CN117305214B - Recombinant bacillus calmette-guerin vaccine and preparation method and application thereof - Google Patents
Recombinant bacillus calmette-guerin vaccine and preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/32—Mycobacterium
Abstract
The invention relates to the technical field of biological medicine, and particularly discloses a recombinant BCG vaccine as well as a preparation method and application thereof. Compared with the original strain, the recombinant BCG vaccine of the invention overexpresses nPPE18, nfadD28, hspX and plcA proteins. The recombinant BCG vaccine of the invention can induce organisms to generate good immunogenicity, can provide higher protectiveness and safety, and can be used as a novel BCG vaccine.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a recombinant BCG vaccine and a preparation method and application thereof.
Background
Tuberculosis (TB) is a disease caused by infection of Mycobacterium TuberculosisMycobacterium tuberculosisMTB), infections caused by, mainly, the respiratory tract pathway, are transmitted from person to person. TB has been one of the leading causes of death from a single infectious disease, and is widespread and high in mortality. Vaccination is the most effective strategy for preventing and controlling infectious diseases. The only TB vaccine approved for use worldwide is BCG vaccineThis is a live attenuated mycobacterium bovis (m. Bovis) vaccine, which was first used in 1921, and is one of the most widely used vaccines worldwide. BCG is effective in preventing childhood severe TB, such as castanospermatic tuberculosis and tubercular meningitis. However, its protective efficacy against pediatric TB gradually decreases over time, and BCG exhibits significantly different levels of protection from crowd to region, varying from 0% to 80% of the protective efficacy against adult TB. In addition, BCG is potentially toxic as an attenuated live vaccine, and may present safety issues in immunocompromised individuals and cannot be used in all populations. Thus, there is an urgent need to develop more effective novel TB vaccines that promote worldwide TB control.
Currently, TB vaccines under investigation include subunit vaccines, live attenuated vaccines, recombinant BCG (rBCG) vaccines and DNA vaccines. The possible reason why BCG protective efficacy gradually decreases with time is that the number of protective immunodominant antigens contained in BCG is limited and stable expression at high levels cannot be sustained. By integrating immunodominant antigen genes into BCG, the preparation of rBCG can perfectly solve this problem. rBCG is an enhanced live vaccine, by taking BCG as a live vaccine carrier, exogenous genes are introduced into BCG, and after the BCG is inoculated into a body, the rBCG can self-reproduce, can continuously express self protein and recombinant protein integrated into rBCG, and continuously stimulates the body to generate adaptive cellular immunity and humoral immunity response. Moreover, the basis of rBCG vaccine construction is BCG, and the rBCG vaccine has higher safety. The rBCG vaccines (MTBVAV and VPM 1002) have entered phase I and phase iii clinical trials, respectively, and rBCG MTBVAC and VPM1002 entering the clinical phase have shown safety and efficacy comparable or better to BCG in relevant animal models, and are very promising new TB vaccine candidates. However, the introduction of an antigen with better immunogenicity into BCG does not enhance the effect of BCG, and sometimes even reduces the effect of the original BCG, for example, see Marques-Neto LM, piwowater ka Z, kanno AI, morae L, trentin MM, rodriguez D, et al Thirty years of recombinant BCG: new trends for a centenary vaccine, expert Rev vaccine 2021, 20:1001-11 and Zhang Lingxia, wu Xueqiong, dong Enjun. Research on the effect of recombinant vaccine of Ag85 a-BCG on tuberculosis, chinese journal of modern medicine 2009, 19:1948-51. Therefore, there is still a need for further research on how to obtain better recombinant BCG.
Disclosure of Invention
One of the purposes of the invention is to provide a novel recombinant BCG vaccine which can improve the protection effect of BCG and better prevent tuberculosis.
In order to achieve the object, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a recombinant bcg that overexpresses npe 18, nfadD28, hspX, and plcA proteins as compared to the starting strain.
Preferably, the recombinant BCG genome of the present invention contains the Rv1196-300, rv2941-369, rv2031c and Rv2351c genes.
The recombinant BCG vaccine of the invention overexpresses fusion protein nPnFHp010, and the amino acid sequence of the fusion protein nPnFHp010 is shown as SEQ ID No. 2.
The genome of the recombinant BCG vaccine contains the coding gene of fusion protein nPnFHp010, and the coding gene of the fusion protein nPnFHp010 is shown as SEQ ID No. 1.
The PPE18 antigen has been shown to have a protective effect on cynomolgus monkey TB as one of the components of the candidate vaccine M72/ASO1E that has now entered phase II clinical trials; the fadD28 protein encodes a fatty acid ligase, possibly associated with the virulence of MTB, and has good immunogenicity.
Most of the vaccines in the current research use antigen full length to prepare the vaccine. However, the part that plays a decisive role in the immune response induced by the body antigen is the epitope, which is the core of the antigenicity of the protein. The antigen epitope protein aggregates the antigen immunodominant epitope, shortens the antigen length, removes redundant sequences in the antigen, improves the immunogenicity of the vaccine, and reduces side effects. The invention provides 2T cell epitope proteins of nPPE18 and nfadD28 proteins, which can greatly improve the immune efficiency of vaccination. Specifically, the invention screens, designs and optimizes the T cell epitope enrichment region of PPE18 and fadD28 protein antigen, and constructs new PPE18 and new fadD28 protein antigen (new PPE18, nPPE18 and new fadD28, nfadD 28). Animal experiments prove that both proteins have good immunogenicity. According to the invention, by shortening the lengths of the antigen fadD28 and the PPE18 and removing the antigen redundant sequence, the constructed nfadD28 and nPPE18 two antigen epitope proteins can gather immunodominant epitopes, so that the immunogenicity of the vaccine is greatly improved and side effects are reduced.
The present invention then provides a method for secreting the antigen HspX from MTB latency (HspX is an MTB latency expressed protein that stimulates the host to produce significant CD 4) + T cell response and protective cytokines such as IL-2 and IL-17, induce Th1 type cellular immune response and humoral response) and active secretory antigens nPPE18, nfadD28 and plcA (plcA is part of MTB phospholipase C and is located in RD7 region, and is deleted in BCG strain, so that the mice can be induced to generate obvious cellular and humoral immune response). The coding genes corresponding to the antigens are respectivelyRv2031c、nRv1196、nRv2941AndRv2351c. Specifically, the fusion antigen composition is a fusion antigen (nPnFHp 010) prepared by sequentially connecting four antigens of nPPE18, nfadD28, hspX and plcA, wherein the two antigens are connected by a flexible peptide segment of Gly-Gly-Ser-Gly-Gly. The nPnFHp010 fusion antigen contains four MTB dominant antigens which are the secretion antigens HspX in the incubation periodRv2031c) Active secretion antigen nfadD28Rv2941-369）、nPPE18（Rv1196-300) And plcA%Rv2351c) Can provide comprehensive and extensive protection for patients with active and latent infections with MTB. The fusion antigen (nPnFHp 010) gene is integrated into BCG, and recombinant BCG vaccine (rBCG-nFHp 010) which can stably express the fusion antigen gene can be obtained.
After MTB enters a human body, more than 1/3 MTB survives in a dormant state in a host, the MTB vaccine under study at present mostly selects MTB active secretion antigen to prepare the vaccine, the immunity protection against MTB in the dormant period cannot be provided, hspX antigen is an MTB latent secretion antigen, and the vaccine developed based on the MTB latent infection can be selected to provide the immunity protection against MTB latent infection. The invention prepares fusion antigen nPnFHp010 by HspX antigen and other 3 active secretion antigens nPPE18, nfadD28 and plcA, integrates the fusion antigen gene into BCG, and the prepared recombinant BCG vaccine rBCG-nPnFHp010 can provide comprehensive protection for organism against MTB infection in active and latent periods.
Specifically, the invention selects an MTB latency secretion dominant antigen HspX @Rv2031c) Three MTB active phases secrete dominant antigen nPPE18Rv1196-300）、nfadD28（Rv2941-369) And plcA%Rv2351c) Wherein, the nPPE18 and the nfadD28 antigen are T cell epitope protein antigens of the nPPE18 and the nfadD28 respectively, an MTB fusion antigen gene nPnFHp010 is designed based on the four antigens, and the fusion gene is integrated into a BCG genome by utilizing an integration plasmid to construct a gene recombinant BCG vaccine capable of expressing the fusion antigen nPnFHp010. The nPnFHp010 contains 4 components, respectively nPPE 18%Rv1196-300）、nfadD28（Rv2941-369）、HspX（Rv2031c) And plcA%Rv2351c) nPnFHp010 fusion antigen geneRv1196-300、Rv2941-369、Rv2031cAndRv2351cthe four genes are sequentially connected from the 5 'end to the 3' end, and the antigen components are connected by flexible peptide segments. And integrating the recombinant BCG into BCG to obtain recombinant BCG vaccine, and immunizing a mouse by using rBCG-nPnFHp010 to evaluate immunogenicity, protectiveness and safety development. The results show that: rBCG-nPnFHp010 can induce mice to generate good immunogenicity, can provide higher protectiveness and safety, is an effective novel TB candidate vaccine, and has potential application value in the aspect of preventing TB infection.
Preferably, in the recombinant BCG vaccine of the present invention, the starting strain is BCG-China.
In a second aspect, the present invention provides a method for constructing the recombinant BCG vaccine described above, comprising the step of transferring plasmids containing the Rv1196-300, rv2941-369, rv2031c and Rv2351c genes into an original strain.
Preferably, the method of the invention comprises the step of transferring a plasmid containing the gene encoding the fusion protein npfhp 010 in the starting strain.
In a third aspect, the invention provides an application of the recombinant BCG vaccine or the recombinant BCG vaccine constructed by the method in preparing a vaccine for preventing MTB infection or a medicine for treating diseases caused by MTB infection.
In a fourth aspect, the present invention provides a vaccine for preventing tuberculosis, comprising the recombinant BCG vaccine described above or the recombinant BCG vaccine constructed by the method described above.
The vaccine of the invention further comprises an adjuvant.
The invention has the advantages that:
the gene recombinant BCG rBCG-nPnFHp010 constructed by the invention has the following advantages: the vaccine has high immunogenicity and reduced side effects, and can provide comprehensive and extensive protection for patients infected with MTB in active and latent periods. Can induce mice to generate good immunogenicity, can provide higher protectiveness and safety, is an effective novel TB candidate vaccine, and has potential application value in the aspect of preventing TB infection.
Drawings
FIG. 1 is a schematic diagram of pMV361-nPnFHp010 recombinant plasmid in example 2 of the present invention.
FIG. 2 shows the mouse serum antibody titers in example 4 of the present invention. In the figure P<0.01。
FIG. 3 shows the secretion of 9 cytokines by spleen lymphocytes of mice after antigen stimulation in example 4 of the present invention. In the figures, a, b, c, d, e, f, g, h, i respectively represent the values of TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF and IL-17 in the supernatant after stimulation of spleen lymphocytes with the corresponding antigens, the asterisks above the two sets of data histograms indicate that the differences between the two sets of data are statistically significant,and->Respectively representP<0.05 sumP<0.01。
FIG. 4 shows the secretion of 9 cytokine levels by spleen lymphocytes from mice after PPD stimulation in example 4 of the present invention. In the figures, a, b, c, d, e, f, g, h, i represent the values of TNF- α, IFN- γ, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF and IL-17, respectively, in the supernatant of spleen lymphocytes stimulated by PPD.
FIG. 5 shows the results of MGIA experiments in example 5 of the present invention. ns indicates that there is no significance in the difference between the two groups,indicating that the inter-group differences are significant, +.>Representation ofP <0.0001。
FIG. 6 shows the pathological results of three organs, namely liver, spleen and lung, of the mice in example 6 of the present invention. A. Three rows B and C represent HE staining results (40×) of liver, spleen and lung pathology sections of mice, respectively.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples below, unless otherwise indicated, are all those available commercially or may be prepared by methods conventional in the art.
The starting strain used for modification in the section of the present invention is BCG China (BCG-China), which is purchased from China food and drug inspection institute.
Example 1 fadD28 and PPE18 protein T cell epitope prediction and selection of T cell epitope enriched region
In the United states of America national center for Biotechnology informationNCBI) database, searching to obtain gene sequences encoding fadD28 and PPE18 proteins, predicting T cell epitopes of fadD28 and PPE18 proteins by using bioinformatics software such as Teprdelct and IEDB, and screening HLA-class II molecules (including HLA-A0201、/>0202、/>0203、/>0206 Better binding with T cell epitope peptide segment, respectively corresponding to the 217 th-339 th amino acid sequence of fadD28 protein and the 201 st-300 rd amino acid sequence of PPE18 protein, splicing the corresponding nucleotide sequences to respectively obtain gene sequences (the numbers on NCBI are respectivelyRv1196-300AndRv2941-369）。
EXAMPLE 2 construction of pMV361-nPnFHp010 recombinant plasmid
(1) Design of nPnFHp010 fusion antigen gene
Querying in NCBI (national center for Biotechnology information) websiteRv1196-300 、Rv2941-369、Rv2031cAndRv2351ca corresponding gene sequence, wherein,Rv1196-300andRv2941-369is thatRv1196AndRv2941the antigen gene is subjected to T cell epitope prediction and optimization, and an epitope enrichment region of the antigen gene is positioned, and the antigen epitope peptide is obtained after splicing. Will beRv1196-300、Rv2941-369、Rv2031cAndRv2351cthe sequence connection is carried out from the 5' end to the 3' end, and ' GGTGGTTCTGGCGGT (SEQ ID NO: 3) ' (the amino acid sequence is ' Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 4) ") is adopted as a flexible connection peptide segment to connect the antigen design fusion antigen genes nPnFHp010. The sequence of the obtained nPnFHp010 fusion gene is shown as SEQ ID NO.1, and the amino acid sequence of the nPnFHp010 fusion antigen is shown as SEQ ID NO. 2.
(2) Construction of pMV361-nPnFHp010 recombinant plasmid
The nPnFHp010 fusion antigen gene with EcoRI at the 5 'end and HindIII cleavage site at the 3' end was synthesized by using the gene synthesis technique, and double cleavage (37℃for 25 min) was performed on the fusion antigen gene and pMV361 vector (purchased from Shanghai Jinofu Biotechnology Co., ltd.) using EcoRI and HindIII restriction enzymes. And (3) respectively carrying out agarose gel electrophoresis identification and gel recovery on the enzyme-digested products, and then connecting by using T4 DNA ligase under the connection condition of 25 ℃ for 30min. The ligation product transformed E.coli DH 5. Alpha. Competent cells, comprising the steps of: taking out 100 mu L of frozen E.coli DH5 alpha competent cells, putting the cells on ice for dissolution, adding 10 mu L of connection products, uniformly mixing, standing in an ice bath for 30min, carrying out heat shock at 42 ℃ for 90s, standing in the ice bath for 2min, adding 800 mu L of non-resistant LB liquid medium into a biosafety cabinet, and culturing for 1h at 140rpm at 37 ℃ of a shaking table. Centrifuging at 4000rpm for 1min after the culture is finished, discarding 400 mu L of supernatant, gently blowing and uniformly mixing the bacterial cell precipitate, and then taking 200 mu L of bacterial liquid to coat on an LB solid plate containing kanamycin, and culturing for 12-16 h in an inverted mode at 37 ℃. Single colony with good growth state is selected and cultured in LB liquid medium containing kanamycin, and the bacterial liquid is taken for plasmid small extraction and then identified by EcoRI and HindIII double enzyme digestion. The enzyme-cut products are identified by agarose gel electrophoresis and then sent to company for sequencing, and the sequencing result is compared with the completely correct bacterial sample by BLAST, and the bacterial sample is cultured in LB liquid medium containing kanamycin for enrichment, and the plasmid is extracted by an alkaline lysis method and stored at-20 ℃. The structure of the pMV361-nPnFHp010 recombinant plasmid is shown in FIG. 1.
Example 3 preparation of rBCG-nPnFHp010 Gene recombinant BCG vaccine
(1) Preparation of BCG competent cells
Scraping a small amount of BCG-China culture from a Roche medium by using an inoculating loop, inoculating the BCG-China culture in a 7H9 liquid medium, standing at 37 ℃ for culturing until the logarithmic phase (about 20 days), centrifuging the bacterial liquid for 15min at 4 ℃ at 4000rpm for 1H in an ice bath after culturing, discarding the supernatant, and collecting bacterial cells. Adding 50mL autoclaved 10% glycerol (precooled on ice in advance) into each 50mL bacterial sediment, lightly blowing and mixing by a pipettor to fully suspend the bacterial, centrifuging at 4000rpm and 4 ℃ for 15min, discarding the supernatant, collecting the bacterial, and repeating washing for 3 times. After the last wash, 1000 μl of 10% glycerol was added to resuspend the bacteria, and the prepared BCG competent cells were aliquoted into EP tubes at 200 μl per tube and stored at-80 ℃ for future use.
(2) Electroporation method for transforming BCG competent cells
The prepared BCG competent cells were taken out from the refrigerator, and were allowed to stand on ice for thawing. 10. Mu.L of the recombinant plasmid successfully constructed in example 1 was pipetted into 200. Mu.L of BCG competent cells, and after 10min of ice bath standing, all were transferred into an electric rotating cup for electric shock transformation (parameters 25kV, 25uF, 1000)) The bacterial liquid after the end of the electrotransformation was transferred to a 1.5mL EP tube, 800. Mu.L of 7H9 liquid medium was added thereto, and resuscitated and cultured in a shaker at 37℃and 180rpm for 24 hours. The resuscitated bacterial solution was centrifuged at 4000rpm for 10min at room temperature, 400. Mu.L of the supernatant was discarded, and 200. Mu.L of the pellet was resuspended and applied to 7H10 solid medium containing kanamycin at a final concentration of 25. Mu.g/mL, and cultured at 37℃until single colonies were grown in the plates. Constructing the gene recombinant BCG rBCG-nPnFHp010 capable of expressing the fusion antigen nPnFHp010.
EXAMPLE 4 immunogenicity evaluation of recombinant BCG rBCG-nPnFHp010
(1) Preparation of rBCG-nPnFHp010 vaccine
Centrifuging rBCG-nPnFHp010 and BCG-China at 4deg.C and 4000rpm respectively to collect bacteria, washing the bacteria with 7H9 liquid medium, suspending the bacteria with sterile PBS, and adjusting the bacteria concentration to 5×10 6 CFU/mL。
(2) Mouse immunization procedure
Experiments were performed on 18 SPF-class female BALB/c mice of 6-8 weeks of age, which were randomly divided into 3 groups of 6, PBS group, rBCG-nPnFHp010 group and BCG-China group (BCG group for short), 200. Mu.L PBS was injected into the PBS group, and 1X 10 was injected into the rBCG-nPnFHp010 group 6 CFU (200 mu L) rBCG-nPnFHp010 bacterial liquid, BCG-China group injection 1X 10 6 CFU (200. Mu.L) BCG-China bacterial solution, 3 groups were immunized 1 time on the back, mice were sacrificed 30 days after immunization, and then eachThe immunogenicity, protection and safety of rBCG-nPnFHp010 were evaluated.
(3) Humoral immunity effect evaluation of rBCG-nPnFHp010
a: blood sample collection
Before primary immunization and before mice sacrifice, blood is collected by an orbital blood collection method, blood is collected, standing is carried out at 37 ℃ for 2h, centrifugation is carried out at 4000rpm for 10min, serum is separated, and the separated serum is stored at-20 ℃ for standby.
b: ELISA method for detecting serum antibody titer
The serum specific antibody titer is detected by enzyme-linked immunosorbent assay (ELISA) as follows:
rBCG-nPnFHp010 and BCG-China whole-cell lysate were diluted to 2. Mu.g/mL with coating buffer (50 mM carbonate buffer, pH=9.0), 100. Mu.L of each well was coated with 96-well ELISA plates, and incubated overnight at 4 ℃. Plates were washed 5 times with 0.01M PBST (PBS solution containing 0.05% tween-20, ph=7.2) and then 100 μl of 3% bsa in hole sealer was added per well and incubated for 2h at 37 ℃. The plates were washed 5 times with PBST, and each serum group was diluted multiple times with PBS at a dilution factor of 2 6 -2 32 The diluted serum was added to each well in sequence and incubated for 1h at 37 ℃. The plates were washed 5 times with PBST and goat anti-mouse IgG antibodies labeled with horseradish peroxidase (HRP) were diluted 1:5000 with PBS. The plates were washed 5 times with PBST, 100. Mu.L of chromogenic substrate TMB was added to each well and incubated at 37℃for 15min.
The reaction was stopped by adding 100. Mu.L of stop solution (2M concentrated sulfuric acid) to each well. Finally, the absorbance at 450 and nm was measured with a conventional micro-pore spectrophotometer. The judgment standard is as follows: if the OD value of the sample is more than or equal to 2.1 times of the OD value of the negative control, the sample is judged to be positive.
The results of serum antibody titer detection are shown in figure 2. The results show that rBCG-nPnFHp010 and BCG can stimulate the organism to produce specific IgG; however, the antibody level of rBCG-nPnFHp010 group (abbreviated as nPnFHp010 in the figure) is obviously higher than that of BCG group (abbreviated as BCG in the figure), and the difference between the two groups has significanceP<0.01）。
(4) Evaluation of cellular immune Effect of rBCG-nPnFHp010
a: isolation of mouse spleen lymphocytes
Mice were sacrificed under anesthesia, immersed in 75% alcohol for 5 minutes, and spleens were aseptically isolated. 200 mesh cell sieves were placed into the wells of a 6 well cell culture plate, 4 mL lymphocyte isolates were added to each well, the mouse spleen was gently ground with the rubber end of a 5mL syringe, and the ground spleen cell filtrate was collected into a 15mL centrifuge tube. 1mL 1640 cell culture medium was slowly added along the side wall of the 15mL centrifuge tube. After gradient centrifugation, intermediate cloud lymphocytes in a 15mL centrifuge tube were collected and washed with 10mL 1640. The supernatant was resuspended in complete 1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin dual antibiotics. Cell concentration was determined by a cell counter and then the spleen cell concentration was diluted to 1X 10 with complete 1640 medium 6 /mL, for use in subsequent experiments.
b: nine extracellular cytokines detected by Luminex method
The Luminex multiple cytokine detection kit is a commercial kit and comprises the following specific operations:
spleen lymphocytes (1X 10) were added to the 96-well cell culture plate at a concentration of 100. Mu.L per well 6 /mL), and 2. Mu.g of the corresponding stimulus (nPnFHp 010 whole-cell lysate, BCG whole-cell lysate, or PPD) was added. Each group was established with one of a sterile PBS-stimulated negative control well and a Canavalia (ConA) (5. Mu.g/mL) -stimulated positive control well, with the plate cover closed and at 37℃with 5% CO 2 The culture is carried out in an incubator for 16-24 hours. After the completion of the culture, 96-well cell plates were centrifuged at 4000rpm for 10min, and supernatants were used for detecting the expression levels of 9 cytokines including IL-2, IFN-gamma, TNF-alpha, IL-12, GM-CSF, IL-17, IL-4, IL-6 and IL-10 by using the Luminex multiple cytokines, and the detection steps were performed according to the instructions.
Specific antigen stimulated Luminex assay results are shown in fig. 3, ppd stimulated Luminex assay results are shown in fig. 4. Experimental results show that compared with the spleen lymphocytes of mice in the BCG group immunized by rBCG-nPnFHp010 after the specific antigen stimulation, the secretion level of IL-6 and IL-17 is obviously increased, and the difference between the two is obviousP<0.05 sumP <0.01 A) is provided; while IL-2, IL-4, IFN-gamma, TNF-alpha, GM-CSF, IL-12 and IL-10 secretion did not significantly differ（P>0.05 A) is provided; compared with the spleen lymphocytes of mice in the BCG group, the spleen lymphocytes of the mice in the rBCG-nPnFHp010 immune group stimulated by PPD have no obvious difference in secretion condition of 9 cytokinesP >0.05). The experimental result shows that rBCG-nPnFHp010 can induce the organism to generate strong humoral and cellular immune response.
Example 5 evaluation of in vitro protective Effect of rBCG-nPnFHp010
The rBCG-nPnFHp010 in-vitro protection effect evaluation is carried out by adopting a mycobacterium tuberculosis in-vitro growth inhibition test (MGIA), and the specific operation is as follows: spleen lymphocytes (1×10) were added at a adjusted concentration per well in a 24-well plate 6 Per mL) 500. Mu.L and 500. Mu.L of MTB standard strain H37Rv (ATCC 27294) containing 50 CFU, 1mL in total. Mixing and placing at 37deg.C and 5% CO 2 The incubator was co-cultured for 4 days. After the end of the incubation, the co-cultures were transferred to centrifuge tubes, centrifuged at 12000rpm for 10 minutes and the supernatant was discarded. While centrifuging, 500. Mu.L of sterile water was added to each well of a 24-well plate, the bottom and side walls were blown off with a pipette and allowed to stand for 5 minutes before transferring to a corresponding centrifuge tube, and after vortexing, allowed to stand for 10 minutes to thoroughly lyse the cells and release intracellular MTB. 50. Mu.L of a 7H 10-coated plate was used, and 50. Mu. L H37 Rv.sup.37 strain solution was directly coated as a blank, and the plate was subjected to colony counting after 2-3 weeks of inversion culture at 37 ℃.
As shown in FIG. 5, the colony numbers of MTB in BCG and rBCG-nPnFHp010 group are obviously lower than those in PBS groupP<0.001 The results show that the spleen cells of the mice immunized by BCG and rBCG-nPnFHp010 can effectively inhibit MTB growth compared with the PBS group; the MTB colony count of rBCG-nPnFHp010 group was slightly lower than that of the BCG group, indicating that rBCG-nPnFHp010 may produce the same or higher protective efficacy as that of BCG.
Example 6 safety evaluation of rBCG-nPnFHp010
After the mice were sacrificed under anesthesia, the liver, spleen and lung of the mice were aseptically isolated, and then fixed in 10% formalin solution for 1 week, after the fixation was completed, conventional paraffin sections were performed, hematoxylin-eosin staining (HE staining), and morphological changes of the three organs were observed under a microscope.
Compared with the control group, each organ of the rBCG-nPnFHp010 group mice is visible by naked eyes in the anatomical structures of three organs: the appearance, the color and the size are normal, and no obvious pathological changes such as hyperplasia, atrophy, edema and the like are seen. Next, the conventional paraffin section experiment results are shown in fig. 6: (1) the liver cells are clear in structure, compact and orderly in arrangement, rich in cytoplasm, large and round in cell nucleus, centered and orderly in arrangement of liver ropes, and have no obvious difference compared with a control group; (2) no obvious abnormality is seen in the white marrow area, the red marrow area and the edge area of spleen cells, the whole morphology of spleen tissue cells is normal, and no obvious difference is seen compared with a control group; (3) the bronchi and alveoli structures of the lung tissues were not abnormal, and a small amount of inflammatory cell infiltration was seen without significant differences from the control group. In short, the structures of all viscera are in the normal morphological range, and obvious pathological changes such as cytoedema, necrosis and the like are not seen.
In a word, compared with a mouse singly inoculated with BCG, the fusion gene recombinant BCG vaccine rBCG-nPnFHp010 provided by the invention has the advantages that the IgG level is obviously improved, and the rBCG-nPnFHp010 is suggested to provide stronger humoral immune response; the spleen cells of the organism are stimulated to secrete 9 kinds of extensive cytokines, wherein the secretion amount of IL-17 is obviously improved, which indicates that rBCG-nPnFHp010 can provide good cellular immune response; the effect of inhibiting MTB proliferation in vitro is slightly higher than that of BCG, which suggests that rBCG-nPnFHp010 has at least the same or stronger protective effect as that of BCG; the pathological result indicates that rBCG-nPnFHp010 has high safety. It can be concluded that: rBCG-nPnFHp010 as a multi-antigen fusion gene recombinant BCG has great potential and good application prospect in the process of preventing the occurrence and development of TB.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (5)
1. Compared with an original strain, the recombinant bacillus calmette-guerin vaccine is characterized in that the genome of the recombinant bacillus calmette-guerin vaccine comprises a coding gene of fusion protein nPnFHp010, and the coding gene of the fusion protein nPnFHp010 is shown as SEQ ID No. 1; the starting strain is BCG-China.
2. The recombinant bacillus calmette-guerin of claim 1 wherein the recombinant bacillus calmette-guerin overexpresses a fusion protein nPnFHp010 having the amino acid sequence shown in SEQ ID No. 2.
3. A method of constructing a recombinant bacillus calmette-guerin as claimed in any one of claims 1-2, comprising the step of transferring a plasmid containing the coding gene of the fusion protein nPnFHp010 in a starting strain.
4. Use of the recombinant bcg vaccine of any one of claims 1-2 or the recombinant bcg vaccine constructed by the method of claim 3 in the manufacture of a vaccine for preventing MTB infection or a medicament for treating a disease caused by MTB infection.
5. A vaccine for the prevention of tuberculosis, comprising the recombinant bcg vaccine according to any one of claims 1-2 or the recombinant bcg vaccine constructed by the method of claim 3.
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