Development And Application Of Novel Vaccine Against Mycoplasmal Pneumonia Of Swine And Monoclonal Antibody

Mycoplasma hyopneumoniae also known as pig enzootic asthma or pneumonia is widely distributed around the world, whose characteristic is chronic, highly contagious, infectious, high morbidity and low mortality. Nowadays, prevention of Mycoplasmal pneumonia of swine inland and international is mainly by using attenuated vaccine, inactivated vaccine and gene engineering vaccine. But these vaccines have a lot of disadvantages such as high cost, side effects, inconvenience immunization, poorly immune defects. New vaccines which are subunit vaccine and live genetic engineering vaccine have become the research directions. Research showed that p36, p46, p65, p97, and Nrdf are the protection antigens of Mycoplasma hyopneumoniae and good candidate antigen in vaccine. This research acquired a huge number of recombinant proteins which are p36, p46, p65 and p97R1-Nrdf of Mycoplasma hyopneumoniae by the genetic engineering technologies. Subunit vaccine against Mycoplasmal pneumonia of swine was developed by these proteins, which is hoping to solve the problem that the preparation of antigens in traditional of subunit vaccine was difficult. Recent studies have confirmed attenuated Salmonella as a vaccine vector has benefits like effectively presenting antigens to induce mucosal immune cells and low cost, low side effects. This study intended to clone antigen genes p36, p46, p65 and p97R1-Nrdf into prokaryotic expression plasmid without resistance to construct the attenuated Salmonella choleraesuis without resistance which could successfully express the exogenous antigens. Evaluated the recombinant strains and made sense to the prevention of Mycoplasma hyopneumoniae. Currently, detection of Mycoplasma hyopneumoniae for the diagnosis is difficult to separate in culture with long time and low detected rate; indirect hemagglutination test have the problem that saving the antigen is difficult; PCR technical demanding high conditions but prone to false negative and false positive and has a high cost. Beause Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculent have common antigens which cause cross reaction in ELISA that has non-specific reaction by using antiserum. Beause the monoclonal antibody is specificity that the antigen-antibody reaction is also specificity, so we can get more accurate results by reducing cross-reaction. 1. The research of subunit vaccine against Mycoplasmal Pneumonia of swineP46 and p65 gene were amplified by the primers desined according to the known sequences in Gene-bank and then cloned into the expression vector pGEX-KG to construct the recombinant plasmid pGEX-46 and pGEX-65. Using site-directed mutagenesis and restructuring expression techniques to obtain the major Mycoplasma hyopneumoniae immunogen membrane protein p46 and p65. Make p46 and p65 protein together with p36 protein which was expression before in our laboratory to prepare the subunit vaccine against Mycoplasmal pneumonia of swine in order to solve the problem that preparing the antigen in the traditional of subunit vaccine.In order to enhance the immune effect of subunit vaccine, the test also make the recombinant p97Rl-Nrdf protein with p36, p46 and p65 protein together to prepared anther subunit vaccine which contained five proteins. Using the protein of p36^ p46 and p65 as group I, that of p36, p46, p65 and p97Rl-Nrdf as group II, Using the Mycoplasma hyopneumoniae bacterin (M+PAC) as group III, PBS added with adjuvants as the control group to immunize BALB/c mice. Detect serum, lungs and the splenic lymphocytes which is stimulated by protein with Mycoplasma pneumoniae antibody, IFN-yand IL-4. The results suggested that group II induced significantly higher antibody than group I and group III(P<0.01) and also induced significantly higher IFN-y production than group III (P<0.05), however, there were no significant differences between group II and group I or group I and group III (P<0.05). IL-4 production was similar among the three group (P<0.05), but was obviously higher than the control group (P<0.01). The antibody level of MP antibody, IFN-y and IL-4, which detected in lung, was group II, group I, group III and the control group in proper order. The splenic lymphocytes results showed that the highest MP antibody and IFN-y level were induced by group II, while the highest IL-4 production was induced by group III. The results showed that the protein expressed by E.coli has good immunogenicity and could induce better immunity in mice. The two subunit vaccines activated both humoral immune and cellular immunity. It shows that the recombinant subunit vaccine containing p36, p46, p65 and p97Rl-Nrdf provided the highest antibody level by simultaneously stimulating humoral immunity and cell immunity. Group I provided almost the same significantly immunologic efficacy with group III in the same way. 2. Genetic engineering vaccine against Mycoplasmal Pneumonia of swinePrimers were designed according to the known sequences in Gene-bank. P46 and p65 gene were amplified and subcloned into pYA3493 which is prokaryotic expression plasmid by using molecular cloning technology to construct the recombinant plasmid pYA-46 and pYA-65. pYA3493 and recombinant plasmids were respectively electroporated into C500 which delete asd gene, resulting in the recombinant Salmonella strain C46(pYA-46), C65(pYA-65) and the empty vector strain CpYA(pYA3493). The expression of proteins were analyzed by Western-blot. The results showed that the recombinant strain C46 and C65 not only retained the parental phenotypic characteristics of strain C500, and can stabilize the expression of genetic and secretion of foreign proteins, their biochemical, growth characteristic and gene were stability. The recombinant strains C46 and C65 with C36 were prepared the corresponding genetic engineering vaccine against Mycoplasmal pneumonia of swine. At the same time combined the C97R1-Nrdf to make another genetic engineering vaccine. The results of the immunogenicity in mice indicated that the group orally immunized with C36, C46, C65, C97R1-Nrdf showed significantly higher Mycoplasma pneumoniae antibody than both the group orally immunized with C36, C46, C65 and the group intramuscular injected with the Mycoplasma hyopneumoniae bacterin (M+PAC) (P<0.01). The group intramuscular injected with C36, C46, C65 showed higher IFN-y production than the group which was injected with the (M+PAC) (P<0.05), but there was no significant difference between the group orally immunized with C36, C46, C65 and the group orally immunized with C36, C46, C65, C97R1-Nrdf (P>0.05). It showed that the highest level of IL-4 was the group orally immunized with C36, C46, C65, higher levels of IL-4 was observed in the group orally immunized with C36, C46, C65, C97R1-Nrdf than the the group injected with the (M+PAC) and the lowest IL-4 level was the group injected with C36, C46, C65. But there were no significant differences among them (P>0.05). The Mycoplasma pneumoniae antibody, IFN-y or IL-4 production of the earch treatment group was obviously higher than the every control group (P<0.01). The Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae which has immunogenicity in mice especially by intramuscular injection could probably serve as a vaccine against mycoplasmal pneumonia of swine. 3. Development and application of Monoclonal Antibody against p36After the recombinant pGEX-36 vector containing the E.coli was induced by IPTG, the soluble GST-p36 protein was purified by glutathione sepharose from the supernatant. Purified the recombinant protein as immunogen for routine immunization and intraperitoneal immunization as the way to strengthen the immune BALB/c mice, spleen cells with SP2/0 myeloma cell fusion by an indirect enzyme-linked immunosorbent assay (ELISA) screening and cloning by limited dilution. After five times cloning got 19 hybridoma monoclonal antibody against p36. Western-blot showed that monoclonal antibody reacted specifically with p36 protein. The supernatants titer were 1:25600 to 1:51200, and ascites titer were 1:13107200 to 1:52428800 by ELISA. The type of antibody subclasses were IgG2b and IgG1,κlight chain. Monoclonal antibody obtained for further study of p36 gene function and the establishment of accurate and specific diagnosis of Mycoplasma hyopneumoniae approach provides a powerful means. Using monoclonal antibody against p36 as primary antibody to established monoclonal antibody immunohistochemistry for the detection of the lung tissue of the blank group challanged with Mhp, the control group and attack the clinical suspected cases. The results showed that the immunohistochemical method was more accurate than the PCR method, which can position accurate with lower cost. It confirmed by the test that the monoclonal antibodies against p36 have high specificity, which course the antigen-specific antibody response and reduce the cross-reaction. So the results are more accurate and specific.