A subunit vaccine for animal abortion chlamydia and its preparation method and application

Abstract

The present invention belongs to the technical field of immunology, and specifically relates to a subunit vaccine for animal abortion chlamydia, and a preparation method and application thereof. The subunit vaccine of the present invention comprises a chlamydia antigen and an adjuvant; the chlamydia antigen comprises one or more combinations of MOMP, OmcB, incA and CPAF. The subunit vaccine of the present invention has good immune effect, high safety, can produce a high level of antibodies, and can prevent and / or treat diseases caused by chlamydia, such as abortion.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology, and specifically relates to an animal abortion chlamydia subunit vaccine and a preparation method and application thereof. Background Art

[0002] Chlamydia is a type of obligate intracellular, Gram-negative bacteria that infects a wide range of hosts, including humans and amebas, and is an important zoonotic pathogen.

[0003] Chlamydia abortus is the main cause of chlamydiosis in sheep, which is also called ovine enzootic abortion (OEA) or enzootic abortion of ewes (EAE). Infection in ewes can lead to suppurative placentitis, premature birth, dead fetuses or weak lambs, and ewes become carriers of chlamydia, excreting a large number of pathogens through the placenta, aborted fetuses or vaginal secretions during pregnancy. Other animals are easily infected with Chlamydia abortus through the respiratory and digestive tracts. In addition, pregnant women may be infected when they come into direct contact with infected animals, leading to abortion, so Chlamydia abortus is a zoonotic pathogen. Although Chlamydia abortus is mainly associated with endemic infections in sheep and goats, sporadic cases can also occur in other host species around the world, such as cattle, pigs, horses, deer and yaks, and even in marine mammals - spotted dolphins. Chlamydia abortus is the leading cause of abortion in goats and sheep worldwide, causing economic losses in most sheep-raising areas. Serological epidemiological surveys show that the chlamydia positivity rate in the UK is 44%, 11.1% in Saudi Arabia, 13.8% in Egypt, 6.6% in Tunisia, 9.7% in Iran, 33.0% in Spain, and 7.2% in Algeria. In recent years, my country has reported a number of cases of Chlamydia abortus infection. The serological positivity rates of Tibetan sheep in Gansu and goats in Hunan were 18.65% and 8.45%, respectively. Li et al. (Li Z, Cao X, Fu B, Chao Y, Cai J, Zhou J. 2015. Identification and characterization of Chlamydia abortus isolates from yaks in Qinghai China. Biomed Res Int, 2015: 658519; Li Z, Cai J, Cao X, Lou Z, Chao Y, Kan W, Zhou J. 2017. Whole-Genome Sequence of Chlamydia abortus Strain GN6 Isolated from Aborted Yak Fetus. Genome Announc, 5(35): e00893-17) successfully isolated Chlamydia abortus from aborted fetuses and vaginal swabs of yaks in Qinghai Province.

[0004] At present, the lack of commercial vaccines for the prevention and control of animal chlamydiosis has led to the control of animal chlamydiosis mainly relying on drug treatment. However, the long-term use of antibiotics will lead to the emergence and even prevalence of drug-resistant strains, which will once again pose a huge threat to animal husbandry and even the lives and health of the public. Therefore, vaccination is the key to the prevention and control of animal abortion chlamydia. Although inactivated vaccines for Chlamydia abortus have been successfully developed at home and abroad, the pathogen needs to be cultured in tissue cells or chicken embryo yolk sac membranes. There are biosafety risks in extracting chicken embryo yolk sac membranes, and the vaccine production process is relatively backward and not suitable for the production lines of modern vaccine manufacturers. So far, there is no commercialized Chlamydia abortus vaccine in China. Therefore, it is very necessary to develop a safe and efficient subunit vaccine for Chlamydia abortus in animals. Summary of the invention

[0005] The purpose of the present invention is to provide an animal abortion Chlamydia subunit vaccine and its preparation method and application. The obtained subunit vaccine has a high antibody level, good immune effect, high safety, can effectively inhibit the proliferation of Chlamydia abortion, reduce pathological damage to placental tissue, and prevent and / or treat diseases caused by Chlamydia abortion.

[0006] The present invention provides an animal abortion Chlamydia subunit vaccine, comprising Chlamydia antigen and adjuvant;

[0007] The chlamydia antigens include a combination of four, three, two or any one of MOMP, OmcB, incA and CPAF.

[0008] Preferably, the adjuvant comprises MONTANIDETE TM ISA201.

[0009] Preferably, the subunit vaccine is in the form of water-in-oil-in-water.

[0010] The present invention also provides a method for preparing the subunit vaccine described in the above technical solution, comprising the following steps:

[0011] expressing the gene encoding the Chlamydia antigen, and purifying it to obtain a purified protein;

[0012] The purified protein is mixed with an adjuvant and emulsified to obtain the subunit vaccine.

[0013] Preferably, the volume ratio of the purified protein to the adjuvant is 1:1.

[0014] Preferably, the emulsification is carried out at 500 r / min; the emulsification time is 5 to 10 min.

[0015] Preferably, the expression step comprises: constructing a recombinant plasmid containing the Chlamydia antigen encoding gene, transforming it into Escherichia coli, and culturing it until the absorbance OD 600 =0.6, add protein inducer, continue culturing for 3-5h, and obtain the Chlamydia antigen.

[0016] Preferably, the protein inducer includes IPTG; the working concentration of the IPTG is 0.8 mM.

[0017] Preferably, the purification method comprises gradient elution.

[0018] The present invention also provides the use of the subunit vaccine described in the above technical solution in the preparation of drugs for preventing and / or treating diseases caused by Chlamydia.

[0019] Beneficial effects:

[0020] The present invention provides an animal abortion chlamydia subunit vaccine, comprising a chlamydia antigen and an adjuvant; the chlamydia antigen comprises a combination of four, three, two or any one of MOMP, OmcB, incA and CPAF. The MOMP, OmcB, incA and CPAF described in the present invention are produced at different developmental stages of chlamydia abortion, and can regulate the life activities of host cells to infect cells and reproduce. The present invention mixes antigen proteins produced at different developmental stages of chlamydia abortion with adjuvants to prepare a subunit vaccine, which can have a good immune effect, high safety, can produce a higher level of antibodies, and prevent the development of chlamydia at different stages, thereby achieving an immune protection effect. The results of the sheep immune protection test show that the animal abortion chlamydia subunit vaccine of the present invention can effectively prevent chlamydia from multiplying and infecting in the body, reduce pathological damage to placental tissue, and the abortion protection rate is 61.5%.

[0021] The present invention is based on MONTANIDE TM ISA 201 is an adjuvant. Compared with traditional oil emulsion, MONTANIDE TM ISA201 emulsion is potent, stable, has low viscosity and is easy to inject. The subunit vaccine prepared can induce short-term and long-term immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 The purification results of MOMP, CPAF, incA and OmcB proteins are shown in Figure 1, where A is MOMP, B is CPAF, C is incA, and D is OmcB protein;

[0024] Figure 2 These are the results of pathological tissue section observation. DETAILED DESCRIPTION

[0025] The present invention provides an animal abortion Chlamydia subunit vaccine, comprising a Chlamydia antigen and an adjuvant; the Chlamydia antigen comprises a combination of four, three, two or any one of MOMP, OmcB, incA and CPAF.

[0026] In the present invention, the chlamydia antigens include a combination of four, three, two or any one of MOMP, OmcB, incA and CPAF, preferably one of incA and CPAF, or a combination of MOMP and OmcB, or a combination of incA and CPAF, or a combination of MOMP, OmcB and CPAF, or a combination of incA, CPAF and MOMP, or a combination of MOMP, OmcB, incA and CPAF, further a combination of MOMP, OmcB, incA and CPAF. The present invention selects different combinations of chlamydia antigens to prepare subunit vaccines according to different developmental stages of abortive chlamydia, which can have good immune effects, high safety, and can produce higher levels of antibodies.

[0027] In the present invention, the adjuvant preferably includes MONTANIDE TM ISA 201. MONTANIDE TM ISA 201 contains no animal ingredients and is a specially refined emulsifier containing a specific concentrated light mineral oil, mannitol from plant sources or pure oleic acid. Compared to traditional oil emulsions, MONTANIDE TM ISA201 emulsion is potent, stable, low in viscosity and easy to inject. The subunit vaccine of the present invention is preferably in the form of water-in-oil-in-water. TM The vaccine prepared by ISA201 and the Chlamydia antigen has good stability, can induce short-term and long-term immunity, and the vaccinated sheep can effectively resist the infection of Chlamydia abortus.

[0028] The present invention also provides a method for preparing the above subunit vaccine, comprising the following steps:

[0029] expressing the gene encoding the Chlamydia antigen, and purifying it to obtain a purified protein;

[0030] The purified protein is mixed with an adjuvant and emulsified to obtain the subunit vaccine.

[0031] The present invention expresses the gene encoding the Chlamydia antigen to obtain the Chlamydia antigen. The expression step of the present invention preferably includes: constructing a recombinant plasmid containing the Chlamydia antigen encoding gene, transforming it into Escherichia coli, culturing it until the absorbance OD600=0.6, adding a protein inducer, and continuing to culture for 3-5 hours to obtain the Chlamydia antigen.

[0032] The starting plasmid of the recombinant plasmid of the present invention is preferably pET-30a. When the chlamydia antigen of the present invention is a variety of combinations among MOMP, OmcB, incA and CPAF, the present invention preferably connects the genes encoding the MOMP, OmcB, incA and CPAF proteins to the starting plasmid respectively to obtain the various combinations of recombinant plasmids pET-30a-MOMP, pET-30a-OmcB, pET-30a-incA and pET-30a-CPAF. The present invention has no strict requirements on the source of the chlamydia antigen encoding gene, and can be amplified using PCR technology. The present invention has no strict requirements on the way in which the MOMP, OmcB, incA and CPAF are connected to the starting plasmid respectively, and conventional operations can be used. The primers used to amplify the genes encoding the MOMP, OmcB, incA and CPAF proteins of the present invention are preferably as shown in Table 1.

[0033] Table 1 Amplification primer sequences

[0034]

[0035] After obtaining the recombinant plasmid, the present invention preferably transforms the recombinant plasmid into Escherichia coli and inoculates it into LB liquid culture medium. When the recombinant plasmid of the present invention is a combination of pET-30a-MOMP, pET-30a-OmcB, pET-30a-incA and pET-30a-CPAF, the present invention preferably transforms the recombinant plasmids pET-30a-MOMP, pET-30a-OmcB, pET-30a-incA and pET-30a-CPAF into Escherichia coli, respectively, and cultivates the transformed Escherichia coli until the absorbance OD 600=0.6, add a protein inducer, continue culturing for 3 to 5 hours, preferably 4 hours, to obtain Chlamydia antigen MOMP, Chlamydia antigen OmcB, Chlamydia antigen incA and Chlamydia antigen CPAF, and chlamydia antigen MOMP, Chlamydia antigen OmcB, Chlamydia antigen incA and Chlamydia antigen CPAF are subsequently purified. The Escherichia coli of the present invention is preferably Escherichia coli BL21 (DE3); the volume ratio of the recombinant plasmid to the Escherichia coli is preferably 8:100. The present invention has no strict requirements on the transformation method, and the operation can be performed according to the instructions. The LB liquid culture medium of the present invention preferably contains kanamycin, and more preferably, 1 mL of kanamycin sulfate solution is added per liter of LB liquid culture medium; the concentration of kanamycin sulfate in the kanamycin sulfate solution is preferably 50-100 mg / mL, more preferably 60-90 mg / mL, more preferably 70-80 mg / mL, and most preferably 75 mg / mL; the volume ratio of the Escherichia coli transformed with the recombinant plasmid to the LB liquid culture medium is preferably (1:50)-(1:500), and more preferably 1:100.

[0036] After the inoculation is completed, the present invention preferably cultures the Escherichia coli, and when the absorbance (OD 600 ) is 0.6, add a protein inducer to the LB culture solution and continue culturing for 3 to 5 hours, preferably 4 hours. The culture temperature of the present invention is preferably 37°C; the rotation speed of the culture is preferably 200r / min. The protein inducer of the present invention preferably includes IPTG, and the working concentration of the IPTG is preferably 0.8mM. The protein inducer of the present invention can induce protein expression.

[0037] After the culture is completed, the present invention preferably performs ultrasonic treatment on the culture and collects the precipitate. The ultrasonic time of the present invention is preferably 20 minutes; the power of the ultrasonic is preferably 250W. The present invention preferably performs ultrasonic treatment for 4 seconds and at intervals of 6 seconds, and the ultrasonic time does not include the interval time. The ultrasonic treatment of the present invention can lyse cells.

[0038] After obtaining the precipitate, the present invention preferably purifies the precipitate to obtain a purified protein. The purification method of the present invention preferably includes gradient elution, and the gradient elution preferably uses an imidazole eluent. After the elution is completed, the present invention preferably collects the eluent that flows out, and the eluent contains the purified protein.

[0039] After obtaining the buffer, the present invention preferably mixes the buffer with urea to renature the protein to obtain the purified protein. The process of mixing the buffer and urea of ​​the present invention preferably includes: placing the buffer in a dialysis bag, then placing the dialysis bag in 8M urea, dialysing for 12 hours, taking out the dialysis bag, placing in 6M urea, dialysing for 12 hours, taking out the dialysis bag, placing in 5M urea for 12 hours, taking out the dialysis bag, placing in 4M urea for 12 hours, taking out the dialysis bag, placing in 3M urea for 12 hours, taking out the dialysis bag, placing in 2M urea for 12 hours.

[0040] After obtaining the purified protein, the present invention preferably mixes the purified protein with an adjuvant, emulsifies, and obtains the subunit vaccine. The volume ratio of the purified protein to the adjuvant of the present invention is preferably 1:1; the emulsification is preferably carried out under 500r / min; the emulsification time is preferably 5 to 10min, and more preferably 6 to 8min. When the chlamydia antigen of the present invention is a combination of MOMP, OmcB, incA and CPAF, the present invention preferably mixes a plurality of purified proteins MOMP, purified protein OmcB, purified protein incA and purified protein CPAF obtained by using recombinant plasmids pET-30a-MOMP, pET-30a-OmcB, pET-30a-incA and pET-30a-CPAF, obtains a mixed protein, and then mixes it with the adjuvant. When the mixed protein of the present invention is a mixture of purified protein MOMP and purified protein OmcB, the mass ratio of the purified protein MOMP and purified protein OmcB is preferably 1:1; when the mixed protein is a mixture of purified protein MOMP, purified protein OmcB, purified protein incA and purified protein CPAF, the mass ratio of purified protein MOMP, purified protein OmcB, purified protein incA and purified protein CPAF is preferably 1:1. The emulsification method of the present invention makes the prepared subunit vaccine milky white, and the emulsification type is water-in-oil-in-water. A small amount of drops on the water surface will not diffuse in a cloudy manner. The pathological tissue section of the mouse immunization on the 7th day showed that the tissue structure of the inoculation site was intact and no inflammatory infiltration occurred, indicating that the inoculated vaccine was safe. The virus was challenged on the 12th day after mating. The normal productivity of mice was 73%, which was significantly higher than 22% of the negative control group. The produced subunit vaccine had a certain protective effect. The subunit vaccine obtained by the present invention has strong stability and good safety.

[0041] The present invention also provides the use of the subunit vaccine in the preparation of drugs for preventing and / or treating diseases caused by chlamydia. The diseases caused by chlamydia in the present invention preferably include one or more of miscarriage, stillbirth, arthritis and conjunctivitis. The subunit vaccine of the present invention has a preventive and / or therapeutic effect on diseases caused by chlamydia in various mammals including pigs, cattle, sheep and yaks. The results of the sheep immune protection test of the present invention show that the subunit vaccine of the present invention has a good immune effect, and all the immunized groups produce a high level of characteristic antibodies, which can effectively eliminate the number of chlamydia in the body and reduce pathological damage to placental tissue. The miscarriage protection rate is 61.5%.

[0042] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Unless otherwise specified, the steps mentioned in the examples are all conventional steps.

[0044] Example 1

[0045] (1) Recovery and testing of seed virus: After the frozen L929 cells were recovered, DMEM medium containing 10% fetal bovine serum was added and cultured in a cell culture incubator with 5% CO2. When the cultured L929 cells grew to about 80%, they were passaged, the medium was discarded, and the cells were washed twice with sterile PBS and 0.25% trypsin was added. After they were fully digested, they were seeded in 6-well cell culture plates (3×10 4 Cells / well) were cultured for 12 hours. The culture medium was discarded, and the cells were washed twice with sterile PBS, and then washed once with serum-free DMEM culture medium. The abortion Chlamydia GN-6 strain (preserved by the Important Zoonotic Bacteria Group of Livestock and Poultry of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) was diluted with serum-free DMEM culture medium to an infection solution with a multiplicity of infection (MOI) of 1. 5 mL of infection solution was added to each well, centrifuged at 37°C, 3000g for 1 hour, and placed in a cell culture incubator for 2 hours. The infection solution was discarded, and the culture medium containing cycloheximide was added to continue culturing for 48 hours. A cell plate was taken for Giemsa staining microscopy. Only a large number of purple-red Chlamydia inclusion bodies were seen in the cells, without any other bacteria contamination. This abortion Chlamydia can be used to infect sheep. The culture medium of uncontaminated cells was discarded, and the cells were washed twice with sterile PBS and repeatedly blown with sterile SPG solution. The cells were collected in a 50 mL centrifuge tube and ultrasonically lysed on an ultrasonic cell disruptor (amplitude 5 Hz, ultrasonication for 2 s, interval 2 s, ultrasonication 20 times in total), centrifuged at 4°C, 3000g for 5 min, and the supernatant was collected and stored at -80°C for use.

[0046] (2) Preparation of protein antigen: Using the genomic DNA of Chlamydia abortus GN-6 extracted in step (1) as a template, and the primer pair OmcB-F / R in Table 1 (the italicized part of Table 1 indicates the restriction site) as amplification primers, the gene encoding OmcB was amplified by PCR amplification technology; the amplified OmcB encoding gene and the pET-30a plasmid after double digestion with EcoRⅤ / XhoⅠ were gel purified and recovered; the purified and recovered products were ligated with T4 ligase at 16°C overnight. The ligation system (20 μL) is as follows: pET-30a 4 μL; target fragment 13 μL; 10×T4 DNA ligase buffer 2 μL; T4 DNA ligase 1 μL. Take 8 μL of the ligation product and transform it into E. coli DH5α competent cells and spread it on LB solid plates containing kanamycin. Culture it at 37°C for 16 hours, pick a single clone, expand the culture, extract the plasmid for PCR and enzyme digestion identification, and send the positive clone to Xi'an Qingke Jersey Biotechnology Co., Ltd. for sequencing. The correctly sequenced recombinant plasmid was named pET-30a-OmcB.

[0047] (3) The correctly identified recombinant plasmid was transformed into the competent Escherichia coli BL21 (DE3), and the bacterial suspension identified as positive by PCR was transferred to LB liquid medium containing kanamycin (1L LB liquid medium + 1mL 50mg / mL kanamycin sulfate) at a ratio of 1:100 (v / v), and cultured at 37°C and 200r / min with shaking until the absorbance OD 600 =0.6, add IPTG with a final concentration of 0.8 mM and continue to culture for 4 h, then sonicate at 250 W for 4 s, with an interval of 6 s, for a total of 20 min, collect the precipitate by centrifugation, add the treated precipitate to a resin with a His tag for purification, elute with 10 mL of imidazole buffer, and collect the outflowing buffer to obtain the purified protein OmcB.

[0048] (4) The purified protein OmcB was subjected to electrophoresis and then stained with Coomassie Brilliant Blue for analysis. Figure 1 As shown in D, the target protein is renatured with different concentrations of urea. Lanes 1 to 8 in the figure represent the purification effect of the protein in the buffer obtained by elution with different imidazole buffers. Specifically, lanes 1 to 8 are eluted with 10 mL of purified protein OmcB, 10 mL of 10 nm imidazole buffer, 20 nm imidazole buffer, 50 nm imidazole buffer, 100 nm imidazole buffer, 200 nm imidazole buffer, 300 nm imidazole buffer and 400 nm imidazole buffer, respectively.

[0049] according to Figure 1It can be seen that after elution with imidazole buffer, a relatively single target band of the same size as expected appeared without any impurity protein, and the eluted protein concentration gradually increased with the increase of the imidazole buffer concentration.

[0050] Example 2

[0051] The same as Example 1, except that in step (2), the primer pair CPAF-F / R in Table 1 (the italicized part in Table 1 indicates the restriction site) is used as the amplification primer, and the gene encoding CPAF is amplified by PCR amplification technology; the amplified CPAF encoding gene and the pET-30a plasmid after double digestion with NdeⅠ / XhoⅠ are gel purified and recovered to obtain the recombinant plasmid pET-30a-CPAF. The Coomassie brilliant blue staining result is as shown in Figure 1 As shown in B, the purified protein CPAF was finally obtained.

[0052] Example 3

[0053] The same as Example 1, except that in step (2), the gene encoding incA is amplified by PCR amplification technology using the primer pair incA-F / R in Table 1 (the italicized part in Table 1 indicates the restriction site); the amplified incA encoding gene and the pET-30a plasmid after double digestion with NdeⅠ / SalⅠ are gel purified and recovered to obtain the recombinant plasmid pET-30a-incA. The Coomassie brilliant blue staining result is as shown in Figure 1 As shown in C, the purified protein incA was finally obtained.

[0054] Example 4

[0055] Same as Example 1, except that in step (2), the gene encoding MOMP is amplified by PCR amplification technology using the primer pair MOMP-F / R in Table 1 (the italicized part of Table 1 indicates the restriction site); the amplified MOMP encoding gene and the pET-30a plasmid after double restriction digestion with EcoRⅤ / XhoⅠ are gel purified and recovered to obtain the recombinant plasmid pET-30a-MOMP. The Coomassie brilliant blue staining result is as shown in Figure 1 As shown in A, the purified protein MOMP was finally obtained.

[0056] Example 5

[0057] Protein antigen emulsification and testing: choose MONTANIDE TM ISA 201 adjuvant was sterilized by high pressure. The purified protein CPAF obtained in Example 2 was mixed with the oil adjuvant MONTANIDE TMISA 201 was mixed at a volume ratio of 1:1 and shaken evenly. Emulsification was carried out for 10 minutes under a shear force of 500 rpm. The emulsified antigen was milky white and the emulsification type was water-in-oil-in-water. A small amount of vaccine was dropped on the water surface without cloud-like diffusion. The CPAF vaccine was obtained and divided into vaccine bottles.

[0058] Example 6

[0059] Same as Example 5, except that the purified protein incA obtained in Example 3 was mixed with the oil adjuvant MONTANIDE TM ISA201 was mixed at a volume ratio of 1:1, shaken evenly, and the incA vaccine was obtained after emulsification, which was then divided into vaccine bottles.

[0060] Example 7

[0061] The same as Example 5, except that the purified protein OmcB obtained in Example 1 and the purified protein MOMP obtained in Example 4 were mixed at a mass ratio of 1:1 to obtain a mixed protein and then mixed with the oil adjuvant MONTANIDE TM ISA 201 was mixed at a volume ratio of 1:1, shaken evenly, and the OmcB+MOMP vaccine was obtained after emulsification, which was then dispensed into vaccine bottles.

[0062] Example 8

[0063] The same as Example 5, except that the purified protein OmcB obtained in Example 1, the purified protein CPAF obtained in Example 2, the purified protein incA obtained in Example 3 and the purified protein MOMP obtained in Example 4 were mixed in a mass ratio of 1:1:1:1 to obtain a mixed protein and then mixed with the oil adjuvant MONTANIDE TM ISA201 was mixed at a volume ratio of 1:1, shaken evenly, and after emulsification, OmcB+MOMP+incA+CPAF vaccine (Mixture vaccine) was obtained and divided into vaccine bottles.

[0064] Test Example 1

[0065] Samples were then taken and mice were inoculated with each bottle of the tested vaccine for sterility, safety and efficacy tests.

[0066] (1) Experimental groups: 120 female mice with consistent growth conditions were randomly divided into 6 groups: positive control group (20 mice), PBS control group (20 mice), MOMP+OmcB group (20 mice), CPAF group (20 mice), incA group (20 mice) and Mixture group (20 mice).

[0067] (2) Experimental methods: On the 0th day of superovulation, 15 female mice with slightly open vulva and pale pink vaginal mucosa were selected from each group and intraperitoneally injected with 8 IU of PMSG. 48 hours later, 8 IU of HCG was intraperitoneally injected. The female mice were caged with male mice raised separately at a 1:1 ratio for synchronous estrus. After 12 hours of caged together, the formation of vulvar plugs was observed. The presence of vulvar plugs indicated successful mating. The number of mice with successful pregnancy in the positive control group, PBS control group, MOMP+OmcB group, CPAF group, incA group, and Mixture group was 11, 10, 12, 11, 9, and 32, respectively.

[0068] The virus challenge was carried out on the 12th day after mating. Specifically, the positive control group was intraperitoneally injected with 100 μL of chicken embryo yolk sac membrane formaldehyde inactivated vaccine (3.2×10 3 IFU); PBS control group intraperitoneally injected 50 μL PBS and 50 μL adjuvant; MOMP+OmcB group intraperitoneally injected 30 μg OmcB+MOMP vaccine obtained in Example 7; CPAF group intraperitoneally injected 30 μg CPAF vaccine obtained in Example 5; incA group intraperitoneally injected 30 μg incA vaccine obtained in Example 6; Mixture group intraperitoneally injected 30 μg Mixture vaccine obtained in Example 8. Pathological tissue sections were observed on the 7th day after immunization. The results are as follows Figure 2 The production status of mice 5 days after inoculation is shown in Table 2.

[0069] Table 2 Mouse production

[0070]

[0071] according to Figure 2 As can be seen from Table 2, the tissue structure of mice at the inoculation site was intact, and no inflammatory infiltration occurred, indicating that the vaccine was safe; the normal production of mice in the MOMP+OmcB group, CPAF group, incA group and Mixture group was higher than that in the PBS treatment group, especially the normal production rate of mice in the Mixture group was 73%, significantly higher than 22% of the negative control group, better than the positive control, and 11 pregnant mice had good appetite and mental state after being vaccinated with the vaccine, and no adverse reactions occurred at the inoculation site. The subunit vaccine obtained by mixing 4 proteins in the present invention can reduce the abortion rate of mice and has a certain protective effect on pregnant mice.

[0072] Test Example 2

[0073] Samples were then taken and each bottle of the tested vaccine was inoculated into sheep to test the sheep's production performance.

[0074] (1) Experimental grouping: A total of 50 sheep aged 8 to 10 months without Chlamydophila abortus, Toxoplasma gondii and Brucella infection were tested using commercial Chlamydophila abortus Indirect Multi-species ELISA kit, Toxoplasma gondii IHA test kit and Brucella bengalensis plate test. The sheep were randomly divided into three groups: Group 1 (Vaccinated) consisted of 20 sheep in the immunized group, Group 2 (Control) consisted of 15 sheep in the non-immunized group, and Group 3 was a blank control group (Sentinel) consisting of 15 sheep. The ewes with congested vaginal mucosa and cervical opening were selected using B-ultrasound. On the 0th day, the progesterone vaginal plug was placed deep in the cervix of the ewe's vagina. The vaginal sponge plug was removed 12 days later. PMSG300IU was injected into the left neck muscle of each ewe. On the 14th day, the breeding rams were semen collected. The fresh goat milk was cooled to 40℃ after 10 minutes of 95℃ water bath. The semen was diluted and then inserted into the external opening of the cervix by about 1-2cm through the insemination device to start insemination. The pregnancy was checked by B-ultrasound instrument 48 days after mating. The pregnancy of sheep was checked by veterinary B-ultrasound instrument 48 days after the synchronous estrus mating. The number of sheep that successfully became pregnant in the immune challenge group, non-immune challenge group and blank control group was 13, 10 and 11 respectively. (2) Experimental method: On the 60th day after mating, each pregnant sheep in the immune challenge group was injected with the Mixture vaccine obtained in Example 8 into the left neck muscle, with a challenge dose of 400 μg; each pregnant sheep in the non-immune challenge group was injected with 500 μL PBS and 500 μL 201 adjuvant into the left neck muscle; the blank control group was not injected with any preparation. The production situation was observed every day starting from the first day after the virus attack. The results showed that the normal production rate of the non-immune virus attack group was 0%, and the abortion rate was 100%. One sheep aborted on the 82nd day of pregnancy, one sheep aborted on the 107th day of pregnancy, and a total of 6 sheep aborted from the 125th to the 135th day of pregnancy. One sheep gave birth to a weak fetus on the 142nd day of pregnancy, which died 35 hours later, and one sheep gave birth to a weak fetus on the 146th day of pregnancy, which died 12 hours later; the normal production rate of the immune virus attack group was 61.5%, and the abortion rate was 38.5%. One sheep aborted on the 104th day of pregnancy, and 4 sheep aborted from the 125th to the 140th day of pregnancy, and the rest gave birth normally; all 11 sheep in the blank control group gave birth normally from the 143rd to the 149th day of pregnancy.

[0075] The subunit vaccine obtained in Example 8 of the present invention has a good immune effect. All the immunized groups produce high levels of specific antibodies, which can effectively eliminate the number of chlamydia in the body and reduce pathological damage to placental tissue. The abortion protection rate is 61.5%.

[0076] The above examples show that the subunit vaccine of the present invention can have a good immune effect, is highly safe, can produce a high level of antibodies, and can prevent miscarriage.

[0077] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention. Sequence Listing <110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences <120> A subunit vaccine for animal abortion chlamydia and its preparation method and application <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <400> 1 aaagatatca tgaaaaaact cttgaaatcg gc 32 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <400> 2 aaactcgagt tagaatctga attgagcatt ca 32 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <400> 3 gaacatatga agctaaaaca aattacagtc t 31 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <400> 4 gtactcgagc gactcagtga ttttgcctgc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 gggtttcata tgacatcgac ggtagaatct 30 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <400> 6 cccaaagtcg acctggtcat atattgggaa g 31 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <400> 7 aaagatatca tgtccaaact catcagacg 29 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <400> 8 aaactcgagt taataaacgt gcgtattttc t 31

Claims

1. An animal abortion Chlamydia subunit vaccine, characterized in that: Includes Chlamydia abortus antigen and adjuvant; The Chlamydia abortus antigens include MOMP, OmcB, incA and CPAF; The mass ratio of MOMP, OmcB, incA and CPAF is 1:1:1:1; The adjuvants include MONTANIDE TM ISA 201.

2. The subunit vaccine according to claim 1, characterized in that The subunit vaccine is in the form of water-in-oil-in-water.

3. The method for preparing the subunit vaccine according to claim 1 or 2, characterized in that: The steps include: Chlamydia abortion antigen MOMP, Chlamydia abortion antigen OmcB, Chlamydia abortion antigen incA and Chlamydia abortion antigen CPAF are purified respectively and then mixed to obtain purified proteins; The purified protein is mixed with an adjuvant and emulsified to obtain the subunit vaccine.

4. The preparation method according to claim 3, characterized in that: The volume ratio of the purified protein to the adjuvant is 1:

1.

5. The preparation method according to claim 3, characterized in that: The emulsification is carried out at 500 r / min; The emulsification time is 5 to 10 minutes.

6. The preparation method according to claim 3, characterized in that: The expression step comprises: constructing a recombinant plasmid containing the abortion Chlamydia antigen encoding gene, transforming it into Escherichia coli, and culturing it until the absorbance OD 600 =0.6, add protein inducer, continue culturing for 3-5 hours, and obtain the Chlamydia abortus antigen.

7. The preparation method according to claim 6, characterized in that: The protein inducer includes IPTG; the working concentration of the IPTG is 0.8 mM.

8. The preparation method according to claim 3, characterized in that: The purification method includes gradient elution.

9. Use of the subunit vaccine according to claim 1 or 2 or the subunit vaccine obtained by the preparation method according to any one of claims 3 to 8 in the preparation of a medicament for preventing and / or treating diseases caused by Chlamydia abortus.

Images