride separation and purification, steps such as preparation promptly obtain adenovirus SARS vaccine.

The SARS vaccine comprises SARS associated coronavirus S gene and and defective adenoviral.Defective adenoviral is 5 type adenovirus, i.e. Ad5 of the C subclass that lacks fully of E1 district.The E3 district of this defective adenoviral can or not lack for disappearance or excalation fully.Dress CMV promoter and BGHpolyA in the defective adenoviral.

Ad-S _F/S _N/S _M/S _C

Be cloned in the adenovirus vector for SARS associated coronavirus S full length gene.

Be cloned into and comprise S for SARS associated coronavirus S gene in the adenovirus vector ₁Domain is in interior sequence.

Be cloned into and comprise S for SARS associated coronavirus S gene in the adenovirus vector ₂Domain is in interior sequence.

Be cloned into and comprise S for SARS associated coronavirus S gene in the adenovirus vector ₁, S ₂(base number is domain: 49～3585) in interior sequence.

Be cloned in the adenovirus vector 3686～3648) and the C end fragment (base number is: for SARS associated coronavirus S gene is striden the film district.

Embodiment 2

Be cloned in the adenovirus vector for SARS associated coronavirus S gene N end fragment.All the other are with embodiment 1.

Embodiment 3

Be cloned in the adenovirus vector for SARS associated coronavirus S gene intermediate segment.All the other are with embodiment 1.

Embodiment 4

Be cloned in the adenovirus vector for SARS associated coronavirus S gene C end fragment.

All the other are with embodiment 1.

Embodiment 5

S gene transformation pShuttle plasmid and evaluation:

With 37 ℃ of water bath condition enzyme action of Xbal+Kpnl, use this enzyme enzyme action pShuttle simultaneously, connect, transformed into escherichia coli DH5a cultivates, and utilizes kanamycin (Kan ^R) the resistance screening positive colony, obtain pShuttle-Sc, pShuttle-S respectively _M, pShuttle-S _N, identify and detect Sequence Identification by conventional agargel electrophoresis, the results are shown in Figure 3, Fig. 4.

Embodiment 6

The Ad-S that the clone is obtained _N/ S _MCarry out the vivoexpression test.

Infect the VeroE6 cell with 2.5～40MOI, discard viral suspension, add cell culture fluid, cultivate under 37 ℃, 5%CO2 condition, collect the cultured cell supernatant in infecting back 48h, detect the expression of Ad-Sn and Ad-Snm respectively with the RT-PCR method, detect the expression of Ad-Sn, the results are shown in Figure 5, Fig. 6 with Western blot method.

Embodiment 7

The Ad-S that the clone is obtained _NCarry out the expression in vivo test.

Subjects is divided into groups in the following manner: Ad-Sn collunarium the group, (contrast of Ad-lacZ collunarium group, annotate anesthesia with 3% pentobarbital abdomen earlier, collunarium 0.5mL/ is only), Ad-Sn tail vein injection group, Ad-lacZ tail vein injection group (contrast, tail vein injection administration 0.5mL/ only), blank group (not doing any processing).Administration in the following manner then: be administered once continuous three weeks weekly; After the administration first time, a week get blood, last 2 week of administration the back put to death animals and get blood and tissue, measure rat anti SARS IgG concentration with the ELISA method, the results are shown in Figure 7, Fig. 8.

Embodiment 8

Green monkey kidney cell (Vero E6) cell is avoided SARS and is attacked

1. inoculation Vero E6 cell is to 96 orifice plates, 2 * 10 ⁴/ hole.

2. after 24 hours, inoculate adenovirus SARS vaccine, method: press 4 times of virus dilution stock solutions with RPMI-1640, earlier culture fluid sucking-off in 96 orifice plates is discarded during inoculation, behind the PBS clean-out opening one time, add different dilution viral liquid, each dilution factor adds 5 holes, 50 μ L/ holes.Simultaneously with the negative contrast of RPMI-1640 (not containing virus).37 ℃, 5%CO ₂, hatch 1 hour under the saturated humidity condition after, add the RPMI-1640 contain 5% new-born calf serum, 200 μ L/ holes, 37 ℃, 5%CO ₂, cultivate under the saturated humidity condition.

3. after 24 hours, inoculate SARS virus, method: RPMI-1640 dilution SARS virus is to 100TCID50, earlier culture fluid sucking-off in 96 orifice plates is discarded during inoculation, behind the PBS clean-out opening one time, add different dilution viral liquid, each dilution factor adds 5 holes, 50 μ L/ holes.Simultaneously with the negative contrast of RPMI-1640 (not containing virus).37 ℃, 5%CO ₂, hatch 1 hour under the saturated humidity condition after, add the RPMI-1640 contain 5% new-born calf serum, 200 μ L/ holes, 37 ℃, 5%CO ₂, cultivate under the saturated humidity condition.

4. after this observed every 12 to 24 hours and record cytopathy situation, each dilution every porocyte pathological changes scoring addition draws the cytopathy index during result of calculation, gets each class mean, and is as follows:

＜110〉Zhongshan Univ. Cancer Cure Center

＜120〉Adenovirus Carrier SARS Vaccines And Its Preparation Method, the application of coronavirus S gene

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<211>3780

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＜213〉virus is planted

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tccgttcgag?atcctaaaac?atctgaaata?ttagacattt?caccttgcgc?ttttgggggt 1740

gtaagtgtaa?ttacacctgg?aacaaatgct?tcatctgaag?ttgctgttct?atatcaagat 1800

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tcaattagca?ttactacaga?agtaatgcct?gtttctatgg?ctaaaacctc?cgtagattgt 2160

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ttttgcacac?aactaaatcg?tgcactctca?ggtattgctg?ctgaacagga?tcgcaaca ca 2280

cgtgaagtgt?tcgctcaagt?caaacaaatg?tacaaaaccc?caactttgaa?atattttggt 2340

ggttttaatt?tttcacaaat?attacctgac?cctctaaagc?caactaagag?gtcttttatt 2400

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gaatgcctag?gtgatattaa?tgctag agat?ctcatttgtg?cgcagaagtt?caatggactt 2520

acagtgttgc?cacctctgct?cactgatgat?atgattgctg?cctacactgc?tgctctagtt 2580

agtggtactg?ccactgctgg?atggacattt?ggtgctggcg?ctgctcttca?aatacctttt 2640

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<220>

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tttctagacc?atgggttgtg?tccttgct

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tttctagacc?atggcatata?ggttcaatg

ccggtaccat?aagttcgttt?atgtgt

Claims

1. A SARS vaccine, characterized in that it comprises a SARS-associated coronavirus S gene and a replication defective adenovirus.

2. The SARS vaccine according to claim 1, characterized in that the defective adenovirus is a type 5 adenovirus of subtype C with complete deletion of the E1 region.

3. The SARS vaccine according to claim 2, characterized in that the defective adenovirus is a type 5 adenovirus of subtype C with complete deletion of the E3 region.

4. The SARS vaccine according to claim 2, characterized in that the defective adenovirus is a type 5 adenovirus of subtype C with partial deletion of the E3 region.

5. The SARS vaccine according to claim 2, characterized in that the defective adenovirus is a type 5 adenovirus of subtype C without deletion of the E3 region.

6. The SARS vaccine according to claim 1 or 2, characterized in that the defective adenovirus contains CMV promoter and BGHpolyA.

7. The SARS vaccine according to claim 1 or 2, characterized in that it comprises the full-length S gene of SARS-associated coronavirus.

8. The SARS vaccine according to claim 1 or 2, characterized in that the sequence cloned into the adenovirus vector is the sequence of the SARS-associated coronavirus S gene including the S1 domain.

9. The SARS vaccine according to claim 1 or 2, characterized in that the sequence cloned into the adenovirus vector is the sequence of the SARS-associated coronavirus S gene including the S2 domain.

10. The SARS vaccine according to claim 1 or 2, characterized in that the sequence cloned into the adenovirus vector is the sequence of the SARS-associated coronavirus S gene including the S1 and S2 domains.

11. The SARS vaccine according to claim 1 or 2, characterized in that the SARS-associated coronavirus S gene transmembrane region and C-terminal fragment with base numbers 3686-3648 are cloned into the adenovirus vector.

12. A preparation method for a SARS vaccine, comprising

(1) Obtain the S gene of SARS-related coronavirus;

(2) Recombining the S gene with the defective adenovirus;

(3) transfect packaging cells;

(4) After amplification, separation and purification, the preparation is made.

13. The SARS vaccine preparation method according to claim 12, characterized in that after the S gene is extracted, it is cloned into the pshuttle plasmid, and then combined with the adenovirus backbone plasmid.

14. The preparation method of SARS vaccine according to claim 12, characterized in that PCR primers are designed according to the S gene sequence as follows:

v1 GGTCTAGAGT TGTGGTTTCA AGTGAT

v2 TTTCTAGACC ATGGGTTGTG TCCTTGCT

V3 TTTCTAGACC ATGGCATATA GGTTCAATG

V4 TAGGTACCAA TGCCAGTAGT GGTG

V5 TTGGTACCTC CGCCTCGACT TT

V6 CCGGTACCAT AAGTTCGTTT ATGTGT

15. The preparation method of SARS vaccine according to claim 12, characterized in that the packaging cell is a cell line or cell strain integrated with the gene of Ad5 E1 region of subtype C adenovirus type 5.

16. The preparation method of SARS vaccine according to claim 15, characterized in that the packaging cells are 293 cells.

17. The preparation method of SARS vaccine according to claim 12, characterized in that the preparation is spray or injection.

18. The application of the S gene of SARS-associated coronavirus in the preparation of a vaccine for preventing SARS.