CN1704118A - Mycobacterium triple nucleic acid vaccine for cattle - Google Patents

Abstract

The invention discloses a mycobacterium bovis triple nucleic acid vaccine, and aims to provide a mycobacterium bovis triple nucleic acid vaccine with better immunogenicity. The Mycobacterium bovis triple nucleic acid vaccine provided by the present invention is to fuse the Ag85B, MPT-83 and MPT-64 genes or the Ag85B, MPT-83 and MPT-64 genes with other nucleic acid sequences respectively or to delete part of the sequence or part of the nucleotide mutated nucleotide sequences that can encode proteins with the same activity as Ag85B, MPT-83 and MPT-64, respectively cloned into eukaryotic expression vectors, and combining the obtained at least two expression vectors formed mixture. The mycobacterium bovis triple nucleic acid vaccine provided by the invention can be used for preventing and/or treating animal tuberculosis, especially preventing and/or treating bovine, human and sheep tuberculosis.

Description

A kind of mycobacterium bovis triple nucleic acid vaccine

technical field

The invention relates to a vaccine, in particular to a mycobacterium bovis triple nucleic acid vaccine.

Background technique

Tuberculosis is a chronic wasting infectious disease that affects both humans and animals. With the increase in the number of AIDS patients and the emergence of drug-resistant strains, the incidence of tuberculosis is increasing year by year. According to foreign relevant data reports, about 1-2 billion populations are infected with mycobacterium tuberculosis in the world at present, and about 3 million people die from tuberculosis every year, and 10 million new cases occur simultaneously. There are currently hundreds of millions of people infected with Mycobacterium tuberculosis in my country, and there are about 6 million tuberculosis patients, and about 256,000 people die each year due to untimely treatment. If TB control measures are not improved as soon as possible, more than 200 million people will develop active pulmonary TB. Bovine tuberculosis is a chronic infectious disease of livestock and can be transmitted to humans. The cross-transmission of tuberculosis between humans and animals is an important reason for the widespread prevalence of tuberculosis, especially in areas where animal husbandry is developed. The cross-transmission of tuberculosis between humans and animals not only poses a threat to human health, but also affects the rapid development of animal husbandry. develop. Worldwide, 50 million cattle are infected with tuberculosis, causing economic losses of $3 billion. The number of cattle on hand in my country has reached 130 million heads, and the positive rate of bovine tuberculosis is calculated at 2%, and each head is calculated at 8,000 yuan, and the direct economic loss can reach more than 20 billion yuan. If dairy cows suffer from tuberculosis, it will not only affect the milk production and nutritional content of milk, but also can be transmitted to humans through various channels. The prevention and control of tuberculosis in livestock mainly relies on PPD intradermal allergy detection, and weed out PPD positive cattle without vaccine. Therefore, the most important thing to do a good job in the prevention and control of bovine tuberculosis is to develop a reasonable and effective tuberculosis vaccine.

Since Calmette and Guerin developed Bacillus Calmette-Guerin (BCG), humans have used the weakened BCG to prevent tuberculosis infection. However, BCG is not effective in many parts of the world, especially for adults, whose protection is easy to lose. The protection efficiency for different groups of people is not consistent, generally 0-85%. Therefore, developing a more comprehensive and effective new vaccine is an important way to control tuberculosis. Genetic engineering technology provides a new way for the development of recombinant subunit vaccines and DNA vaccines. DNA vaccines have both the safety of recombinant subunit vaccines and the high efficiency of inducing a comprehensive immune response from live attenuated vaccines. One of the hot spots (Bonato VLD, LimaVMF, Tascon R E, et al. Identification and Characterization of ProtectiveT Cells in hsp64 DNA-Vaccinated and Mycobacterium tuberculosi-Infected Mice. Infect. Immun. 1998, 66: 169-175).

Studies have shown that Mycobacterium tuberculosis secretory proteins can stimulate the body to produce a protective immune response, induce antibody production and induce CD4 ⁺ and CD8 ⁺ T cell-mediated immune responses. During the tuberculosis infection experiment, the early recognition of the patient's lymphocytes is mainly some secreted proteins, such as the Ag85 complex distributed in most mycobacteria, in which Ag85B has the activity of mycolate transferase, and the mycobacterial cell wall It is related to synthesis and participates in the pathogenic process after being combined with human fibronectin. It ranks first in the secreted protein of mycobacteria. In a series of purified antigen tests, this antigen has the strongest antigenicity. MPT-83 is a lipoglycosylated protein that attaches to the cell surface to expose epitopes (Wiker H G, uptyuioiloii S, Hewinson R G, Russell W P, and Harboe M. Heterogenous expression of the related MPB70 and MPB83proteins distinguishing various substrains of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv. Scand J. Immunol. 1996, 43:374-380). This protein is also a sero-dominant antigen found in cattle infected with M.bovis, and can only be detected in infected T lymphocytes, but not in BCG immunized animal cells (O'Loan CJ , Pollock J M, Hanna J, and Neill S D. Immunoblot analysis of humoral immune responses to Mycobacterium bovis in experimentally infected cattle: early recognition of a 26-kilodalton antigen. Clin. Diagn. Lab. Immunol. 1994, 1: 608-611; Vordermeier H M, Cockle P C, Whelan A, Rhodes S, Palmer N, Bakker D, and Hewinson R G. Development of diagnostic reagents to differentiate between Mycobacterium bovis BCG vaccination and M. bovis infection in cattle. Clin. Diagn. Lab. Immunol. 1999, 6 : 675-682). MPT64 is also a component of the M. tuberculosis complex. It is a 23KD protein that exists in strains of human Mycobacterium tuberculosis, virulent Mycobacterium bovis and some BCG vaccines (Oettinge T, Andersen A B, Cloning and B-Cell-epitopemapping of MPT64 from Mycobacterium tuberculosis H37Rv. Infection and Immunity. 1994, 62:2058-2064). Purified native MPT64 stimulated T cell responses in M. tuberculosis-infected mice and tuberculosis patients. In addition, natural MPT64 can also trigger DTH response in guinea pigs activated by Mycobacterium tuberculosis (Oettinger T, Holm A, Mtoni I M, et al. Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis.Infection and Immunity. 1995, 63:4613-4618). Therefore, the genes encoding these proteins have become the first choice for nucleic acid vaccines.

There are many foreign research reports on Mycobacterium tuberculosis nucleic acid vaccine. In 1994, Lowrie et al. used Mycobacterium leprae 65kDa heat shock protein gene DNA vaccine to immunize mice, and the results showed that the DNA vaccine had a similar protective effect to BCG (Lowrie DB, Tascon R E, Colston M J, et al. Towards a DNA vaccine against tuberculosis. Vaccine, 1994, 12:1537-1540).

In 1996, Huygen et al prepared the Ag85 DNA vaccine, and the mice immunized with the Ag85 DNA vaccine produced humoral and cellular responses and obtained obvious protection (Huygen K, Content J, Denis O, et al.Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat. Med. 1996, 2: 893-897).

In 1999, Zhong M et al. used DNA vaccines encoding ESAT-6, MPT-83, KatG or HBHA proteins, and experiments proved that they all induced specific humoral immunity, produced a large amount of r-interferon and had obvious protective responses (Zhong M , Howard A, Kelley C, et al. Immunogenicity of DNA Vaccines Expressing Tuberculosis Proteins Fused to Tissue Plasminogen Activator Signal Sequences. Infect. Immun. 1999, 67: 4780-4786).

In 2000, Morris evaluated the immune response after administration of monovalent and polyvalent Mycobacterium tuberculosis DNA vaccines. Monovalent DNA vaccines include DNA vaccines encoding MPT-63 and MPT-83, and multivalent combined DNA vaccines include ESAT-6, MPT-83, MPT-63, and KatG. The protection is stronger (Morris S, Kelley C, Howard A, et al. The immunogenicity of single and combination DNA vaccines against tuberculosis. Vaccine. 2000, 18: 2155-2163).

In 2000, Vordermeier etc. used mycobacterial antigen MPT70, MPT83, Ag85-A DNA vaccine to immunize cattle, the results showed that: MPT83 DNA immunized cattle can induce CD4 ⁺ cell response and humoral immune response (Vordermeier H M, Cockle P J, Whelan A O , et al. Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skintest. Vaccine, 2000, 19: 1246-1255).

Although a lot of research has been done on the development of Mycobacterium tuberculosis nucleic acid vaccines at home and abroad, the protective power of the obtained nucleic acid vaccines is not high enough. In 2001, Fan Xionglin et al. reported that the specific antibody titer of the Mycobacterium tuberculosis Ag85B secreted protein single-gene vaccine was only 1:200 (Fan Xionglin, Xu Zhikai, Li Yuan, etc. Construction and immunogen of the Mycobacterium tuberculosis Ag85B secreted protein gene vaccine Sexual Research, Chinese Journal of Tuberculosis and Respiratory Medicine, 2001, 24:548-550). In 2001, the specific antibody titer of Mycobacterium tuberculosis Ag85B secreted protein monogene vaccine reported by You Li et al. was 1:100 000 after the third immunization for 45 days (You Li, Zhao Yueran, Gao Chunyi et al., Mycobacterium tuberculosis Ag85B Ag85B The construction of DNA vaccine and its immune effect. Chinese Journal of Tuberculosis and Respiratory Medicine, 2001, 24: 736-739). The development of a more comprehensive and effective new nucleic acid vaccine is of great significance to effectively control the spread and prevalence of tuberculosis.

Mycobacterium bovis is similar to Mycobacterium tuberculosis in terms of growth characteristics, chemical composition and virulence potential. Mycobacterium bovis is mainly a pathogenic bacteria of cattle, which causes tuberculosis infection in cattle. infected with milk contaminated with this bacterium. However, if it is inhaled from the respiratory tract, it can also cause the same infection as Mycobacterium tuberculosis, which is difficult to distinguish.

The animal expression vector pJW4303, whose physical map is shown in Figure 7, has a cytomegalovirus (CMV) early gene promoter, which is a strong promoter that ensures efficient transcription of the target gene in muscle cells. The vector also carries the extracellular secretion signal sequence of tissue plasminogen activator, and the exogenous gene product downstream of this signal sequence can be successfully secreted extracellularly, increasing the interaction between antigen and macrophages. Exposure opportunities accelerate the proteasome-dependent degradation of endogenous synthetic antigens, leading to enhanced cell-mediated responses induced in vivo. In addition, pJW4303 contains 11 CpG sequences, which play an important role in DNA vaccine-induced immune response, can directly activate B cells in the absence of T cells, stimulate the secretion of immunoglobulin and interleukin-6, and directly Activate monocytes, macrophages and dendritic cells, up-regulate the expression of co-stimulatory molecules, help to produce a variety of Th1 cytokines, and improve protective immunity.

Invention content

The purpose of the invention is to provide a mycobacterium bovis triple nucleic acid vaccine with better immunogenicity.

The Mycobacterium bovis triple nucleic acid vaccine provided by the present invention is to fuse Ag85B, MPT-83 and MPT-64 genes or the three genes of Ag85B, MPT-83 and MPT-64 with other nucleic acid sequences respectively or to delete partial sequences Or part of the nucleotide mutated nucleotide sequence that can encode the same active protein as Ag85B, MPT-83 and MPT-64, respectively cloned into eukaryotic expression vectors, and the resulting expression vectors are combined to form a mixture .

The above vaccine also contains adjuvant DDA and/or MPL and/or Quil-A and/or RIBI adjuvant and/or saline (physiological saline) or other adjuvants, such as aluminum adjuvant and Freund's adjuvant. The weight ratio of mycobacterium bovis triple nucleic acid vaccine to DDA in the vaccine can be 1-3:1, preferably 3:1.

Wherein, the promoter of the expression vector can be the cytomegalovirus (CMV) early gene promoter carried by pJW4303 or other eukaryotic expression promoters, such as the mouse housekeeping gene promoter.

The above-mentioned Mycobacterium bovis triple nucleic acid vaccine is preferably a polyvalent Mycobacterium bovis nucleic acid vaccine formed by cloning the MPT-64, MPT-83 and Ag85B genes into the eukaryotic expression vector pJW4303 respectively; wherein, the Mycobacterium bovis triple The nucleic acid vaccine is preferably a mycobacterium bovis triple nucleic acid vaccine formed by combining expression vectors of MPT-64, MPT-83 and Ag85B in the same weight ratio.

The mycobacterium bovis triple nucleic acid vaccine of the present invention can be used for preventing and/or treating animal tuberculosis, especially preventing and/or treating human, bovine and sheep tuberculosis.

The Gene Bank accession numbers of the three genes are: MPT-64: X75361; MPT-83: X94579; Ag85B: X62398.

In the present invention, the structural genes encoding Mycobacterium tuberculosis Ag85B, MPT-83 and MPT-64 secreted proteins or the whole gene sequence including the signal peptide are directionally cloned into the eukaryotic expression vector pJW4303, transformed into TOP10 Escherichia coli, and the plasmid DNA is extracted by Restriction endonuclease identification and sequence analysis confirmed that the complete sequences of the structural genes (including signal peptides) of the above three secreted proteins were correct. Simultaneous intramuscular injection of three recombinant expression plasmids and DDA to immunize cattle resulted in a significant increase in the specific IgG antibody response levels of Ag85B, MPT-83 and MPT-64 in cattle, and the antibody level reached its peak at the end of 2 months after the third immunization. The PPD-B skin test test shows that immunization with the mycobacterium bovis triple nucleic acid vaccine of the present invention can trigger a strong systemic immune response, but cannot cause bovine tuberculin PPD skin test reaction. The results of comparing the levels of γ-interferon before and after the tracheal challenge of cattle with Mycobacterium bovis triple nucleic acid vaccine showed that cattle immunized with Mycobacterium bovis triple nucleic acid vaccine can produce effective immunity against Mycobacterium bovis and stimulate significant The high level of T cell response, the content of IFN-γ reached the highest at the end of the second month after the third immunization. The detection of the proliferation response of bovine peripheral blood mononuclear cells (lymphocyte proliferation assay) showed that all bovine blood lymphocytes immunized with the Mycobacterium bovis triple nucleic acid vaccine had a strong stimulation of Ag85B, MPT-64 and MPT-83. proliferative response. The histopathological changes of the lung tissue in the trachea of the immunized and control cattle were observed 4 months after the challenge. The results showed that the fibrosis of the lung parenchyma of the control cattle immunized with the empty vector pJW4303 DNA was 50-70%. Fibroblasts and lymphocytes increased, and the lung sections of BCG-immunized bovines showed obvious fibrosis in the lung parenchyma, with more fibroblasts and lymphocytes. The bovine lung slices immunized with the mycobacterium bovis triple nucleic acid vaccine of the present invention have normal lung tissue, clear alveoli and a small amount of fibroblasts and lymphocytes. Mycobacterium bovis was cultured in the lung, liver and lymph node tissues of the trachea of the immunized and control cattle 4 months after challenge, and the number of live bacteria was counted after 8 weeks. Significantly decreased (p<0.05). The number of bacteria [(1.87 ± 1.40) × 10 ¹ ] in the lungs of cattle immunized by the Mycobacterium bovis triple nucleic acid vaccine of the present invention was reduced by more than 7,000 times than that of the empty vector control group, and the protection efficiency was 80%. The protection efficiency of the positive control BCG group was 60%, the protection efficiency of the negative control group injected with empty vector was 16%, and the protection efficiency of the injection of normal saline was 0%. These results fully prove that the Mycobacterium bovis triple nucleic acid vaccine of the present invention has good protection efficiency for cattle. The mycobacterium bovis triple nucleic acid vaccine of the present invention not only enhances the body fluid response of the immunized animals, but also enhances the cell-mediated immune response, and the latter is an important prerequisite for improving the efficiency of the nucleic acid vaccine. In addition, the adjuvant DDA selected in the present invention not only improves the immune efficiency of the vaccine, but also makes the immune response to Th1-response.

Compared with live BCG vaccines and subunit vaccines, the mycobacterium bovis triple nucleic acid vaccine of the present invention has other potential advantages: easy production, strong stability, relatively safe, and does not cause sensitive reactions to tuberculin. In addition, HIV immunocompromised patients can be immunized, whereas the BCG vaccine cannot be used in such patients. The adjuvant DDA used in the present invention greatly improves the protection efficiency of the vaccine. The mycobacterium bovis triple nucleic acid vaccine of the invention significantly improves the protection efficiency of animals, especially large animals (such as cattle, sheep, etc.), and does not cause tuberculin sensitive reactions, thereby enhancing the safety of the vaccine. The invention has important theoretical significance and application value for improving the immunity of animals and humans to mycobacterium bovis, more effectively controlling the spread and prevalence of tuberculosis, prospering animal husbandry and enhancing human physique.

Description of drawings

Figure 1 is a histogram of PPD-B skin test after cattle immunization

Figure 2 is the change curve of IgG antibody response in the body of cattle after triple nucleic acid vaccine immunization and Mycobacterium bovis challenge

Figure 3A is a bar graph of the content of IFN-γ in bovine whole blood cultures 4 weeks after the last immunization with the triple nucleic acid vaccine

Figure 3B is a histogram of the content of IFN-γ in bovine whole blood cultures 8 weeks after the final immunization with the triple nucleic acid vaccine

Figure 3C is a histogram of the content of IFN-γ in bovine whole blood cultures 4 weeks after the bovine tracheal challenge immunized with the triple nucleic acid vaccine

Fig. 4 is the change curve of IFN-γ in bovine whole blood culture after immunization with triple nucleic acid vaccine

Figure 5 is a histogram of the value-proliferating response of the whole blood culture of cattle after immunization with the triple nucleic acid vaccine

Fig. 6A is a photomicrograph (×10) of lung tissue sections of cattle in the pJW4303 control group 16 weeks after challenge with Mycobacterium bovis.

Fig. 6B is a photomicrograph of the lung tissue section of cattle in the pJW4303 control group after 16 weeks of Mycobacterium bovis challenge (×40)

Figure 6C is a photomicrograph of the lung tissue section of cattle in the pJW4303 control group after 16 weeks of Mycobacterium bovis challenge (×100)

Figure 6D is a photomicrograph of the lung tissue section of cattle in the BCG immunized group after 16 weeks of Mycobacterium bovis challenge (×10)

Figure 6E is a photomicrograph of the lung tissue section of cattle in the BCG immunized group after 16 weeks of Mycobacterium bovis challenge (×40)

Figure 6F is a photomicrograph of the lung tissue section of cattle in the BCG immunized group after 16 weeks of Mycobacterium bovis challenge (×100)

Figure 6G is a photomicrograph of the lung tissue section of cattle in the triple nucleic acid vaccine immunization group after 16 weeks of Mycobacterium bovis challenge (×10)

Figure 6H is a photomicrograph of the lung tissue section of cattle in the triple nucleic acid vaccine immunization group after 16 weeks of Mycobacterium bovis challenge (×40)

Figure 6I is a photomicrograph (×100) of lung tissue sections of cattle in the nucleic acid triple vaccine immunization group after 16 weeks of Mycobacterium bovis challenge

Figure 7 is the physical map of animal expression vector pJW4303

Detailed ways

Embodiment 1, the construction of eukaryotic expression vector

The Ag85B, MPT-83 and MPT-64 genes of M. tuberculosis were amplified by PCR and cloned into the eukaryotic expression vector pJW4303 for expression in eukaryotic cells.

The basic steps of eukaryotic expression vector construction are:

(1) Using the Mycobacterium tuberculosis genomic DNA extracted by conventional methods as a template, PCR amplification of Ag85B, MPT-83 and MPT-64 genes and directional cloning into the tissue plasminogen activator (TPA) signal on the pJW4303 vector downstream of the sequence, a fusion protein is formed. The primer sequences used for amplification are as follows:

Ag85B: 5'-AAATGGGGCACAGCTAGCCATATGACAGACGTGAGCC-3', and 5'-ACTAGGATCCTAAGCAACCTTCGGTTGATCCCGTCAGC-3';

MPT-83: 5'-ATTGCTAGCATGATCAACGTTCAG-3', and 5'-TATGGATCCCGAACGTTACTGT-3';

MPT-64: 5'-TAGAGTACTGCTAGCGTGCGCATCAAGATCTTC-3', and 5'-TAGAGTACTGGATCCTAGGCCAGCATCGAGTCG-3'. The 5' end primers all contain NheI sites, and the 3' end primers all contain BamHI sites. The PCR product was subjected to agarose gel electrophoresis and recovered according to the instructions of the QIAquick Gel Extraction Kit to obtain the target gene.

(2) Digest the gel-purified target gene product and pJW4303 vector DNA with Nhe I and BamH I respectively, treat with T4 DNA ligase for 16 hours (16°C), and take 5 μl of the ligated product (total volume 20 μl) to transform into competent cells Escherichia coli DH5α strain. A single colony of transformed bacteria was picked to extract the plasmid, and sequence analysis was carried out after Nhe I and BamH I double enzyme digestion to confirm the correctness of the inserted fragment sequence. Transform the recombinant vector containing the correct reading frame into TOP10 Escherichia coli, screen the recombinant strain again in agar containing Amp (100 μg/ml), use QIAGEN Plasmid Maxi Kit and Mega Kit to prepare a large amount of plasmid DNA, and dilute it to a concentration with DDA 1-2 μg/μl, quantified by ultraviolet spectrophotometer, and obtained recombinant plasmids pJW-Ag85B, pJW-MPT-83 and pJW-MPT-64.

Embodiment 2, immunize cattle with triple DNA vaccine and detect the protective efficiency of the vaccine to cattle

1) Immunization of cattle by triple DNA vaccine

The 16 cows were randomly divided into four groups: Mycobacterium bovis triple nucleic acid vaccine immunization group, BCG immunization group and pJW4303 empty vector control group each with five heads, and the remaining one head was inoculated with normal saline as a non-immune control group. 500 μg of DDA was heated to 80°C to form micelles, and after cooling to room temperature, it was mixed with 1500 μg of plasmid DNA (500 μg of each plasmid) encoding Ag85B, MPT-64 and MPT-83 antigens respectively (recombinant plasmids pJW-Ag85B, pJW-MPT-83 and pJW-MPT-64), and then carry out neck muscle immunization to the cattle in the nucleic acid vaccine immunization group, and each head is inoculated with 2000 μg each time. The cattle of the empty vector control group were immunized with the neck muscle of 1500 μg of pJW4303 plasmid DNA and 500 μg of DDA treated as above, 2000 μg per head each time. In the BCG immunized group, 1×10 ⁶ CFU BCG was subcutaneously inoculated in the first and third injections, and 1×10 ⁶ CFU BCG was subcutaneously inoculated in the second injection. The cow serving as a non-immune control was inoculated with 2 ml of normal saline each time. All inoculations were performed at monthly intervals for a total of three visits. Four weeks after the third immunization, antiserum was isolated by conventional methods.

2) Enzyme-linked immunoassay (ELISA) to detect the antigen-specific immune response of cattle after nucleic acid vaccine immunization

100 μl (10 μg/ml) of purified antigens Ag85B, MPT-64, and MPT-83 dissolved in 0.1M carbonate buffer (pH9.6) were applied to polystyrene 96-well microtiter plates (Nunclon, Roskilde, Denmark) respectively. ) wells were coated overnight at 4°C. The plate was washed with PBS solution saturated with skimmed milk and containing 0.05% Tween-20 at 37° C. for 2 hours, and washed 5 times in total. Starting from 1:100, serum samples were serially diluted two-fold with PBS-Tween buffer (containing 0.5M NaCl), and then added to two duplicate 96-well plate wells at 100 μl/well. Each plate was incubated at 37°C for 2 hours with constant shaking, and then washed as described above. After washing the plate, horseradish peroxidase-labeled goat anti-bovine antiserum IgG, IgG1 and IgG2 (Serotec Ltd, Oxford, UK) were added to each well at 1:50000 dilution in PTN, and continued at 37 Incubate for 2 hours at °C. After washing the plate, the substrate solution 3,3',5,5'-tetramethylbenzidine (Chemicon, Harrow, UK) was added and incubated at 37°C for 10 minutes. After stopping the reaction with 2M H ₂ SO ₄ , the color response was analyzed by the OD450 value read by a microplate reader (BIO-RAD, Model 550, Japan). The results are expressed as the ratio of optical density (OD), which is the ratio between the OD450 of an experimental sample and the OD450 of the 0-day sample. A reliable Ab response was shown when OD≥2. Samples with OD values between 1.5-1.9 were scored as weak Ab positives. The experimental results are shown in Table 1, showing that the cattle immunized with the triple nucleic acid vaccine produced a significant IgG response four weeks after the third injection; four of the five triple nucleic acid vaccine immunized cattle produced a strong response to the MPT-64 antigen, Anti-MPT-64 antibodies could be detected between 1:100 and 1:800 dilutions; three of the five triple nucleic acid vaccine immunized cattle produced positive reactions to the Ag85B antigen, and the antibody titers ranged from 1:100 to 1:400; four of the five cattle immunized with the triple nucleic acid vaccine produced significant IgG responses to the MPT-83 antigen, with titers ranging from 1:100 to 1:800. All three antigens induced the same antibody titers in two independent experiments. Meanwhile, cattle immunized with BCG did not develop specific humoral responses in three immunizations. In addition, five cattle immunized with empty vector pJW4303 plasmid DNA also did not induce humoral responses against the antigen.

Table 1. The titer of specific antibody IgG produced by humoral immune response induced by immunizing cattle with the triple nucleic acid vaccine encoding Ag85B, MPT-83, MPT-64 and adjuvant DDA antigen Four weeks after the third immunization, the total antibody IgG titers of cattle with different numbers 2 3 4 5 6 Ag85BMPT-64MPT-83 --- 1:4001:8001:200 1:4001:8001:400 -1:1001:800 1:1001:2001:100

3) Detection of delayed hypersensitivity reaction after immunization of cattle - PPD-B skin test test

All 16 cattle were tested for intradermal tuberculin by injection with bovine PPD (PPD-B) four weeks after the final immunization. The size of the response induced by B-PPD was measured on day 3-4 after injection of bovine PPD. Cattle immunized with BCG served as a positive control. Results are shown as the average response size of 5 triple nucleic acid vaccine immunized cattle (or 5 BCG immunized cattle per group). The results are shown in Figure 1, indicating that the DTH responses of the 5 cattle immunized with BCG to PPD were 3.3-6mm, and the average response and standard deviation (4.56±2.27) obtained from these cattle were higher than the corresponding values of the triple DNA vaccine immunized group (0.8±0.8) (P≤0.01). Neither the triple DNA vaccine immunization group nor the empty vector pJW4303DNA immunization control group produced a DTH response equal to or greater than 0.82mm. Therefore, immunization with the triple nucleic acid vaccine can elicit a strong systemic immune response, but it cannot cause an allergic reaction to bovine tuberculosis PPD. Compared with the triple DNA vaccine immunization group and the empty vector pJW4303 DNA immunization group, P≤0.05 (Mann-Whitney test).

4) Mycobacterium bovis challenge experiment

At the 17th week after the first immunization, 1×10 ⁷ CFU of virulent Mycobacterium bovis M.bovis (purchased from the Tuberculosis Research Center of the 309th Hospital of the Chinese People’s Liberation Army) was used to treat the cattle of each immunization group and one non-immune cattle All underwent tracheal intubation challenge experiments. That is, an endotracheal tube with a length of 80 cm and a very thin cannula was inserted deep into the trachea of an anesthetized animal, and 1.5 ml of bacterial solution containing 1×10 ⁷ CFU of virulent Mycobacterium bovis M.bovis was injected through the cannula and used 2ml of normal saline was added.

5) Trend analysis of serum IgG antibody response before and after cattle challenge

The assay method of antibody titer is the same as that described in 2). All immunized and challenged cattle were tested for IgG antibody responses. Results are expressed as mean optical density. The results are shown in Figure 2, indicating that at the time of challenge, compared with the non-immune control group, the cattle in the triple DNA vaccine immunized group had significantly higher antibody levels, and reached the eighth week after the third immunization. Highest. Antibody titers in all immunized cattle showed a slight decrease at week 8 post-challenge and a further decrease at week 16.

6) Determination and comparison of gamma-interferon levels before and after cattle challenge

Whole blood cultures were performed in 96-well plates (0.2 ml/well). 1 ml of heparinized blood taken from the jugular vein was mixed with an equal volume of antigen solution (10 μg/ml) for culture. A portion of blood was incubated with solution without antigen as a negative control. The supernatant was collected after culturing in a 5% CO ₂ incubator at 37° C. for 24 hours, and γ-interferon (IFN-γ) was measured with BOVIGAM ^™ Bovine γ Interferon Test Kit (CSL, Victoria, Australia). The results are shown in Figure 3A, Figure 3B, Figure 3C and Figure 4, indicating that in the fourth week after the third immunization, compared with the empty vector and other control groups, the T cells of five triple nucleic acid vaccine immunized cattle stimulated Ag85B specific antigen production Significant response (P<0.05), but did not show a significant response to MPT-64 and MPT-83 specific antigen stimulation (Figure 3A); and after 8 weeks after the third immunization, the triple nucleic acid vaccine immunization group was more effective than the empty vector The control group showed a higher level of T cell response, in which the average response of the immune group to Ag85B and MPT-64 was significantly higher than that of the control group (P<0.01), and the response to MPT-83 also showed Difference (P<0.05) (Fig. 3B, 3C). Among them, the results are represented by optical density index ODI (antigen-containing OD450:antigen-free OD450) ± SD; 5 cows in each group; Mann-Whitney test was used for statistical analysis, compared with the response of the pJW4303 immunized group, P<0.05. Figure 4 shows that immunization with the triple nucleic acid vaccine elicited a strong response in the cattle, and the content of IFN-γ reached the highest at the end of the second month after the triple immunization.

7) Detection of the proliferation response of bovine peripheral blood mononuclear cells

Detection experiments were performed 2 months after the last immunization. The specific process is: the heparinized bovine blood is first diluted 1:4 with medium, and the medium is composed of: RPMI-1640 and contains 5% CPSR-1 (Sigma, Poole, UK), non-essential amino acids (Sigma), 5×10 ^-5 M mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate; then distribute 100 μl per well into a flat-bottomed 96-well plate, wherein each well contains the antigen solution dissolved in the culture medium ( The concentrations of Ag85B solution, MPT-64 solution and MPT-83 solution were all 5 or 10 μg/ml) (100 μl/well, repeated three times). Each plate was cultured for 6 days, and the cells without any treatment and those treated with Con A (10 μg/ml) were used as controls. Proliferation response was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) staining method, that is, after 72 hours of incubation, 20 μl of MTT was added to each well for further incubation for 4 hours. Finally 150 μl DMSO was added to each well. After mixing the dark precipitate by pipetting, read the absorbance value at 650nm. The results are shown in Figure 5, indicating that compared with the empty vector control group, all bovine blood immunized with the triple nucleic acid vaccine produced a strong response to the stimulation of Ag85B, MPT-64 and MPT-83. In contrast, bovine blood immunized with BCG did not detect any in vitro proliferation response that was significantly different from that of the empty vector control group after PPD stimulation. The proliferative response of control cattle did not appear to be elicited by the antigen. Among them, the results are represented by optical density index ODI (OD450 of culture containing antigen: OD450 of culture without antigen)±SD. 0.5 cattle/group. Statistical analysis was performed by Mann-Whitney test, P<0.01 compared with the response of the pJW4303 immunized group.

8) Pathological analysis of cattle after challenge

All cattle underwent extensive pathological examination at week 16 post-challenge. Lymph nodes are removed and sliced into thin slices for examination. The lungs were first palpated for nodules and sliced into approximately 1 cm thick slices to observe damage. The found lesions were examined histopathologically for tuberculosis. When doing histopathological examination, samples of left and right anterior shoulder lymph nodes, left and right anterior femoral lymph nodes, mesenteric lymph nodes and hilar lymph nodes were collected from all cattle. Additional samples were taken from sites of injury found in other lymph nodes or organs. Tissues used for histopathological analysis were first fixed in neutral 10% formaldehyde buffer, then embedded in paraffin, stained with hematoxylin and eosin, and made into 6 μm sections for observation under a microscope. The results are shown in Table 2 and Figures 6A-6I. Table 2 shows that tuberculosis lesions were found in the organs of all cattle in the pJW4303 control group, while only 1 cattle in the triple nucleic acid vaccine immunized group had damage. The lung injury was significantly different between the nucleic acid vaccine immunization group and the empty vector control group, and the score of the immunization group (0.5±1.0) was lower than that of the control group (2.6±1.7). Lung tuberculosis lesions were also found in 3 out of 5 cows in the BCG immunized group, and the mean lung lesion score (1.8±1.8) was also lower than that in the empty vector control group. All lesions in the M. bovis triple nucleic acid vaccine immunization group, BCG immunization group, pJW4303 empty vector control group and non-immunization control group had no yellow calcium caseation center. Lesions in the non-immunized control group and the immunized group did not differ significantly in size. Additionally, five pJW4303 empty vector control cows with microscopic tuberculosis lesions had culture-positive lungs. Two cows in the BCG group had culture-positive lungs, and one of the BCG-vaccinated cows had organ damage but was culture-negative for M. bovis. Figures 6A-6I show that all the immunized cattle had lower inflammation than the control group, and the results obtained from the triple nucleic acid vaccine and BCG vaccine immunized cattle were significantly different from those of the empty vector control group. different. The alveolar tissues of cattle treated with the empty vector pJW4303 showed abnormal and fibrotic pulmonary organ damage, about 70% of the lung parenchyma was involved but no granuloma or caseous necrosis appeared (Fig. Lymphocyte infiltration mixed with phagocytes (Fig. 6B&C). In contrast, lung fibrosis in BCG-immunized cattle affected only a small portion of the lung parenchyma (approximately 15-20%), was small and compact (Fig. 6D), and had a greater number of lymphocytes throughout the injury site (Fig. 6E&F). The alveolar tissue of cattle immunized with the triple nucleic acid vaccine appeared intact and clear (Fig. 6G), with a small amount of infiltration of lymphocytes mixed with epithelioid macrophages (Fig. 6H&I). The extent of lymphocytic infiltration varied greatly between groups, with statistically significant smaller lymphocytic infiltration in all immunized cattle than in controls at each site.

Table 2. Macroscopic lung damage and scores in cattle challenged with Mycobacterium bovis group Number of cows with lung damage/total number of cows lung injury score ^a BCGAg85B, MPT-64, MPT-83pJW4303 3/51/5*5/5 1.8±1.80.5±1.0*2.6±1.7

^a , Mean lung injury scores±SE of immunized and control cattle. Lesion score: 4, ≥100 small lesions or a lesion with a diameter >60mm; 3, 25-99% of small lesions or a 20-60mm diameter lesion; 2, 10-24% of small lesions; 1, 1- 9% minor lesions; 0, no lesions.

^* , significantly different from the empty vector control group (P<0.05).

9) Analysis of the protective effect of the triple nucleic acid vaccine on the genetic immunity of cattle against the challenge experiment

All cattle were slaughtered 16 weeks after challenge. Four thoracic lymph nodes (left and right bronchus and anterior and posterior mediastinal lymph nodes) were removed. Tissue samples were homogenized with a Tenbroeck grinder (Wheaton, Millville, NJ), treated in 0.75% cetylpyridium chloride for 1 hour, centrifuged at 3,500×g for 20 minutes, and then processed according to the literature (Buddle, BM, GWde Lisle, A. Pfeffer, and FEAldwell. 1995. Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG. Vaccine 13: 1123-1130) to isolate Mycobacterium bovis. 10-fold serial serial dilution of lung tissue homogenate spread on Lowenstein-Jensen medium for culture. M. bovis colonies were counted after 8 weeks. The results are shown in Table 3, which indicated that the number of bacteria in the BCG-immunized cattle was 2,000-fold lower ((6.82±0.11)×10 ¹ ) than that of the pJW4303 control group ((1.38±1.31)×10 ⁵ ). Neither of the cattle treated with empty vector DNA showed any protective effect. The number of bacteria ((1.87±1.40)×10 ¹ ) in the lungs of cattle immunized with the triple nucleic acid vaccine was more than 7,000 times lower than that of the empty vector control group.

Table 3. Analysis of the protective efficiency of bovine lungs after tracheal challenge with different vaccines and empty vector DNA

(Taking the number of surviving bacteria as an indicator) vaccine Lung CFU±SD/organ ^* BCGAg85B, MPT-64, MPT-83pJW4303 (6.82±0.11)×10 ¹ (1.87±1.40)×10 ¹ (1.38±1.31)×10 ⁵

^* Geometric mean of bacterial numbers (CFU/gram tissue) ± SD.

Sequence Listing

<110> Peking University

<120> A Mycobacterium bovis triple nucleic acid vaccine

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<141> Application date 2004-05-31

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Claims (8)

1. A Mycobacterium bovis triple nucleic acid vaccine, which is to fuse Ag85B, MPT-83 and MPT-64 genes or the three genes of Ag85B, MPT-83 and MPT-64 with other nucleic acid sequences respectively or to delete part of the sequence or Partial nucleotide mutated nucleotide sequences that can encode proteins with the same activity as Ag85B, MPT-83 and MPT-64 are respectively cloned into eukaryotic expression vectors, and the obtained expression vectors are combined to form a mixture. 2. The Mycobacterium bovis triple nucleic acid vaccine according to claim 1, characterized in that: said vaccine also contains adjuvant DDA and/or MPL and/or Quil-A and/or RIBI adjuvant and/or physiological Saline and/or aluminum adjuvant and/or Freund's adjuvant. 3. The Mycobacterium bovis triple nucleic acid vaccine according to claim 2, characterized in that the ratio of nucleic acid vaccine to DDA in said Mycobacterium bovis triple nucleic acid vaccine is 1-3:1 by weight. 4. The Mycobacterium bovis triple nucleic acid vaccine according to claim 3, characterized in that the ratio of nucleic acid vaccine to DDA in said Mycobacterium bovis triple nucleic acid vaccine is 3:1. 5. The Mycobacterium bovis triple nucleic acid vaccine according to claim 1, wherein the expression vector contains a promoter. 6. The Mycobacterium bovis triple nucleic acid vaccine according to claim 5, characterized in that: the promoter is the early gene promoter of cytomegalovirus (CMV) or mouse housekeeping gene carried by pJW4303. 7. The Mycobacterium bovis triple nucleic acid vaccine according to any one of claims 1 to 6, characterized in that: the Mycobacterium bovis triple nucleic acid vaccine is a combination of Ag85B, MPT-83 and MPT-64 genes The Mycobacterium bovis triple nucleic acid vaccine formed by cloning into the eukaryotic expression vector pJW4303 and combining the expression products. 8. The Mycobacterium bovis triple nucleic acid vaccine according to claim 7, characterized in that: the Mycobacterium bovis triple nucleic acid vaccine is a combination of the expression vectors of Ag85B, MPT-83 and MPT-64 in the same weight ratio The Mycobacterium bovis triple nucleic acid vaccine formed.

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