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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<210>1
<211>3780
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atgtttattt?tcttattatt?tcttactctc?actagtggta?gtgaccttga?ccggtgcacc 60
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tactatcctg?atgaaatttt?tagatcagac?actctttatt?taactcagga?tttatttctt 180
ccattttatt?ctaatgttac?agggtttcat?actattaatc?atacgtttgg?caaccctgtc 240
atacctttta?aggatggtat?ttattttgct?gccacagaga?aatcaaatgt?tgtccgtggt 300
tgggtttttg?gttctaccat?gaacaacaag?tcacagtcgg?tgattattat?taacaattct 360
actaatgttg?ttatacgagc?atgtaacttt?gaattgtgtg?acaacccttt?ctttgctgtt 420
tctaaaccca?tgggtacaca?gacacatact?atgatattcg?ataatgcatt?taattgcact 480
ttcgagtaca?tatctgatgc?cttttcgctt?gatgtttcag?aaaagtcagg?taattttaaa 540
cacttacgag?agtttgtgtt?taaaaataaa?gatgggtttc?tctatgttta?taagggctat 600
caacctatag?atgtagttcg?tgatctacct?tctggtttta?acactttgaa?acctattttt 660
aagttgcctc?ttggtattaa?cattacaaat?tttagagcca?ttcttacagc?cttttcacct 720
gctcaagaca?tttggggcac?gtcagctgca?gcctattttg?ttggctattt?aaagccaact 780
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gagagaaaaa?aaatttctaa?ttgtgttgct?gattactctg?tgctctacaa?ctcaacattt 1080
ttttcaacct?ttaagtgcta?tggcgtttct?gccactaagt?tgaatgatct?ttgcttctcc 1140
aatgtctatg?cagattcttt?tgtagtcaag?ggagatgatg?taagacaaat?agcgccagga 1200
caaactggtg?ttattgctga?ttataattat?aaattgccag?atgatttcat?gggttgtgtc 1260
c
ttgcttgga?atactaggaa?cattgatgct?acttcaactg?gtaattataa?ttataaatat 1320
aggtatctta?gacatggc
aa?gcttaggccc?tttgagagag?acatatctaa?tgtgcctttc 1380
tcccctgatg?gcaaaccttg?caccccacct?gctcttaatt?gttat
tggcc?attaaatgat 1440
tatggttttt?a
caccactac?tggcattggc?taccaacctt?acagagttgt?agtactttct 1500
tttgaacttt?taaatgcacc?ggccacggtt?tgtggaccaa?aattatccac?tgaccttatt 1560
aagaaccagt?gtgtcaattt?taattttaat?ggactcactg?gtactggtgt?gttaactcct 1620
tcttcaaaga?gatttcaacc?atttcaacaa?tttggccgtg?atgtttctga?tttcactgat 1680
tccgttcgag?atcctaaaac?atctgaaata?ttagacattt?caccttgcgc?ttttgggggt 1740
gtaagtgtaa?ttacacctgg?aacaaatgct?tcatctgaag?ttgctgttct?atatcaagat 1800
gttaactgca?ctgatgtttc?tacagcaatt?catgcagatc?aactcacacc?agcttggcgc 1860
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cat
gtcgaca?cttcttatga?gtgcgacatt?cctattggag?ctggcatttg?tgctagttac 1980
catacagttt?ctttat
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aatatgtaca?tctgcggaga?ttctactgaa?tgtgctaatt?tgcttctcca?atatggtagc 2220
ttttgcacac?aactaaatcg?tgcactctca?ggtattgctg?ctgaacagga?tcgcaaca
ca 2280
cgtgaagtgt?tcgctcaagt?caaacaaatg?tacaaaaccc?caactttgaa?atattttggt 2340
ggttttaatt?tttcacaaat?attacctgac?cctctaaagc?caactaagag?gtcttttatt 2400
gaggacttgc?tctttaataa?ggtgacactc?gctgatgctg?gcttc
atgaa?gcaatatggc 2460
gaatgcctag?gtgatattaa?tgctag
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gctatgcaaa?
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gatatcctt
t?cgcgacttga?t
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ggcacttctt?ggtttattac?acagaggaac?ttcttttctc?cacaaataat?tactacagac 3300
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gatcctctgc?aacctgagct?tgactcattc?aaagaagagc?tggacaagta?cttcaaaaat 3420
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gatgactctg?agccagttct?caagggtgtc?aaattacatt?
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<213〉artificial sequence
<220>
<223〉primer (a pair of) that is used for pcr amplification spike gene N end that designs according to spike gene base sequence
<400>2
ggtctagagt?tgtggtttca?agtgat
taggtaccaa?tgccagtagt?ggtg
<210>3
<211>50
<212>DNA
<213〉artificial sequence
<220>
<223〉primer (a pair of) that is used for pcr amplification spike gene M end that designs according to spike gene base sequence
<400>3
tttctagacc?atgggttgtg?tccttgct
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<213〉artificial sequence
<220>
<223〉according to the primer that is used for pcr amplification spike gene M end of spike gene base sequence design (to)
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tttctagacc?atggcatata?ggttcaatg
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