CN117186247A - Mycobacterium tuberculosis multi-antigen fusion protein, encoding gene and application - Google Patents
Mycobacterium tuberculosis multi-antigen fusion protein, encoding gene and application Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicine, in particular to a mycobacterium tuberculosis multi-antigen fusion protein, a coding gene and application thereof. The mycobacterium tuberculosis multi-antigen fusion protein consists of an Rv3875 protein antigen, an Rv3874 protein antigen and a nRV0350 protein antigen. All three protein antigens can induce protective immune response against mycobacterium tuberculosis, and nRV0350 protein antigen is taken as a latency antigen, and is combined with other two early secretion antigens to form fusion protein, which is more beneficial to preventing the reactivation of new infection or latency infection of tuberculosis in teenagers and adult people. Experiments show that the fusion protein provided by the invention has good immunogenicity, can induce stronger cellular immunity and humoral immunity than BCG vaccine in mice, has long-term protection effect, and does not show stronger toxicity than BCG vaccine.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a mycobacterium tuberculosis multi-antigen fusion protein, a coding gene and application thereof.
Background
While bcg provides a desirable protective effect against extrapulmonary invasive tuberculosis in pediatric applications, the protective immunity it induces during infants declines with age, resulting in an inability to prevent infection Mtb in adolescents and adults. The reduced protective power of BCG on teenagers and adults may be one of the main causes of the widespread tuberculosis. Therefore, the development of a novel tuberculosis vaccine that can provide a higher protective effect in teenagers and adult people is considered as a primary task in the global tuberculosis prevention task.
Studies have shown that the re-breeding of BCG in the adult population does not enhance its prophylactic effect, further demonstrating our need for the development of novel tuberculosis vaccines. The current novel tuberculosis vaccine mainly comprises recombinant bacillus calmette-guerin vaccine, adjuvant protein subunit vaccine, viral vector vaccine, DNA vaccine, inactivated vaccine and the like. Wherein, the adjuvant protein subunit vaccine, the viral vector vaccine and the DNA vaccine can only carry a limited number of antigens due to the limitation of self characteristics, and cannot induce comprehensive immune response aiming at more antigens. Therefore, the use of the bacillus calmette-guerin vaccine in clinic is mainly used for the intensive inoculation of bacillus calmette-guerin vaccine. Clinical trial results of MVA85A viral vector vaccine showed that although MVA85A could induce a strong Th 1-type immune response in humans, it did not show better protective effect as a booster vaccine for BCG than BCG alone. While the inactivated vaccine ensures the safety to a certain extent, the capability of inducing protective immunity in the population with normal immune function may be weak due to the characteristic of non-engraftable replication. At present, no novel tuberculosis vaccine shows the protective effect of far beyond BCG vaccine.
The construction of recombinant bacillus calmette-guerin for expressing mycobacterium tuberculosis related antigen by using bacillus calmette-guerin as carrier is an important strategy in the development and research of new tuberculosis vaccine at present. Among these, the selection of effective protective Mycobacterium tuberculosis-related antigen combinations is critical in the development of novel recombinant BCG. At present, the biggest obstacle to development of high-efficiency novel tuberculosis vaccines is that we have not clearly defined what type of immunity can exert long-acting anti-tuberculosis protection in human bodies.
In view of this, the present invention has been made.
Disclosure of Invention
In view of the technical problems existing in the background technology, the invention provides the following technical scheme:
in a first aspect, the invention provides a mycobacterium tuberculosis multi-antigen fusion protein, which consists of an Rv3875 protein antigen, an Rv3874 protein antigen and a nRV0350 protein antigen, wherein the amino acid sequence of the nRV0350 protein antigen is shown as SEQ ID NO. 5.
In the early stage of research and screening of potential effective antigens of mycobacterium tuberculosis, it is found that the Rv3875 protein antigen (ESAT-6), the Rv3874 protein antigen (CFP-10) and the RV0350 protein (DnaK) are dominant antigens of mycobacterium tuberculosis and can induce the generation of protective immune response. Therefore, ESAT-6, CFP-10 and DnaK are selected as potential candidate antigen components for constructing the recombinant BCG vaccine.
The invention uses the bioinformatics analysis platform such as TEprediction and IEDB to analyze the antigen of the mycobacterium tuberculosisRv0350Namely, dnaK protein is used for T cell epitope prediction, and 122 ammonia with the length is selectedPeptide chain of DnaK protein of basic acid T cell epitope concentration region new DnaK (nDnaK) and obtaining nucleotide chain of corresponding length 366 base pairs encoding epitope concentration region nDnaKRv0350-366And is named asnew Rv0350(nRv0350)。nRv0350The amino acid sequence of the coded protein (namely nRV0350 protein antigen) is shown as SEQ ID NO. 5.
The mycobacterium tuberculosis multi-antigen fusion protein consists of an Rv3875 protein antigen, an Rv3874 protein antigen and a nRV0350 protein antigen. All three protein antigens can induce protective immune response against mycobacterium tuberculosis, and nRV0350 protein antigen is taken as a latency antigen, and is combined with other two early secretion antigens to form fusion protein, which is more beneficial to preventing the reactivation of new infection or latency infection of tuberculosis in teenagers and adult people.
According to the invention, the antigen DnaK is used as a main vaccine component of mycobacterium tuberculosis for the first time, and experiments show that the fusion protein provided by the invention shows good immunogenicity, can induce stronger cellular immunity and humoral immunity than BCG vaccine in mice, has a long-term protection effect, and does not show stronger toxicity than BCG vaccine.
Preferably, the amino acid sequence of the mycobacterium tuberculosis multi-antigen fusion protein is shown as SEQ ID NO. 2.
In a second aspect, the invention provides a gene encoding the mycobacterium tuberculosis multi-antigen fusion protein, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
In a third aspect, the present invention provides a biological material for expressing the mycobacterium tuberculosis multi-antigen fusion protein, wherein the biological material is selected from an expression vector and engineering bacteria, and the biological material comprises a gene with a nucleotide sequence shown as SEQ ID NO. 1.
Preferably, the biological material is recombinant bcg. The recombinant bacillus calmette-guerin uses bacillus calmette-guerin as a vector to express related antigens, so that the original advantages of the bacillus calmette-guerin can be maintained, for example: BCG vaccine can be safely inoculated in infants; BCG vaccine is highly protective against invasive tuberculosis in infants; bcg can induce long lasting antigen stimulation; the inherent adjuvant activity of BCG vaccine; BCG vaccine is easy to produce and low in cost. Therefore, the development of the novel recombinant BCG vaccine has wide prospect.
In the present invention, the construction method of the recombinant BCG vaccine preferably comprises:
s1, connecting the genes to a shuttle vector to obtain a recombinant plasmid carrying fusion genes;
s2, transforming the recombinant plasmid in the step S1 into bacillus calmette-guerin, and screening to obtain the recombinant bacillus calmette-guerin for stably expressing the mycobacterium tuberculosis multi-antigen fusion protein.
Preferably, the shuttle vector is pMV361; and/or, the BCG vaccine is BCG-China strain.
In a more specific embodiment of the present invention, the construction method of the recombinant BCG vaccine is as follows: three gene fragments were generatedRv3875、Rv3874、nRv0350(linker ligation of each gene fragment) was used to construct a fusion gene, and the fusion gene was ligated to pMV361 shuttle vector for expression of multi-antigen fusion protein ECD003. Then, the pMV361-ECD003 recombinant plasmid carrying the fusion gene is transformed into BCG-China by an electrotransformation method. Positive clones were selected by 7H10 containing kanamycin (25. Mu.g/ml), and the obtained positive clones were verified by PCR and sequencing techniques to obtain the recombinant BCG-ECD003 of interest. Wherein the conditions of the electrotransformation are preferably: 20-30kV,20-30uF,900-1100 Ω; more preferably 25kV,25uF, 1000. OMEGA.
The invention uses the successfully constructed recombinant BCG-ECD003 to immunize BALB/c mice, uses BCG as a positive control group, uses PBS and an adjuvant as a blank control group and a negative control group respectively, adopts the means such as enzyme-linked immunosorbent assay (Elispot), multiplex microsphere Luminex technology, flow cytometry analysis (FCM), enzyme-linked immunosorbent assay (ELISA), in-vitro Mycobacterium Growth Inhibition Assay (MGIA) and the like to evaluate and analyze the immunogenicity and the protective effect, and proves that the recombinant BCG-ECD003 which is constructed based on BCG and expresses fusion antigen ECD003 has good immunogenicity, can induce stronger cellular immunity and humoral immunity than the BCG in the mice, has long-term protective effect and does not show stronger virulence than the BCG. Meanwhile, the recombinant BCG-ECD003 selects various immunodominant antigens expressed in different infection stages of the mycobacterium tuberculosis, so that the problem of poor protective effect of the current BCG in adults is solved, and the novel recombinant BCG is hopeful to become a candidate vaccine for tuberculosis prevention.
In a fourth aspect, the invention provides the use of said mycobacterium tuberculosis multi-antigen fusion protein, or said gene, or said biomaterial, in the preparation of a mycobacterium tuberculosis vaccine.
In a fifth aspect, the present invention provides a mycobacterium tuberculosis vaccine, the active ingredient of which comprises the mycobacterium tuberculosis multi-antigen fusion protein; or the mycobacterium tuberculosis multi-antigen fusion protein is prepared by expressing the genes or the biological materials.
In a sixth aspect, the present invention provides the use of said mycobacterium tuberculosis multi-antigen fusion protein, or said gene, or said biomaterial, in the manufacture of a mycobacterium tuberculosis detection reagent or a medicament for the treatment of a mycobacterium tuberculosis disease.
Advantageous effects
The invention provides a mycobacterium tuberculosis multi-antigen fusion protein, which consists of an Rv3875 protein antigen, an Rv3874 protein antigen and a nRV0350 protein antigen, wherein the amino acid sequence of the nRV0350 protein antigen is shown as SEQ ID NO. 5. All three protein antigens can induce protective immune response against mycobacterium tuberculosis, and nRV0350 protein antigen is taken as a latency antigen, and is combined with other two early secretion antigens to form fusion protein, which is more beneficial to preventing the reactivation of new infection or latency infection of tuberculosis in teenagers and adult people. Experiments show that the fusion protein provided by the invention has good immunogenicity, can induce stronger cellular immunity and humoral immunity than BCG vaccine in mice, has long-term protection effect, and does not show stronger toxicity than BCG vaccine.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be described below.
FIG. 1 shows the construction of recombinant plasmid pMV361-ECD003 and rBCG-ECD003 of example 2 of the present invention. Wherein, the A diagram is a schematic diagram of a pMV361-ECD003 shuttle plasmid; panel B shows agarose gel electrophoresis of amplified PCR products of rBCG strains transformed with pMV361-ECD003 shuttle vector, wherein: lane 1, dna Marker; lane 2, PCR products amplified from BCG-China; lane 3, PCR products amplified from rBCG-ECD003.
FIG. 2 shows the results of an analysis of the type of immune response induced by rBCG-ECD003 in mouse spleen cells in example 3 of the present invention. Frequency of BALB/c mouse spleen cells PPD or CED003 specific IFN-gamma (panel A in FIG. 2) and IL-4 (panel B in FIG. 2) secreting cells after 4 and 12 weeks of immunization of mice.
FIG. 3 shows the results of an assay for rBCG-ECD003 induced secretion of multiple cytokines by mouse spleen cells in example 3 of the present invention. After 4 and 12 weeks of immunization of mice, the secretion levels of various cytokines were analyzed in isolated spleen cells. Mouse spleen cells were re-stimulated with fusion proteins ECD003 (FIG. 3A) or PPD (FIG. 3B) and the concentration of cytokines (IFN-. Gamma., TNF-. Alpha., IL-2, IL-12, IL-4, IL-10) was analyzed by multiplex cytokine assay.
FIG. 4 shows the induction of CD4 in mice by rBCG-ECD003 at 12 weeks after immunization in example 3 of the present invention + T cell subpopulation analysis results. Analysis of CD4 in mouse spleen cells 12 weeks after immunization + A subpopulation of T cells. FIG. 4A is a panel of fusion protein ECD 003-specific CD4 + T cell subpopulation profile; panel B of FIG. 4 shows PPD-specific CD4 + T cell subpopulation profile.
FIG. 5 is the result of the humoral immunogenicity analysis of rBCG-ECD003 in example 3 of the present invention. Mice serum IgG titers were detected by ELISA technique 4,8, 12 weeks after immunization. FIG. 5A shows anti-fusion protein ECD003IgG titers; panel B in FIG. 5 shows anti-PPDIG titers. Results in log 2 Antibody titer forms are presented.
FIG. 6 is the in vitro protective evaluation result of rBCG-ECD003 in example 4 of the present invention. In FIG. 6Panel A and B are the colony counts of mice after 4 and 12 weeks post immunization, respectively, after spleen cells were isolated and co-cultured with Mtb H37 Rv. Results in log 10 CFU form presentation.
FIG. 7 is a result of evaluating the safety of rBCG-ECD003 in example 5 of the present invention. The pathological sections of liver, spleen, and lung were taken from left to right for each immunized group, 4 weeks and 12 weeks after immunization.
Detailed Description
The invention builds a novel recombinant bacillus calmette-guerin for expressing immunodominant antigens containing various mycobacterium tuberculosis expressed in different infection stages based on the bacillus calmette-guerin, and has long-acting comprehensive protection for tuberculosis of teenagers and adult people.
Because BCG vaccine can not provide good protection effect in adult population, and can not control reactivation of mycobacterium tuberculosis in tuberculosis latent infection population, development of an effective tuberculosis vaccine for adult population is urgently needed at present. The recombinant BCG vaccine can inherit the higher protection effect, safety, low price and other characteristics of the BCG vaccine in infants based on the BCG vaccine, so that the recombinant BCG vaccine has better application and development prospects. It has not been determined that tuberculosis protective antigens and unreasonable antigen combination selection may be the cause of the relatively lagging development of current novel recombinant bcg. Due to the nature of the latent in vivo infection of mycobacterium tuberculosis, its expression of early antigens can be down-regulated during the latent in vivo infection and result in a failure of the immune response of the body to mycobacterium tuberculosis. The design strategies of commonly employed purely immunodominant antigen-based tuberculosis vaccines may be inadequate to prevent reactivation of latent infection with mycobacterium tuberculosis in the adult population.
When the protective mycobacterium tuberculosis antigen is screened, three dominant antigens (ESAT-6, CFP-10 and DnaK) are screened by taking the capability of inducing Th1 type cellular immunity as a main standard. The genes encoding ESAT-6 and CFP-10 are located in the RD-1 region and are associated with Mycobacterium tuberculosis virulence, absent in all BCG strains at present. In the current clinical trials, there is also no vaccine to select the latent antigen DnaK as the main component of the vaccine.
The invention provides a recombinant BCG vaccine rBCG-ECD003 for expressing fusion protein ECD003. The fusion protein ECD003 includes two early secretion antigens ESAT-6 and CFP-10 and also includes one segment of binding latency antigen nDnaK (122 amino acid long peptide chain T cell epitope concentration region of DnaK protein). The use of a multi-stage antigen combination of early expressed antigens for tuberculosis infection and preferential expressed antigens for latent infection may be more beneficial in preventing the reactivation of new or latent infection of tuberculosis in adolescents as well as in the adult population.
Specifically, the recombinant protein ECD003 expressed by the rBCG-ECD003 of the invention is expressed by the rBCG-ECD003 respectivelyRv3875、Rv3874、nRv0350The ESAT-6, CFP-10 and nDnaK fusion antigens encoded by the three gene fragments are formed, and the three mycobacterium tuberculosis gene sequences are connected through a specific linker.
Wherein the amino acid sequence of ESAT-6 is:
MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA（SEQ ID NO: 3）。
the amino acid sequence of CFP-10 is:
AEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF（SEQ ID NO: 4）。
the amino acid sequence of nDnaK is:
VDADKNPLFLDEQLTRAEFQRITQDLLDRTRKPFQSVIADTGISVSEIDHVVLVGGSTRMPAVTDLVKELTGGKEPNKGVNPDEVVAVGAALQAGVLKGEVKDVLLLDVTPLSLGIETKGGV（SEQ ID NO: 5）。
the nucleotide sequence of the coded recombinant protein ECD003 is as follows:
ATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAGCGCAATCCAGGGAAATGTCACGTCCATTCATTCCCTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAGGGTGTCCAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATGGCTTCGACCGAAGGCAACGTCACTGGGATGTTCGCAGGTGGTTCTGGCGGTGCAGAGATGAAGACCGATGCCGCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTCGACGGCAGGTTCGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGAGGAGCAGCAGCAGGCGCTGTCCTCGCAAATGGGCTTCGGTGGTTCTGGCGGTGTCGACGCCGACAAGAACCCGTTGTTCTTAGACGAGCAGCTGACCCGCGCGGAGTTCCAACGGATCACTCAGGACCTGCTGGACCGCACTCGCAAGCCGTTCCAGTCGGTGATCGCTGACACCGGCATTTCGGTGTCGGAGATCGATCACGTTGTGCTCGTGGGTGGTTCGACCCGGATGCCCGCGGTGACCGATCTGGTCAAGGAACTCACCGGCGGCAAGGAACCCAACAAGGGCGTCAACCCCGATGAGGTTGTCGCGGTGGGAGCCGCTCTGCAGGCCGGCGTCCTCAAGGGCGAGGTGAAAGACGTTCTGCTGCTTGATGTTACCCCGCTGAGCCTGGGTATCGAGACCAAGGGCGGGGTGTAG（SEQ ID NO: 1）。
the amino acid sequence of the coded protein is as follows:
MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAGGSGGAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGFGGSGGVDADKNPLFLDEQLTRAEFQRITQDLLDRTRKPFQSVIADTGISVSEIDHVVLVGGSTRMPAVTDLVKELTGGKEPNKGVNPDEVVAVGAALQAGVLKGEVKDVLLLDVTPLSLGIETKGGV（SEQ ID NO: 2）。
the invention predicts the possible T cell epitope of the latent antigen DnaK in human by a series of bioinformatics software, selects the concentrated region nDnaK of the antigen epitope and removes the redundant sequence of the original antigen. Thus optimizing the disadvantage of the antigen that it is less immunogenic due to the presence of redundant sequences. The immune effect of the novel recombinant BCG rBCG-ECD003 immunized mice in the invention at different time points is evaluated. Experimental results show that rBCG-ECD003 exhibited a greater capacity to induce Th1 type immune responses than BCG at 4 weeks and 12 weeks after vaccination, whereas the rBCG-ECD003 immune group induced Th2 type immune responses were only transient in elevation at 4 weeks after immunization. Compared with BCG immunized group, spleen cells of rBCG-ECD003 immunized mice can produce more Th1 type cytokines such as IFN-gamma, TNF-alpha, IL-2 and IL-12 related to protection, while spleen cells of rBCG-ECD003 group secrete Th2 type cytokines IL-4, IL-6 and IL-10 only at 4 weeks after immunization have significantly increased compared with BCG group. At the same time, higher levels of innate immune-related cytokine IL-17 and GM-CSF were seen in the rBCG-ECD003 immunized group 4 weeks after the re-stimulation with PPD.Flow cytometry analysis results showed rBCG-ECD003 immunized group spleen CD4 + T cells have single positive IL-2 significantly higher than the BCG group + CD4 of (2) + T cell ratio and biscationic IFN-gamma + IL-2 + CD4 + T cell ratio. ELISA results show that rBCG-ECD003 immunized mice produce significantly higher IgG titers than BCG group fusion antigen ECD003 specific and PPD specific antibodies in the middle and long term. Meanwhile, in a mycobacterium tuberculosis in-vitro growth inhibition experiment (MGIA) experiment, although the immune short-term rBCG-ECD003 and BCG can not obviously inhibit the in-vitro growth of the mycobacterium tuberculosis, the immune long-term rBCG-ECD003 and BCG both show a certain protective force, and the rBCG-ECD003 shows stronger capacity of inhibiting the in-vitro growth of the mycobacterium tuberculosis than the BCG. Finally, the vaccine safety evaluation results show that rBCG-ECD003 does not exhibit virulence compared to the parental BCG vaccine. By combining the results, the recombinant BCG-ECD003 which is constructed based on BCG and expresses the fusion antigen ECD003 has good immunogenicity, can induce stronger cellular immunity and humoral immunity than BCG in mice, has long-term protection effect, and does not show stronger virulence than BCG. Meanwhile, the recombinant BCG-ECD003 selects various immunodominant antigens expressed in different infection stages of the mycobacterium tuberculosis, so that the problem of poor protective effect of the current BCG in adults is solved, and the novel recombinant BCG is hopeful to become a candidate vaccine for tuberculosis prevention.
The invention introduces the gene for encoding the fusion protein ECD003 into BCG-China to obtain the rBCG-ECD003 of the BCG by genetic engineering technology. By analyzing and verifying the immunogenicity and the in vitro protection effect, the result shows that: the novel rBCG-ECD003 provided by the invention can generate long-acting protective immune response against tuberculosis infection multi-stage antigens, can be used as a novel tuberculosis vaccine, and has good application potential.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the materials, reagents and the like used are all commercially available.
Example 1 DnaK protein T cell epitope prediction and selection of T cell epitope enriched region
Retrieval of Mtb H37Rv Gene by the national center for Biotechnology information (National Center for Biotechnology Information)Rv0350Is a gene sequence of (a). Analysis of pairs by NetMHCII pan3.2 Server, SYFPEITHI, TEpredict and IEDB bioinformaticsRv0350Analysis of the Gene sequence predicts its potential human CD4 + T and CD8 + T cell epitope distribution. Human CD4 + T cell epitope prediction using HLA-DRB1 in HLA-class II alleles0101、DRB1/>0301、DRB1/>0401、DRB1/>0701、DRB1/>0802、DRB1/>0901、DRB1/>1101、DRB1/>1302、DRB1/>1501 is a prediction constraint. Human CD8 + T cell epitope prediction using HLA-A +.in HLA-class I alleles>0201、/>0202、/>0203、/>0206 is a prediction constraint. And collecting epitope distribution conditions of all potential antigens DnaK T cells which are predicted and analyzed, selecting 260 th amino acid to 379 th amino acid of a DnaK peptide chain as a DnaK T cell epitope enrichment region, and naming the peptide chain as nDnaK.
EXAMPLE 2 construction of recombinant plasmid pMV361-ECD003 and recombinant BCG rBCG-ECD003
1. Construction of recombinant plasmid pMV361-ECD003
Obtaining Mycobacterium tuberculosis H37RV from retrieval results from the national center for biotechnology informationRv3874、Rv3875、Rv0350Is a gene sequence of (a). Obtaining corresponding nucleotide sequence based on DnaK peptide chain T cell epitope enrichment region nDnaKnRv0350. The three genes are synthesized by using the gene synthesis technology (Beijing Optimaceae)Rv3874、Rv3875AndnRv0350the nucleotide sequences of the genes are connected in sequence, a specific Linker is used for connection among the genes, the sequence of the Linker gene is GGTGGTTCTGGCGGT (SEQ ID NO: 6), the corresponding amino acid sequence of the Linker is GGSGG (SEQ ID NO: 7), and EcoRI and HindIII enzyme cutting sites are designed at two ends of the fusion gene. The recombinant plasmid pMV361-ECD003 (schematic view is shown in FIG. 1A) was constructed by ligating the two ends of the fusion gene sequence to EcoRI and HindIII of the multiple cloning site of the E.coli-Mycobacterium shuttle vector pMV361, respectively. The ligation product was transformed into E.coli DH 5. Alpha. (Beijing full gold organism), positive clones were screened by LB solid plates containing kanamycin (25. Mu.g/ml) and their sequence correctness was verified by sequencing techniques.
2. Construction of rBCG-ECD003 of recombinant BCG vaccine
Subsequently, the recombinant plasmid pMV361-ECD003 after sequencing verification was transferred into BCG-China strain (ATCC 27294) by electrotransformation (Biorad, electric shock conditions of 25kV,25uF,1000Ω), and the bacterial liquid after electrotransformation was spread on 7H10 solid plate containing kanamycin (25. Mu.g/ml) for 3-4 weeks. Finally, positive clones after kanamycin resistance screening are selected, the recombinant plasmid pMV361-ECD003 is verified through specific primers (the primer sequences are shown in table 1), the target gene is transferred into BCG-China (the result is shown in a B diagram in fig. 1), and the recombinant BCG rBCG-ECD003 is obtained through sequencing identification again.
TABLE 1
EXAMPLE 3 evaluation of the immunogenicity of recombinant BCG rBCG-ECD003
1. BALB/c mice grouping and immunization
The experiment adopts 6-8 week old female BALB/c mice without specific pathogen (Beijing Veitzerland) as immune subjects, wherein the mice with the immune detection points of 4 weeks are divided into 3 groups of 6 animals, namely 4 weeks of immune PBS group, BCG group and rBCG-ECD003 group respectively. Mice with 12 weeks of immunodetection point were divided into 3 groups of 6 animals each, which were immunized with 12 weeks of PBS, BCG, and rBCG-ECD003 groups, respectively. Each of the immunodetection point BCG group and rBCG-ECD003 group was subjected to multipoint immunization at 200. Mu.l/live bacteria amount of 1X 10 in the 0.0 Zhou Fenbie th intradermal layer 6 BCG-China or rBCG-ECD003 physiological saline suspension of CFU. Each immunodetection spot PBS group was multi-spot immunized with 200 μl/PBS alone in the 0.0 Zhou Fenbie intradermal.
2. Cell immunogenicity detection
(1) Spleen lymph isolation
Groups of mice with immunodetection spots of 4 weeks and 12 weeks were sacrificed at 4 weeks and 12 weeks, respectively, and spleens of the mice were removed by aseptic manipulation and temporarily immersed in sterile EP tubes containing RPMI-1640 (Gibco, usa). The spleen was then placed on a 200 mesh nylon mesh and 4ml of lymphocyte separation medium (Beijing daceae) was added dropwise, and the mouse spleen was gently ground with a plunger. The spleen cell suspension was transferred to a centrifuge tube and added to 500 μl of serum-free medium for low-speed gradient centrifugation, and the intermediate cloud cell layer was carefully aspirated. Then, 10ml of serum-free medium was added for washing, and the cells were collected by centrifugation. Rear part (S)Spleen lymphocytes subsequently used in ELISPOT assays, luminex multiplex cell assay techniques, and flow cytometry were resuspended in complete RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin dual antibiotics. Spleen lymphocytes subsequently used in mycobacterial in vitro growth inhibition assays were resuspended in complete RPMI-1640 medium without diabody. Cell concentration was measured by a cell technology instrument and adjusted to 1X 10 6 cells/ml was used for the subsequent experiments.
(2) ELISPOT detection of IFN-gamma, IL-4 secreting T cells
Isolated spleen lymphocytes were added to 96-well plates (1X 10) in a pre-coated mouse IFN-gamma or IL-4ELISPOT kit (Beijing Daidae) 5 Individual spleen cells/well). Antigen PPD or fusion protein ECD003 (2. Mu.g/well) was then added, PHA and RPMI-1640 complete medium as positive control and negative control. Biotinylated corresponding antibody and avidin HRP reagent were added after 20 hours of incubation. Finally, AEC solution is added for color development, and the instrument is used for detecting the number of spots, so that the result is analyzed statistically.
The results of mouse secretion of IFN-. Gamma.splenocytes ELISPOT are shown in FIG. 2, panel A, and the results of mouse secretion of IL-4 splenocytes ELISPOT are shown in FIG. 2, panel B. The results showed that the number of IFN-gamma secreting splenocytes and IL-4 secreting splenocytes from the PBS immunized group remained at very low levels after 4 weeks and 12 weeks of immunization, in vitro stimulation with PPD or ECD003 antigen. rBCG-ECD003 induced significantly higher frequency of PPD-specific IFN-gamma secreting cells at 4 weeks (P < 0.01) and 12 weeks (P < 0.01) than the parental BCG-China. Similar results were observed in stimulation with ECD003, with rBCG-ECD003 exhibiting higher levels of IFN-gamma secreting cells at both 4 weeks (P < 0.05) and 12 weeks (P < 0.01). For Th2 type cytokine IL-4, rBCG-ECD003 after PPD (P < 0.01) or ECD003 (P < 0.01) restimulation at week 4 post immunization induced an increase in the number of IL-4 secreting cells over BCG. Whereas at 12 weeks, PPD-specific or ECD 003-specific IL-4 response levels in the rBCG-ECD003 vaccinated group were similar to those in the BCG immunized group, indicating that rBCG-ECD 003-induced Th 2-type responses decreased over time during the trial. Overall, rBCG-ECD003 vaccination produced a strong Th 2-type immune response in the short term, but in the long term it induced an immune response in the mouse spleen that was predominantly biased towards a Th 1-type immune response.
(3) Luminex multiple cytokine detection technology
The isolated mouse spleen lymphocyte suspension was used at 1X 10 5 Individual cells/well were seeded in U-96 well plates and stimulated with 10 μg/well PPD, ECD003, RPMI-1640 medium as negative control. After incubation at 37℃for 24 hours, the supernatant was harvested by centrifugation at 400g for 5 minutes. By Luminex multiplex cytokine analysis (U.S. R&D) Cytokine production was examined, including IFN-gamma, TNF-alpha, IL-2, IL-12, IL-4, IL-10, IL-6, IL-17, and GM-CSF. The ECD003 restimulation results are shown in FIG. 3, panel A, and the PPD restimulation results are shown in FIG. 3, panel B.
To further investigate the comprehensive immune status of mice vaccinated with rBCG-ECD003, this example compares the ability of individual groups of mice spleen cells to secrete various cytokines at 4 and 12 weeks post-immunization, and the results are shown in figure 3, panels a and B. At 4 and 12 weeks post immunization, PBS group results were kept at minimum in each cytokine assay except TNF- α. After stimulation with ECD003, the Th1 cytokines IFN-. Gamma.and IL-2 produced by the rBCG-ECD003 group at 4 weeks (all P < 0.01) and 12 weeks (all P < 0.01) after immunization were significantly higher than those produced by the BCG group, whereas TNF-. Alpha.levels were elevated only at 12 weeks after immunization compared to those produced by the BCG group (P <0.05; panel A in FIG. 3). Meanwhile, the release of IL-12 cytokine is stronger (P < 0.05) in 4 weeks after immunization of mice inoculated with rBCG-ECD003, the secretion level of IL-6 in the rBCG-ECD003 group is obviously increased (P < 0.05) compared with that in BCG vaccine group at 4 weeks after immunization, and the secretion levels of IL-4 and IL-10 are not obviously increased. There was no significant difference in IL-17 and GM-CSF levels between the three groups. rBCG-ECD003 increased secretion levels of various Th1 cytokines including IFN-gamma, IL-2 and IL-12 in vaccinated mice after PPD re-stimulation, compared to the parental BCG at 4 weeks (average P < 0.01) and 12 weeks (IFN-gamma P <0.01; IL-2P <0.05; IL-12P < 0.05), respectively (FIG. 3, panel B). Although there was no statistical difference in TNF- α levels between BCG and rBCG-ECD003 groups at 4 weeks post-immunization, an increase in TNF- α levels was observed at 12 weeks post-immunization for rBCG-ECD003 groups (P < 0.01). The Th2 cytokines IL-4 (P < 0.01), IL-6 (P < 0.05) and IL-10 (P < 0.05) were higher in the rBCG-ECD003 group than in the BCG group only at 4 weeks post-immunization, while there was no statistical difference between the two groups at 12 weeks post-immunization. In terms of innate immunity-related cytokines, rBCG-ECD003 induced higher levels of IL-17 (P < 0.05) and GM-CSF (P < 0.01) at 4 weeks post-immunization compared to bcg group, but no statistical differences between the two groups at 12 weeks post-immunization. These data indicate that both short-term and long-term rBCG-ECD003 can enhance the secretion of various Th1 cytokines in mice, and that the secretion of Th2 cytokines, which is stronger in the immunized short-term rBCG-ECD003 than in BCG, also decreases over time. Although rBCG-ECD003 did not exhibit a greater ability to induce innate immunity-related cytokines over the long-term immunization, rBCG-ECD003 induced greater secretion of innate immunity-related cytokines over the short-term immunization.
(4) Multifunctional CD4 detection by flow cytometry + T cell
For detection of immune long-term specific cytokine secreted CD4 + T cell responses, intracellular cytokine staining. At 12 weeks post immunization, single cell suspensions of isolated spleen lymphocytes were stimulated with 10 μg of fusion protein ECD003 or PPD for 8 hours (37 ℃,5% CO 2 ) The protein transport inhibitor Brefeldin a (BD in the united states) was then added. Cells were stained with LIVE/DEAD fixed cells and with fluorophore conjugated antibodies directed against the surface markers anti-CD 3, anti-CD 4 (BD in the united states). Then, intracellular staining was performed with fluorophore-conjugated antibodies to IFN-gamma, TNF-alpha and IL-4 (BD in the United states). Finally, cells were washed with PBS and fixed with 4% paraformaldehyde. The samples were analyzed by flow cytometry. The ECD003 restimulation results are shown in FIG. 4, panel A, and the PPD restimulation results are shown in FIG. 4, panel B.
To verify in more detail the long-term induction of CD4 by recombinant BCG + T immune response, mice spleen multifunctional CD4 was assessed 12 weeks after vaccination + A subpopulation of T cells. After ECD003 stimulationPBS group, BCG group and rBCG-ECD003 all preferentially induced single positive IFN-gamma in the spleen of mice + CD4T cell subpopulations in which BCG induced the highest proportion of IFN-gamma + CD4T (panel a in fig. 4). In addition, rBCG-ECD003 induced ECD003 specific single positive IL-2 + T cell (P)<0.01 TNF-alpha) + T cell (P)<0.05 A) is significantly higher than BCG. BCG-induced biscationic IL-2 in a biscationic T cell population + TNF-α + CD4T cells and IFN-gamma + TNF-α + The proportion of CD4T cells was slightly higher, while vaccination with rBCG-ECD003 vaccine induced more ECD 003-specific IFN-gamma in the spleen than BCG + IL-2 + CD4T cells (P)<0.05). Similar results were also observed under PPD stimulation as after ECD003 stimulation (panel B in fig. 4), i.e. IFN- γ in three different immunized groups of mice + T cell subpopulations are responsible for the secretion of cytokine CD4 + T dominates. The present invention also found a large number of single positive IL-2 in mice vaccinated with rBCG-ECD003 vaccine + CD4T cells, the number of which is significantly higher than that of PBS group (P<0.01 Group (P) and BCG<0.01). IFN-gamma at a frequency of occurrence in each group + TNF-α + CD4 + T cells, but without statistical differences. In addition, mice vaccinated with rBCG-ECD003 produce PPD-specific IFN-gamma + IL-2 + CD4 + T double positive cell proportion was increased compared to BCG (P<0.01 Although rBCG-ECD003 did not exhibit IFN-gamma induction + IL-2 + TNF-α + Powerful ability of T three positive cell subsets. These results indicate that rBCG-ECD003 induces CD4 more favorable for anti-tuberculosis than BCG + Ability of T cell subsets.
3. Humoral immunogenicity detection
(1) Mouse serum isolation
Serum antibody assays were performed on mice of PBS, BCG, and rBCG-ECD003 groups at 12 weeks of immunodetection, at 4 weeks, 8 weeks, and at 12 weeks of three time points. Three groups of mice were blood collected by eye 200 μl before immunization, 4 weeks, 8 weeks, and 12 weeks, respectively. The collected blood was allowed to stand at room temperature for 2 hours for coagulation, and then serum was separated by centrifugation at 3000 rpm for 5 minutes. Stored at-20 ℃ for subsequent serum antibody titer detection.
(2) ELISA detection of serum IgG antibody titres
A96-well enzyme-labeled version (Corning, U.S.A.) was coated with PPD or the fusion protein ECD003 protein at 200 ng/well using 0.05mol/L Na2CO3-NaHCO3 buffer (pH 8.5) at 4℃overnight. Plates were washed with 0.05% Tween20 PBS (PBST), then blocked with 200. Mu.l/well of 5% nonfat milk powder in PBS for 2 hours at 37℃and washed 5 times with PBST. Serum was diluted 2-fold in 96-well microplate (dilution ratio from 2 6 To 2 17 ) Serum samples were added continuously, incubated at 37 ℃ for 2 hours, and then washed. Incubate for 2 hours at 37℃and then wash with PBST. After addition of 100. Mu.l/ml horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (Boolone, suzhou) (1:5000 dilution) and incubation at 37℃for 1 hour, 100. Mu.l Tetramethylbenzidine (TMB) was added to each well and incubated at 37℃for 15 minutes. By adding 50. Mu.l/well of H 2 SO 4 (0.3M) terminate the reaction and read absorbance A at a wavelength of 450nm on ELISA plate reader 450 . anti-ECD 003IgG titres are shown in FIG. 5, panel A, and PPD specific IgG titres are shown in FIG. 5, panel B. The results showed that both BCG and rBCG-ECD003 induced antibodies specific for antigen ECD003, and that the antibody levels were gradually increased within 12 weeks after immunization (panel a in fig. 5). Notably, compared to BCG group, rBCG-ECD003 group was found to be stable at 8 weeks (P<0.01 And 12 weeks (P)<0.05 Higher levels of IgG titers were produced. In addition, BCG immunized mice produced only a level of IgG response to PPD at three time points, with BCG vaccine groups at 8 weeks (median Log 2 10.67 And 12 weeks (median Log 2 10.83 IgG titers at 8 weeks (median Log 2 13.50；P<0.05 And 12 weeks (median Log 2 13；P<0.05 The level of IgG antibodies raised against PPD was significantly greater. (panel B in FIG. 5). In summary, rBCG-ECD003 enhances strong IgG-mediated immunity over the mid-long term, which may help the body to limit the growth of Mtb in vivo.
EXAMPLE 4 rBCG-ECD003 protective evaluation of rBCG
Mycobacterium in vitro growth inhibition assay (MGIA)
To evaluate the protective efficacy of rBCG-ECD003, the present example performed a mouse spleen cell in vitro mycobacterial growth inhibition assay. Spleen lymphocytes (1X 10) suspended in 1ml of antibiotic-free complete RPMI-1640 medium containing 10% fetal bovine serum ( 5 Well) and Mycobacterium tuberculosis virulent strain H37Rv (500 CFU/well) were co-cultured in 24 well plates at 37℃for 72 hours. The co-cultures were harvested in centrifuge tubes and after centrifugation at 12000rpm for 10 minutes the supernatant was carefully removed by a pipette. Subsequently, 500. Mu.l of sterile water was added to the 24-well plate to lyse the cells adhered to the wall, and incubated at room temperature for 10 minutes. After blow-off, the lysates were transferred to corresponding centrifuge tubes containing the co-cultures. The centrifuge tube was pulsed vortexed and 50 μl of bacterial suspension was plated onto Middlebrook 7H10 agar containing TCH (5 μg/ml). The low-concentration TCH can specifically inhibit the growth of BCG vaccine without affecting the growth of mycobacterium tuberculosis H37Rv, so that the influence of residual BCG vaccine inoculation in the spleen of a mouse can be eliminated. Three weeks later, colony forming units on Middlebrook 7H10 agar were counted and experimental data were log 10 CFU indicates that the results of the 4-week MGIA experiment after immunization are shown in panel a of fig. 6, and the results of the 12-week MGIA experiment after immunization are shown in panel B of fig. 6.
The results of the mycobacterial in vitro growth inhibition experiments showed that, 4 weeks after immunization, the amount of lotus was similar and maintained at a higher level for all immunized groups, and that neither BCG nor rBCG-ECD003 showed significant protection compared to PBS (panel a in fig. 6). Notably, but at 12 weeks, the BCG group and rBCG-ECD003 group were significantly improved in inhibiting mycobacterial growth compared to the PBS group. In addition, compared with the mice inoculated with the parent BCG, the control capacity of spleen cells of the mice inoculated with rBCG-ECD003 on mycobacterium growth is also obviously improved (P < 0.01). In conclusion, compared with the parent BCG vaccine, the intradermal injection of rBCG-ECD003 can improve the long-term control capacity of the spleen lymphocytes of mice on the growth of the mycobacterium tuberculosis, and meanwhile, the intradermal injection of rBCG-ECD003 is also suggested to provide stronger protection capacity against the mycobacterium tuberculosis for organisms compared with BCG.
EXAMPLE 5 evaluation of rBCG-ECD003 safety of rBCG vaccine
This example performed pathological analysis on liver, spleen and lung samples of immunized mice to assess the safety of recombinant BCG strains. The pathology analysis was determined by H & E staining. Organs including lung, spleen and liver were harvested after 4 weeks and 12 weeks of mice were sacrificed after immunization and fixed with 4% paraformaldehyde, embedded in paraffin blocks. After cutting into serial sections, the treated organs were stained with H & E and observed, and the pathological results are shown in fig. 7.
There were no obvious pathological changes in the liver, spleen and lung of the PBS group at 4 and 12 weeks post immunization. While mice vaccinated with rBCG-ECD003 and BCG found certain histopathological lesions in the liver after 4 and 12 weeks, including hepatocyte necrosis, cytoplasmic loosening, and liver plate disorders. In contrast, no significant pathological changes were observed in the spleens of both groups. Only partial infiltration and thickening of alveolar spaces were observed in the lungs of BALB/c mice from rBCG-ECD003 and BCG groups, indicating moderate tissue damage. Overall, pathology analysis showed that intradermal immunization of rBCG-ECD003 and BCG resulted mainly in liver damage in infected mice, both without destroying the basic structure of the liver. Thus rBCG-ECD003 expressing various mycobacterium tuberculosis virulence factors did not exhibit significantly higher virulence in the major organs of the mice than the parental bcg.
The results demonstrate that rBCG-ECD003 significantly improves the long-term induction capacity of the parent BCG vaccine on Th1 type immune response of mice, and enhances the secretion capacity of various protective related cytokines including IFN-gamma, IL-2 and TNF-alpha of mice. Whereas at 12 weeks post immunization, rBCG-ECD003 induced CD4 + T cells are biased towards CD4 which is beneficial to the anti-tuberculosis capability of the organism + A subpopulation of T cells. In addition, rBCG-ECD003 also showed superior IgG-mediated humoral immunity. The in vitro protective assay of rBCG-ECD003 also suggests the advantage of rBCG-ECD003 for long-term control of Mycobacterium tuberculosis growth. Finally, the toxicity level of the recombinant BCG vaccine is not obviously improved compared with that of the parent BCG vaccine.
In summary, the recombinant BCG-ECD003 can be used as a potential candidate vaccine for replacing BCG vaccine, and the rBCG-ECD003 can express two antigen ESAT-6, CFP-10 secreted by Mycobacterium tuberculosis early and epitope enrichment region peptide nDnaK of latent antigen DnaK, so that the situation that the current BCG vaccine is poorly protected in teenagers and adult people is hopeful to be improved.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. The mycobacterium tuberculosis multi-antigen fusion protein is characterized by comprising an Rv3875 protein antigen, an Rv3874 protein antigen and a nRV0350 protein antigen, wherein the amino acid sequence of the nRV0350 protein antigen is shown as SEQ ID NO. 5.
2. The mycobacterium tuberculosis multi-antigen fusion protein according to claim 1, wherein the amino acid sequence of the mycobacterium tuberculosis multi-antigen fusion protein is shown as SEQ ID No. 2.
3. A gene encoding the mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 1.
4. A biological material for expressing the mycobacterium tuberculosis multi-antigen fusion protein of claim 1, the biological material being selected from the group consisting of expression vectors, engineering bacteria, characterized in that the biological material comprises the gene of claim 3.
5. The biomaterial of claim 4, wherein the biomaterial is recombinant bcg; the construction method of the recombinant BCG vaccine comprises the following steps:
s1, connecting the gene of claim 3 to a shuttle vector to obtain a recombinant plasmid carrying a fusion gene;
s2, transforming the recombinant plasmid in the step S1 into bacillus calmette-guerin, and screening to obtain the recombinant bacillus calmette-guerin for stably expressing the mycobacterium tuberculosis multi-antigen fusion protein.
6. The biomaterial of claim 5, wherein the shuttle vector is pMV361; and/or, the BCG vaccine is BCG-China strain.
7. The biomaterial of claim 5 or 6, wherein the transformation is performed by electrotransformation, and wherein the conditions of the electrotransformation are: 20-30kV,20-30uF,900-1100 omega.
8. Use of a mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2, or a gene according to claim 3, or a biomaterial according to any one of claims 4-7 in the preparation of a mycobacterium tuberculosis vaccine.
9. A mycobacterium tuberculosis vaccine, characterized in that the effective component of the mycobacterium tuberculosis vaccine comprises the mycobacterium tuberculosis multi-antigen fusion protein of claim 1 or 2; or the mycobacterium tuberculosis multi-antigen fusion protein prepared by expressing the gene of claim 3 or the biological material of any one of claims 4-7.
10. Use of a mycobacterium tuberculosis multi-antigen fusion protein according to claim 1 or 2, or a gene according to claim 3, or a biomaterial according to any one of claims 4-7, in the manufacture of a mycobacterium tuberculosis detection reagent or a medicament for the treatment of a mycobacterium tuberculosis disease.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1467221A (en) * | 2002-07-14 | 2004-01-14 | 虹 王 | Preparation and application of tubercle bacillus CFP-10, ESAT-6 and CD40L fusion bacterin |
| WO2008140478A2 (en) * | 2006-11-01 | 2008-11-20 | Immport Therapeutics, Inc. | Compositions and methods for immunodominant antigens |
| CN101822829A (en) * | 2010-04-07 | 2010-09-08 | 四川大学 | Recombinant BCG vaccine for tuberculosis prevention |
| CN102573896A (en) * | 2009-05-13 | 2012-07-11 | 因波特医疗股份有限公司 | Compositions and methods for immunodominant antigens of mycobacterium tuberculosis |
| CN104327159A (en) * | 2014-10-29 | 2015-02-04 | 南方医科大学 | Mycobacterium tuberculosis specific CD8+T cell epitope peptide P45 and application thereof |
| CN116726155A (en) * | 2023-03-31 | 2023-09-12 | 中国疾病预防控制中心传染病预防控制所 | Construction, expression, purification and application of tuberculosis subunit vaccine |
-
2023
- 2023-11-07 CN CN202311469377.7A patent/CN117186247A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1467221A (en) * | 2002-07-14 | 2004-01-14 | 虹 王 | Preparation and application of tubercle bacillus CFP-10, ESAT-6 and CD40L fusion bacterin |
| WO2008140478A2 (en) * | 2006-11-01 | 2008-11-20 | Immport Therapeutics, Inc. | Compositions and methods for immunodominant antigens |
| CN102573896A (en) * | 2009-05-13 | 2012-07-11 | 因波特医疗股份有限公司 | Compositions and methods for immunodominant antigens of mycobacterium tuberculosis |
| CN101822829A (en) * | 2010-04-07 | 2010-09-08 | 四川大学 | Recombinant BCG vaccine for tuberculosis prevention |
| CN104327159A (en) * | 2014-10-29 | 2015-02-04 | 南方医科大学 | Mycobacterium tuberculosis specific CD8+T cell epitope peptide P45 and application thereof |
| CN116726155A (en) * | 2023-03-31 | 2023-09-12 | 中国疾病预防控制中心传染病预防控制所 | Construction, expression, purification and application of tuberculosis subunit vaccine |
Non-Patent Citations (1)
| Title |
|---|
| 刘新民等: "《中华医学百科大辞海 第1卷 内科学》", 北京：军事医学科学出版社, pages: 594 * |
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