CN101020055B - SARS vaccine based on replicative vaccinia virus vector - Google Patents

Abstract

The present invention relates to recombinant SARS vaccine expressing SARS-CoV nucleocapsid protein and protuberant protein and its use. The vaccine is constructed based on replicative vaccinia virus, such as vaccinia virus Tiantan strain. The present invention provides the carrier for constructing the SARS vaccine. The present invention provides also the DNA vaccine encoding SARS nucleocapsid protein and protuberant protein. The present invention also relates to immunizing process with the DNA vaccine and the AIDS vaccine.

Description

SARS vaccine based on replicating vaccinia virus vector

technical field

The invention relates to the field of antiviral immunology. More specifically, the present invention relates to a vaccine against SARS-CoV based on a replication-type vaccinia virus vector and its preparation method and use. the

Background technique

Since the first case of infectious atypical pneumonia was discovered in Guangdong Province, China in November 2002, the infectious disease was once widespread all over the world. The World Health Organization issued a warning to the world in March 2003 and named it severe acute respiratory syndrome (severe acute respiratory syndrome, SARS). SARS mainly spreads through the respiratory tract, is highly contagious, and has a mortality rate as high as 5%-15%, seriously endangering people's life and health. The World Health Organization announced on April 16, 2003 that the pathogen causing SARS belonged to a new variant of coronavirus and was named "SARS-CoV" (Peiris J S M, Lai S T, Poon M L L, et al .Coronavirus as a possible cause of severe acute respiratory syndrome. The Lancet. 2003, April 8). Through the joint efforts of scientists from many countries, including China, the sequencing of the SARS-CoV genome has been completed. Sequence analysis shows that the SARS-CoV genome contains 5 main open reading frames (ORFs), which encode DNA polymerase proteins respectively. , spike protein (S protein), envelope protein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (NC protein). the

The spike protein is the main cell receptor binding protein of coronavirus, and its amino acid changes can significantly affect the virulence of the virus. In addition, the functional site prediction of the SARS-CoV spike protein revealed that there may be multiple antigenic determinants in the protein, and the spike protein of the currently prevalent SARS-CoV in the world has a high degree of conservation, which can be used as an important target for vaccine research. Target (Walgate R.SARS vaccine race: US and European groups moving forward, but WHO would rather put SARS "back in the box", Available May2 at http://www.Biomedcentral.Com/news/20030502/03). the

Nucleocapsid protein is another important structural protein in coronaviruses. It is located in the core part of virus particles and is of great significance for the accurate assembly of virus particles. Chinese researchers obtained lymphocytes from the convalescent blood of SARS patients, and the humanized Fab antibody obtained by genetic engineering can specifically bind to the SARS-CoV nucleocapsid protein, which shows that the nucleocapsid protein is also an important antigen of SARS-CoV Site (Du Runlei, Yu Jianshi, Liang Mifang, etc. Preliminary study on human-derived anti-severe acute respiratory syndrome (SARS) virus genetically engineered antibody. Acta Virus. 2003, 19(2): 104-108). the

Although the SARS epidemic has been effectively controlled around the world, the possibility of its re-emergence cannot be ruled out. Anti-SARS-CoV vaccine is the most effective way to prevent SARS epidemic. At present, the SARS whole-virus inactivated vaccine developed in China has entered the clinical trial stage. The vaccine carries all the antigens of the virus and has good immunogenicity. However, from the perspective of SARS pathology, the inactivated SARS whole virus vaccine has the potential danger of causing the body's autoimmune response; in addition, the conditions for producing this type of vaccine are high, and there are hidden dangers in terms of biological safety. Therefore, developing a new generation of safe and effective SARS genetically engineered vaccines is also a top priority. the

Currently available vaccine types include the following: traditional vaccines (inactivated and live attenuated vaccines), synthetic peptide and protein subunit vaccines, DNA vaccines, and live vector vaccines. Compared with other types of vaccines, the advantages of live vector vaccines are reflected in: (1) It can actively infect target tissues or cells, which improves the efficiency of foreign genes entering cells; (2) The vector itself has an adjuvant effect, which can induce cytokines and chemokine production; (3) Most can induce long-term immune responses. In the field of live vector vaccines against SARS, non-replicating vectors are currently used. the

Contents of the invention

The object of the present invention is to provide a new vaccine and immunization method against SARS-CoV infection, so that a kind of solution is provided for the problems existing in the above-mentioned prior art. the

One object of the present invention is to provide a vaccine against SARS-CoV based on replicating vaccinia virus, which comprises replicating vaccinia virus as a vector, and the thymidine kinase (TK) region of the replicating vaccinia virus genome is inserted into the gene encoding SARS-CoV. Polynucleotides of the nucleocapsid and spike proteins of CoV. the

In a preferred embodiment of the present invention, the replicating vaccinia virus is the Tiantan strain of vaccinia virus, and preferably the vaccine does not contain a selectable marker gene. the

In a preferred embodiment of the present invention, the polynucleotide encoding the nucleocapsid protein and the protuberance protein of SARS-CoV inserted into the TK region of the vaccinia virus Tiantan strain is codon-optimized and suitable for high expression in mammalian cells. Efficiency expression. In a specific embodiment, the polynucleotide encoding the nucleocapsid protein of SARS-CoV inserted into the TK region of the vaccinia virus Tiantan strain has the nucleotide sequence shown in SEQ ID NO: 1 and/or Or the polynucleotide of the spike protein of described encoding SARS-CoV has the nucleotide sequence as shown in SEQ ID NO:2. the

The vaccine of the present invention may further comprise suitable pharmaceutically acceptable adjuvants and/or carriers. the

Another object of the present invention is to provide a DNA vaccine against SARS-CoV comprising a vector comprising a polynucleotide encoding the nucleocapsid protein of SARS-CoV operably linked to a promoter and/or Or a polynucleotide encoding the spike protein of SARS-CoV. In a specific embodiment of the DNA vaccine of the present invention, the polynucleotide encoding the nucleocapsid protein of SARS-CoV has a nucleotide sequence as shown in SEQ ID NO: 1, and the polynucleotide encoding SARS-CoV The polynucleotide of the prominence protein has the nucleotide sequence shown in SEQ ID NO:2. the

Another object of the present invention is to provide a method of immunization against SARS-CoV, which includes administering to individuals an immune effective dose of the present invention, which uses replicating vaccinia virus, especially the Tiantan strain of vaccinia virus, as a carrier against SARS-CoV. vaccine. The immunization methods of the invention may also include administering to the individual one or more DNA vaccines against SARS-CoV, such as the DNA vaccines of the invention described herein, prior to administering the vaccine. the

The present invention also provides an immunization kit comprising one or more DNA vaccines against SARS-CoV, such as the DNA vaccine of the present invention as described herein, which also includes the replicating vaccinia virus of the present invention as Vector vaccine against SARS-CoV. Optionally, the kit further includes instructions to use the one or more DNA vaccines against SARS-CoV for primary immunization, and then boost with the vaccine against SARS-CoV using replicating vaccinia virus of the present invention as a carrier Immunization schedule instructions. the

In addition, the present invention also provides a universal transfer vector pVTT 1.0 of vaccinia virus, the preservation number of which is CGMCC No.1458. the

Description of drawings

Figure 1: shows the construction route of the universal transfer vector pVTT1.0 of vaccinia virus. the

Figure 2: shows the construction route of the transfer plasmid pVTT-NS. the

Figure 3: Shows the identification results of vaccinia virus universal transfer vector pVTT1.0 enzyme digestion analysis. Lane M shows the molecular weight marker DL15000 DNA Marker (purchased from Dalian Bao Biological Engineering Co., Ltd.); lanes 1, 2, and 3 show the results of KpnI, NdeI or EcoRV digestion of vaccinia virus universal transfer vector pVTT1.0, respectively. the

Figure 4: PCR amplification of the coding sequence of SARS-CoV nucleocapsid protein and the coding sequence of PCR fusion amplification promoter PE/L+P7.5 and SARS-CoV nucleocapsid protein. Swimming lane M is the DNA Marker of molecular weight marker DL2000 (purchased from Dalian Bao Biological Engineering Co., Ltd.); Swimming lane 1 shows the band of the coding sequence of the SARS-CoV nucleocapsid protein amplified by PCR; Swimming lane 2 shows the PCR fusion amplification promoter P Bands of E/L+P7.5 and coding sequence of SARS-CoV nucleocapsid protein. the

Figure 5: Shows the identification results of T-NC plasmid restriction analysis. Lane 1 shows the result of double digestion of T-NC plasmid with Spe I and Not I; lane 2 shows the result of single digestion of T-NC plasmid with Sal I. the

Figure 6: Shows the identification results of T-NC+P+S plasmid digestion analysis. Swimming lane M is the DNA Marker of molecular weight marker DL15000 (purchased from Dalian Bao Biological Engineering Co., Ltd.); Swimming lanes 1-4 show that the picked T-NC+P+S plasmid (No. Cut the result. the

Figure 7: Shows the identification results of vaccinia virus universal transfer vector pVTT1.0 and vaccinia virus transfer vector pVTT-NS enzyme digestion analysis. Lane M shows the molecular weight marker DL15000 DNA Marker (purchased from Dalian Bao Biological Engineering Co., Ltd.); Lane 1 shows the result of Sac II and Spe I double digestion of vaccinia virus universal transfer vector pVTT1.0; Swimming lane 2 shows the vaccinia virus transfer vector Sac II and Spe I double digestion results of pVTT-NS. the

Figure 8: It shows that 9 sixth-generation white virus clones were randomly selected from the fifth-generation rVTT-NS, and the viral DNA template was extracted, and the SARS-CoV nucleocapsid protein code was amplified with NC primer 1 and NC primer 2. Sequence result test. Among them, lane M shows the DNA Marker of molecular weight marker DL15000 (purchased from Dalian Bao Biological Engineering Co., Ltd.); lane N shows the PCR amplification result of wild-type virus negative control; Virus clone genome PCR amplification NC protein coding sequence (1.2Kb) result of SARS-CoV. the

Figure 9: It shows that 9 sixth-generation white virus clones were randomly selected from the fifth-generation rVTT-NS, and the viral DNA template was extracted, and the S protein code of SARS-CoV was amplified using S primer 1 and S primer 2 as primers Sequence result test. Lane M shows the DNA Marker of molecular weight marker DL15000 (purchased from Dalian Bao Biological Engineering Co., Ltd.); Lane P shows the positive control result using plasmid pSK-S as a template; Lane N shows the PCR amplification result of the wild-type virus negative control Swimming lanes 1-9 show the S protein coding sequence (3.6Kb) result of randomly picking 9 sixth-generation leukovirus clone genome PCR amplification SARS-CoV. the

Figure 10: Shows that each generation of rVTT-NS infected CEF cells, the cells and supernatant were harvested after 48 hours, and the results of Western Blot analysis were performed with human polyantiserum (provided by the Center for Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention). Lane N shows the immunoblotting results of wild-type virus negative control; Lane M shows the pre-stained protein Marker; Lane 1, 3, 5, and 6 show the immunity of CEF cells infected with rVTT-NS at passages 1, 3, 5, and 6, respectively Hybrid bands. the

Fig. 11 shows ELISPOT detection of T lymphocyte responses secreting IFN-γ after stimulation with N epitope peptide (A) and S antigen epitope peptide (B) in vitro. 1×10 ⁶ mouse splenocytes per well were stimulated with NC protein stimulating peptide and S protein stimulating peptide for 30 hours, and the number of T lymphocytes secreting IFN-γ was detected.

Fig. 12: represents the titer level of NC protein (A) and S protein (B) specific antibody IgG of experimental group animal serum SARS-CoV. the

Figure 13: Schematic diagram of the structure of SARS-CoV DNA vaccines pDRVISV1.0-S and pDRVISV1.0-N. the

save

The transfer plasmid pVTT 1.0 was deposited on September 19, 2005 in the General Microorganism Center (CGMCC) of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC No.1458. the

Plasmid pSC65 was deposited on February 24, 2004 in the General Microorganism Center (CGMCC) of China Committee for Culture Collection of Microorganisms, and the preservation number is: CGMCC No.1097. the

Plasmid pSK-N was deposited on September 19, 2005 in the General Microorganism Center (CGMCC) of China Committee for Culture Collection of Microorganisms, and the preservation number is: CGMCC No.1459. the

Plasmid pSK-S was deposited on September 19, 2005 in the General Microorganism Center (CGMCC) of China Committee for Culture Collection of Microorganisms, and the preservation number is: CGMCC No.1457. the

Detailed ways

The vaccine against SARS-CoV of the present invention is constructed based on the replicative vaccinia virus vector, which is different from the conventionally used non-replicative vaccinia virus vector, such as MVA (Modifiedvirus Ankara), NYVAC (New York Vaccinia) and ALVAC ( avipoxvirus canarypox) (Paoletti E. Applications of poxvirus vectors to vaccination: Anupdate.Proc.Natl.Acad.Sci.USA.Vol.93, pp.11349-11353). In the vaccine of the present invention, polynucleotides encoding SARS-CoV nucleocapsid protein (NS) and spike protein (S) are inserted into the TK region of the replicative vaccinia virus vector, which can trigger individuals to produce anti-SARS - Protective immune response to CoV. the

The term "replicative" refers to a vaccinia vector that can replicate in humans. In a preferred embodiment of the present invention, the replicative vaccinia virus vector is vaccinia virus TianTan strain (vaccine virus TianTan strain, VTT). The Tiantan strain of vaccinia virus has made great contributions to the eradication of smallpox in China, and its application in the research of live vector vaccines is also very active. It has the advantages of good safety, convenient vaccination, and no need for adjuvants. the

Preferably, the polynucleotide encoding the nucleocapsid protein and protrusion protein of SARS-CoV inserted into the TK region of the replicative vaccinia virus genome can be codon-optimized and modified. The term "codon optimization transformation" refers to the reverse translation of the amino acid sequence of a polypeptide back into a nucleotide sequence by selecting the most preferred genetic codon for humans according to the genetic code usage frequency in the human genetic code preference table , so that the nucleotide sequence is suitable for high-efficiency expression in human and mammalian cells. In the present invention, through the codon optimization strategy, according to the known amino acid sequence of the nucleocapsid protein and the protruding protein of SARS-CoV (with reference to the amino acid sequence of the SARS-CoV Hong Kong strain HKU-39849 isolate published by Genebank), reverse The translation can obtain the nucleotide sequence encoding the nucleocapsid protein and the protuberance protein modified by codon optimization. Small adjustments and modifications can be made to the obtained nucleotide sequence, using the degeneracy of the genetic code to eliminate redundant restriction enzyme recognition sites, and eliminate potential nucleic acid secondary structures and other sequences that are not conducive to gene synthesis. In a specific embodiment, the polynucleotide encoding the nucleocapsid protein of SARS-CoV has the nucleotide sequence shown in SEQ ID NO: 1, and the polynucleotide encoding the protrusion protein of SARS-CoV Acid has the nucleotide sequence shown in SEQ ID NO:2. the

In the present invention, polynucleotides encoding the nucleocapsid protein and the protruding protein of SARS-CoV are inserted into the thymidine kinase (TK) gene in the vaccinia virus genome by a suitable method such as homologous recombination to form Genetic recombination of replicating vaccinia viruses. Preferably, the vaccine of the invention, ie said recombinant replicating vaccinia virus, does not contain a selectable marker gene. the

Therefore, the present invention provides a universal transfer vector of vaccinia virus, so as to recombine the target gene into the TK region of vaccinia virus genome DNA. In a specific embodiment, the universal transfer vector of vaccinia virus of the present invention is the transfer plasmid pVTT 1.0 (CGMCC No.1458) containing double selection markers of neo gene and lacZ gene. The transfer plasmid vector contains the following elements: ① Three selection markers: Amp resistance gene, lacZ gene and neo gene. The lacZ gene initiated by the p7.5 promoter is used for blue-white screening of recombinant vaccinia virus, and the neo gene initiated by the PE6 promoter is used for the purification of recombinant vaccinia virus with both a screening marker and a gene of interest (in G418 The proliferation of vaccinia virus wild strains under the action will be inhibited), in order to enable the neo gene to play a better role, the tail of the neo gene has a 200bp poly(A) sequence. ② Homologous arm tkL and tkR sequences: tkL and tkR are partial fragments of vaccinia virus thymidine kinase (TK), which are homologous sequences of intermolecular homologous recombination between vaccinia virus and transfer plasmid. ③lacZ' sequence: This 200bp sequence is completely homologous to the 200bp sequence at the tail of the lacZ gene, so that the recombinant vaccinia virus with both the selection marker and the target gene undergoes intramolecular homologous recombination, thereby losing the selection marker. ④ Early and late promoter pE/L of vaccinia virus. ⑤Multiple cloning site, located downstream of the promoter pE/L.

By using the universal transfer vector pVTT 1.0 of the present invention, the target gene can be recombined into the TK region of the vaccinia virus genome DNA, and the recombined vaccinia virus genome does not contain a selection marker gene. Therefore, the present invention also provides a method for constructing a vaccine against SARS-CoV based on replication-type vaccinia virus, said method comprising: using the transfer plasmid pVTT 1.0 (CGMCC No.1458) to encode the SARS-CoV nucleocapsid protein ( NS) and spike protein (S) polynucleotides were placed under the promoter pE/L of pVTT 1.0 to construct the recombinant plasmid pVTT-NS; pVTT-NS undergoes homologous recombination with vaccinia virus Tiantan strain in chicken embryo cells, making The target gene of SARS-CoV was recombined into the TK region of vaccinia virus genomic DNA together with the double selection marker neo gene and lacZ gene; under the selection pressure of antibiotic G418, the low-melting point agarose layer with X-gal and neutral red was added Spot, pick the blue recombinant vaccinia virus containing both the target gene and the screening marker, and go through three rounds of single-spot purification; then, under no G418 pressure selection, the blue recombinant vaccinia virus itself will be due to a small segment of about 200bp in the transfer plasmid. Intramolecular homologous recombination of the lacZ' fragment and the complete lacZ gene, thereby losing the neo gene and the lacZ gene, thereby obtaining a recombinant vaccinia virus Tiantan strain that only contains the coding sequences of the nucleocapsid protein and the protruding protein of SARS-CoV. the

The vaccine of the present invention may further comprise suitable pharmaceutically acceptable adjuvants, carriers and/or excipients, and suitable adjuvants, carriers and excipients are known in the art. the

The present invention also provides a DNA vaccine against SARS-CoV, said DNA vaccine comprising a vector comprising a polynucleotide encoding a nucleocapsid protein of SARS-CoV operably linked to a promoter and/or Or a polynucleotide encoding the spike protein of SARS-CoV. Vectors for constructing DNA vaccines are known in the art, such as eukaryotic expression vector pcDNA3.1. After the target gene is obtained, the sequence of the target gene can be connected to a suitable vector by known methods to construct a DNA vaccine. In a specific embodiment of the DNA vaccine of the present invention, the polynucleotide encoding the nucleocapsid protein of SARS-CoV has a nucleotide sequence as shown in SEQ ID NO: 1, and the polynucleotide encoding SARS-CoV The polynucleotide of the prominence protein has the nucleotide sequence shown in SEQ ID NO:2. the

Another object of the present invention is to provide a method of immunization against SARS-CoV, which includes administering to an individual an immune effective amount of the present invention based on the replicative vaccinia virus, especially the anti-SARS-CoV vaccine based on the Tiantan strain of vaccinia virus. vaccine. The term "effective amount" refers to the amount of the vaccine of the present invention that is sufficient to stimulate an individual to produce cellular immunity and/or humoral immunity against pathogens. The specific administration amount, administration speed and administration time will depend on the individual's condition, and can be determined by a doctor according to situation to judge. The immunization methods of the invention may also include administering to the individual one or more DNA vaccines against SARS-CoV, such as the DNA vaccines of the invention described herein, prior to administering the vaccine. The inventors found that the initial immunization was carried out by using a DNA vaccine containing the coding sequence of SARS-CoV nucleocapsid protein and/or protrusion protein, and then the immunization scheme of boosting immunization with SARS-CoV Tiantan strain recombinant vaccinia virus vaccine (i.e. Prime -boost strategy), which can successfully induce the body to produce high levels of humoral and cellular immune responses and high titers of neutralizing antibodies. the

The present invention also provides an immunization kit comprising one or more DNA vaccines against SARS-CoV, such as the DNA vaccine of the present invention as described herein, which also includes the replication-competent vaccinia virus of the present invention. Vaccines against SARS-CoV. Optionally, the kit further includes instructions for primary immunization with the one or more DNA vaccines against SARS-CoV, followed by booster immunization with the replicative vaccinia virus-based vaccine against SARS-CoV of the present invention Instructions for vaccination procedures. the

The present invention will be further elaborated below in conjunction with specific examples. the

Example

Example 1: Construction of vaccinia virus universal transfer vector pVTT 1.0

1. Construction of recombinant plasmid pSC-neo

Plasmid pIRESneo (purchased from Clontech Company) was first digested with XhoI, then digested with SmaI (reaction temperature: 25°C), filled in with Klenow enzyme, and the 1.2kb target fragment neo-polyA was recovered; the transitional vector plasmid pSC65 (preservation number: CGMCC No.1097) was digested with BglII, filled with Klenow enzyme, treated with dephosphorylase (CIAP), and the vector was recovered; the two were connected at 16°C for 4 hours, and transformed into E. coli TOP10. Multiple single colonies were picked, a small amount of plasmid was extracted, identified by XbaI and PstI, and the correct recombinant clone was named pSC-neo. the

2. Artificially synthesized vaccinia virus vector early promoter PE6 and lacZ fusion fragment. the

The gene was synthesized by overlapping PCR (Overlapping PCR). First, the positive and negative strands of the sequence PE6 and lacZ fusion polynucleotide fragments are listed separately, and then the positive and negative strand sequences are divided into 50bp oligonucleotides according to the length of the gene (the most 5' end of the two strands One length is about 25bp), except for the sequences at both ends, each positive strand and negative strand sequence has about 25bp complementarity with the oligonucleotides of the two complementary strands. Eight oligonucleotides are mixed with the same number of complementary oligonucleotides to form an annealing system, and the temperature is first raised in the PCR tube and then slowly annealed. Using the annealed product as a template, PCR amplification was performed with paired upstream and downstream primers to obtain a 429bp fusion polynucleotide fragment of PE6 and lacZ. The synthetic vaccinia vector early promoter PE6 and lacZ fusion fragment was connected to the T-easy universal sequencing vector to obtain the plasmid pT-lacZ'-PE6, and the sequencing result was in line with the expected design (SEQ ID NO: 9). the

3. Obtain the vaccinia virus transfer vector pVTT 1.0. the

The plasmid pT-lacZ'-PE6 prepared as above was digested with SmaI+HindIII, and the 0.4kb lacZ'-PE6 fragment was recovered; it was connected to the plasmid pSC-neo to obtain the vaccinia virus transfer vector pVTT 1.0 (see Figure 1 for the construction process). After identification by KpnI, NdeI and EcoRV, the results are shown in Figure 3. the

Example 2: Codon optimization of the nucleocapsid protein and protrusion protein coding sequence of SARS-CoV and construction of DNA vaccine:

1. Genetic code optimization. According to the genetic codon usage frequency in the human genetic code preference table, select the most preferred genetic codon, according to the amino acid sequence of the nucleocapsid protein and protrusion protein of SARS-CoV (refer to the SARS-CoV Hong Kong strain HKU-39849 published by Genebank isolate amino acid sequence), back translated back to nucleotide sequence. the

2. Modification and adjustment of gene sequence. Small adjustments and modifications, using the degeneracy of the genetic code to eliminate redundant restriction enzyme recognition sites, eliminate potential nucleic acid secondary structures and other sequences that are not conducive to gene synthesis. the

3. Artificially synthesize the target gene. Genes were synthesized by overlapping PCR. First, the positive and negative strands of the nucleocapsid protein and prominence gene sequences are listed separately, and then the positive and negative strand sequences are divided into 50bp oligonucleotides according to the length of the gene (the length of the most 5' end of the two strands is about 25bp), except for the sequences at both ends, each positive strand and negative strand sequence has about 25bp complementarity with the oligonucleotides of the two complementary strands. Every 10 or so oligonucleotides are mixed with the same number of complementary oligonucleotides to form an annealing system, and the temperature is first raised in the PCR tube and then slowly annealed. Using the annealed product as a template, PCR amplification was performed with paired upstream and downstream primers to obtain a gene fragment of about 500 bp. Multiple overlapping fragments are mixed first, then heated up and annealed, using the annealing system as a template, PCR amplification is performed with primers at both ends, and longer gene fragments can be obtained. Fragments over 1Kb can be spliced by PCR through preset restriction sites to obtain the full-length gene sequence. the

4. Link the target gene of the nucleocapsid protein (N) and the protuberance protein (S) of synthetic encoding SARS-CoV to the pSK universal sequencing vector, and the sequencing results meet the expected design (N and S of SARS-CoV through codon optimization) The target gene sequences are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2). The plasmid pSK-S was obtained, which was deposited on September 19, 2005 in the General Microorganism Center (CGMCC) of China Committee for Culture Collection of Microorganisms, and the preservation number is: CGMCC No.1457. Plasmid pSK-N was deposited on September 19, 2005 in the General Microorganism Center (CGMCC) of China Committee for Culture Collection of Microorganisms, and the preservation number is: CGMCC No.1459. the

Then SmaI+SalI double enzyme digestion plasmid pSK-S and pSK-N, the polynucleotide of the nucleocapsid protein (NC) and the protuberance protein (S) of the encoding SARS-CoV that obtains is connected to SmaI+SalI double enzyme digestion treatment In the DNA vaccine vector pDRVISV1.0 (Chinese patent application: 200410028280.3), the DNA vaccines pDRVISV1.0-S and pDRVISV1.0-N of SARS-CoV were obtained (Figure 13). the

Example 3: Construction of target gene expression element and vaccinia virus transfer vector

1. PCR amplification of fusion promoter PE/L+P7.5 sequence

Design primers:

P7.5 primer 1: 5'-GAAGATCTGTCGACTTCGAGCTTATTT-3' (SEQ ID NO: 3);

PE/L Primer 2: 5'-GAGAATTCGTTTAAACCGATGC-3'(SEQ ID NO: 4)

The pE/L+p7.5PCR amplification reaction uses the kit of Dalian Bao Biological Engineering Co., Ltd., and the reaction system is as follows:

Plasmid pSC65 (the deposit number of plasmid pSC65 is: CGMCC No.1097.) 1 μl, each 1 μl of forward and reverse primers (P 7.5 primer 1, PE/L primer 2), 5 μl of 10×Pyrobest buffer, dNTP mixture (each 2.5 mM) 5 μl, Pyrobest DNA polymerase (5 U/ml) 0.5 μl, ddH ₂ O 37.5 μl. PCR reaction conditions: pre-denaturation at 94°C for 2min; 30 cycles at 94°C for 30s, 58°C for 30s, and 72°C for 30s; 72°C for 7min; 4°C.

The extension product of pE/L+p7.5 PCR amplification reaction was purified and recovered with E.Z.N.ACycle-Pure Kit from Omega Company. the

2. PCR amplification of the nucleocapsid protein (NC) coding sequence of SARS-CoV

Design primers:

NC primer 1: 5'-CATCGGTTTAAACGAATTCTCACCATGAGCGATAATGGCCC-3' (SEQ ID NO: 5);

NC Primer 2: 5'-CC GGATCC TTATCAGGCCTGTGTAGAATC-3' (SEQ ID NO: 6)

The PCR amplification reaction of the NC gene of SARS-CoV uses the kit of Dalian Bao Biological Engineering Co., Ltd., and the reaction system is as follows: 

Plasmid pSK-N (the storage number of plasmid pSK-N is: CGMCC No.1459) 1 μl, forward and reverse primers (NC primer 1, NC primer 2) each 1 μl, 10×Pyrobest buffer 5 μl, dNTP mixture (each 2.5 mM) 5 μl, Pyrobest DNA polymerase (5 U/ml) 0.5 μl, ddH ₂ O 37.5 μl.

PCR reaction conditions: pre-denaturation at 94°C for 2 minutes; 30 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min; 72°C for 7 min; 4°C. the

The extension product of the NC PCR amplification reaction was purified and recovered with the E.Z.N.A Cycle-Pure Kit of Omega Company (see SEQ ID NO: 1 for the nucleocapsid protein coding sequence). the

3. PCR fusion amplification promoter PE/L+P7.5 and the nucleocapsid protein coding sequence of SARS-CoV

The reaction system is as follows:

pE/L+p7.5 PCR reaction recovery template 5 μl, SARS-CoV nucleocapsid protein coding gene PCR reaction recovery template 5 μl, forward and reverse primers (P 7.5 primer 1, NC primer 2) each 1 μl, 10×Pyrobest buffer 5 μl, dNTP mixture (2.5 mM each) 5 μl, Pyrobest DNA polymerase (5 U/ml) 0.5 μl, ddH ₂ O 37.5 μl.

PCR reaction conditions: 94°C pre-denaturation for 2 minutes; 94°C for 30s, 58°C for 30s, 72°C for 1min 30s, a total of 30 cycles; 72°C for 7min; 4°C. the

The fusion PCR amplification product of PE/L+P7.5 and the nucleocapsid protein coding sequence of SARS-CoV was purified and recovered with E.Z.N.A Cycle-Pure Kit from Omega Company. The results are shown in Figure 4. The product was connected to the T-easy universal vector of Promega Company to obtain the T-NC plasmid, and the sequencing result was correct. The corresponding restriction endonucleases were used to analyze and identify, and the results of enzyme digestion and identification were shown in Figure 5. the

4. Acquisition of S and NC gene expression elements of SARS-CoV

Use SalI and SacI double enzyme digestion to connect the pSK-S (the preservation number of the plasmid pSK-S is: CGMCC No.1457) vector of the artificially synthesized SARS-CoV spike protein coding sequence (SEQ ID NO: 2), and digest it to obtain The spike protein coding sequence of SARS-CoV was connected to the T-NC plasmid treated with SalI and SacI double enzyme digestion, and the plasmid T-NC+P+S with the S and NC gene expression elements of SARS-CoV was obtained (constructed The process is shown in Figure 2). Plasmids were extracted, analyzed and identified by corresponding restriction endonucleases, see Figure 6 for the results of enzyme digestion and identification. the

5. Construction of the S and NC gene vaccinia virus transfer vector plasmid pVTT-NS of SARS-CoV

The plasmid T-NC+P+S carrying the target gene expression element was digested with SpeI and SacII. The enzyme-digested fragments of the S and NC gene expression elements of SARS-CoV were recovered using the gel recovery kit from Omega, and then ligated into the vaccinia virus universal transfer vector pVTT 1.0, which was treated with SmaI and SacII double enzymes, to obtain SARS-CoV-carrying fragments. S. The vaccinia virus transfer vector plasmid pVTT-NS of the NC gene expression element (see Figure 2 for the construction process). The plasmid was extracted, analyzed and identified by corresponding restriction endonucleases, and the results of the enzyme digestion and identification were shown in Fig. 9 respectively. the

Example 4: Construction and screening of the Tiantan strain recombinant vaccinia virus vaccine rVTT-NS containing the nucleotide sequence of the nucleocapsid protein and the protuberance protein encoding SARS-CoV

The vaccinia virus Tiantan strain infected 80% of chicken embryo cells CEF with 0.1～0.01pfu/cell virus amount, and after the cells were adsorbed for 1～1.5h, the recombinant plasmid pVTT- In CEF cells transfected with NS, the S and NC gene expression elements of SARS-CoV and the double selection markers of neo gene and lacZ gene were homologously recombined into the TK region sequence of vaccinia virus genomic DNA. The first three rounds of selection of recombinant vaccinia virus are after 400ug/ml G 418 pressurized selection, and the low-melting point agarose with X-gal and neutral red is used to spread the spots, so that the recombinant vaccinia virus containing both the target gene and the selection marker can be selected. blue recombinant vaccinia virus (the wild strain without recombination was inhibited by the presence of G418). Then, under the pressure selection without antibiotic G418, the blue recombinant vaccinia virus itself will have intramolecular homology with the complete lacZ gene due to a small segment of lacZ' fragment about 200 bp upstream of the S and NC gene expression elements of the transferred plasmid. Recombination, thereby losing the neo gene and lacZ gene and obtaining the recombinant vaccinia virus containing only the S and NC gene expression elements of SARS-CoV, and using low-melting point agarose with X-gal and neutral red to spread the spots, you can pick Take the white recombinant virus containing only the gene of interest. After five rounds of single-spot purification of the white spot virus obtained from the primary screening, a single cloned recombinant vaccinia virus vaccine rVTT-NS containing the S and NC gene expression elements of SARS-CoV can be obtained. the

Example 5: Detection of rVTT-NS passage stability by PCR and Western blot

After rVTT-NS was subcultured, 9 sixth-generation leukoviruses were randomly selected, and the viral DNA template was extracted (viral genome extraction kit from Hangzhou Weitejie Biotechnology Co., Ltd.), and the NC target gene was amplified with NC primer 1 and NC primer 2 respectively. (Refer to Example 3 for primer sequences and methods); with S primer 1: 5'-CTCTACGTAGCGGCCGCTAACCATGTTTATCTTCTGCTG-3' (SEQ ID NO: 7); and S primer 2: 5'-TCC CCCGGG TTATCAGGTGTAG-3' (SEQ ID NO: 8 ) is the primer used to amplify the S target gene, and the reaction system is as follows: rVTT-NS DNA 5 μl; forward and reverse primers (S primer 1, S primer 2) 1 μl each; 10×Pyrobest buffer 5 μl; dNTP mixture (2.5 mM each) 5 μl; LA DNA polymerase (5 U/ml) 0.5 μl; ddH ₂ O 32.5 μl.

PCR reaction conditions: pre-denaturation at 94°C for 2min; 30 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 3min; 72°C for 7min; 4°C. the

Agarose gel electrophoresis showed that the positive target bands were amplified in the randomly selected virus genomes, the NC gene was 1.2kb; the S gene was 3.6kb. As shown in Figures 6 and 7. the

The fifth-generation rVTT-NS infected CEF cells, harvested the cells and supernatant 48 hours later, and performed Western Blot analysis with human polyantiserum (provided by the Capital Institute of Pediatrics), and a specific positive reaction band appeared, and the NC protein was 4.4KDa ; The S protein is 120KDa, indicating that the constructed rVTT-NS vaccine can stably express the target gene, as shown in FIG. 10 . the

Example 6: Prime-Boost immunization experiment using DNA vaccine containing NC and S coding sequences of SARS-CoV and Tiantan vaccinia virus vaccine rVTT-NS

1. Prime-Boost immunization strategy of DNA vaccine containing NC and S coding sequences of SARS-CoV and Tiantan vaccinia virus vaccine rVTT-NS. the

In this example, 6-8 week-old BALB/c (H-2d) female mice (19-25 grams in weight, purchased from China Institute for the Control of Pharmaceutical and Biological Products) were used to test the efficacy of the vaccine of the present invention. The DNA vaccine containing the NC and S target genes of SARS-CoV of Example 2 was prepared into a 1 mg/ml injection with 1×PBS. Recombinant vaccinia virus rVTT-NS 1×10 ⁸ pfu/mL. Four groups of 6 mice were immunized. The vaccination strategies of each immunization group are shown in Table 1. DNA vaccine was injected into the tibialis anterior muscle at 100ug/rat/time (50ug per hind limb). The dose of rVTT-NS recombinant vaccinia virus was 10 ⁷ pfu/mouse/time. Immunoassays were performed at week 10. As a control, vaccinia virus Tiantan strain with pCDNA empty vector and no target gene inserted was used.

Table 1. Prime-Boost immunization regimen for DNA vaccine against SARS-CoV and Tiantan vaccinia virus vaccine rVTT-NS:

2. ELISPOT was used to detect the response of T lymphocytes secreting IFN-γ after stimulation with epitope peptide in vitro. the

The ELISPOT experiment for detecting IFN-γ used a kit from U-CyTech Company in the Netherlands, and for specific procedures, refer to the instruction manual of U-CyTech Company.刺激多肽为S蛋白的16条肽(S1，VFNATKFPSVYAWERKKI；S2，SVYAWERKKISNCVADY；S3，STFFSTFKCYGVSATKL；S4，KCYGVSATKLNDLCFSNV；S5，NIDATSTGNYNYKYRYLR；S6，NYNYKYRYLRHGKLRPF；S7，RASANLAATKMSECVL；S8，AATKMSECVLGOSKRVDF；S9，LMSFPQAAPHGVVFLHV；S10，APHGVVFLHVTYVPSQER) and 1 peptide of NC protein (N1, QIGYYRRATRRVRGGDGK). The results show that the initial immunization of single-shot or double-shot SARS-CoV (NC and S gene) DNA vaccine, and then booster immunization of mice with the vaccinia virus vaccine against SARS-CoV of the present invention, can induce the body to produce a high level of T lymphocyte immune response , the specific results are shown in Figure 11. the

3. Detection of specific IgG-binding antibodies against SARS-CoV nucleocapsid protein and protrusion protein in immunized mouse serum

In order to detect the level of specific humoral immunity induced by SARS-CoV recombinant vaccinia virus single immunization and booster immunization, mouse serum was collected at week 10 and tested with SARS-CoV nucleocapsid protein and spike protein antigens (National Institutes of Health Vaccine Research Donated by the center) coated with microtiter plates, indirect ELISA method was used to detect the levels of IgG antibodies specific to SARS-CoV nucleocapsid protein and spike protein. The specific IgG antibody titers of SARS-CoV nucleocapsid protein and protuberance protein in each immune group are listed in Figure 12. the

4. SARS-CoV neutralizing antibody titer in the sera of immunized mice. the

Plaque reduction neutralization test was used to measure the neutralizing antibody in the sera of immunized mice to evaluate the immune effect. Vero-E6 cells were used to inoculate 2-well plastic cell culture plates, and two layers of agarose-containing medium were added. A plaque test was established using neutral red as a stain. The anti-SARS-CoV BJ 01 strain neutralizing antibody was determined based on the plaque that can be reduced by 50%. The test results of each group are shown in Table 2. the

Table 2 The titer of neutralizing SARS-CoV antibody in the serum of experimental group animals in vitro:

Immunization experiment group Neutralizing antibody titer negative control group ＜1:20 Two-dose SARS-CoV NC+S DNA vaccine group 1:446 Single-needle SARS-CoV NC+S plus vaccinia rVTT-NS group 1:1280 Double-needle SARS-CoV NC+S plus vaccinia rVTT-NS group 1:1807 Single injection vaccinia rVTT-NS group 1:181

The above embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Obviously, those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and essence of the present invention. Therefore, these modifications and changes are also within the protection scope of the present application. the

sequence listing

<110> Chinese Center for Disease Control and Prevention

Center for STD and AIDS Prevention and Control

<120> SARS vaccine based on replicating vaccinia virus vector

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Claims (9)

1. A vaccine against SARS-CoV based on replicating vaccinia virus, which comprises replicating vaccinia virus as a carrier, wherein the thymidine kinase (TK) region of the vaccinia virus genome is inserted into the encoding SARS-CoV nucleocapsid protein and The polynucleotide of the spike protein, and the replication-competent vaccinia virus is the Tiantan strain of vaccinia virus. 2. The vaccine of claim 1, wherein said vaccine does not contain a selectable marker gene. 3. The vaccine according to claim 1 or 2, wherein the polynucleotide is codon-optimized and suitable for high-efficiency expression in mammalian cells. 4. The vaccine of claim 3, wherein the polynucleotide encoding the SARS-CoV nucleocapsid protein has the nucleotide sequence shown in SEQ ID NO: 1 and/or the polynucleoside encoding the SARS-CoV protrusion protein Acid has the nucleotide sequence shown in SEQ ID NO:2. 5. The vaccine according to any one of claims 1-4, further comprising a pharmaceutically acceptable adjuvant and/or carrier. 6. Use of the vaccine according to any one of claims 1-5 in the preparation of a vaccine composition for preventing SARS-CoV infection. 7. An immunization kit comprising the vaccine according to any one of claims 1-5. 8. The kit of claim 7, further comprising one or more DNA vaccines against SARS-CoV. 9. A universal transfer plasmid pVTT 1.0 of vaccinia virus, which was deposited in the General Microorganism Center (CGMCC) of China Committee for Culture Collection of Microorganisms on September 19, 2005, and the preservation number is CGMCC No.1458.

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