Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig

Development and Application of Novel Vaccine Against Mycoplasmal Pneumonia of Swine and Monoclonal Antibody

Author MaFengYing
Tutor HeQiGai
School Huazhong Agricultural University
Course Preventive Veterinary Medicine
Keywords Swine mycoplasma pneumonia Subunit vaccine Genetically engineered live vaccine Monoclonal antibodies Immunohistochemistry
CLC S858.28
Type Master's thesis
Year 2011
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Swine mycoplasma pneumonia, also known as swine asthma or swine enzootic pneumonia, widely distributed, is characterized by a chronic, highly contagious, infectious, high morbidity and low mortality. Commercialization prevent swine mycoplasma pneumonia vaccine rabbits attenuated, inactivated vaccine and subunit vaccine. Existing vaccine, there is the high cost, the immune pathway inconvenience ineffective immune defects. The novel vaccine subunit vaccine as well as the development of genetically engineered live vaccine has become an ideal research directions. The M. hyopneumoniae p36, p46, p65, p97, and Nrdf gene is the protective antigen, is excellent vaccine candidate antigen. This study through genetic engineering-related technology, a large number to get swine pneumonia mycoplasma p36, p46, of p65 and p97R1-Nrdf1 recombinant protein, which prepared the corresponding swine mycoplasma pneumonia sub-unit vaccines, in order to solve the traditional sub-unit vaccine antigen preparation difficult the problem . Recent studies have confirmed that the attenuated Salmonella vaccine vector has effectively presenting antigen to induce cellular and mucosal immunity, low cost, and side effects is low. This study intends the antigen gene p36, p46, p65 and p97R1-Nrdf clone to prokaryotic expression plasmid with no resistance, and successfully built effective expression of exogenous antigen non-resistant recombinant attenuated Salmonella choleraesuis, and recombinant strains preliminary evaluation, and laid the foundation for the prevention and treatment of swine mycoplasma pneumonia. Currently, the diagnostic methods for the detection of Mycoplasma hyopneumoniae isolation and culture is difficult, and take long, and the detection rate is low; indirect hemagglutination antigen hard to save; PCR technical conditions prone to false negative and false positive testing costs; ELISA Mycoplasma hyopneumoniae and Mycoplasma hyorhinis flocculent Mycoplasma common antigen, leading to the increase in non-specific cross-reactivity. While mAb can specifically enhance the antigen-antibody reaction, so that decrease in cross-reaction, thereby to ensure the accuracy of test results. The main contents are as follows: 1 of Mycoplasma hyopneumoniae subunit vaccine known primers were designed according to the Gene-Bank, amplification of p46 and p65 protein gene, the fragment was cloned into the expression plasmid pGEX-KG recombinant plasmid pGEX -46 and pGEX-65. Mycoplasma hyopneumoniae immunogen membrane proteins p46 and p65 by site-directed mutagenesis and recombinant expression. The combination of p36 protein the construct expression before the p46 and p65 protein laboratory, which prepared the the corresponding swine mycoplasma pneumonia subunit vaccine. In order to further improve the effect of swine mycoplasma pneumonia subunit vaccine, this test will series recombinant expression p97R1-Nrdf protein and P36, P46 and p65 protein composition together to prepare another five kinds of protein-containing swine mycoplasma pneumonia alkylene subunit vaccine, in order to achieve a better immune effects. Set up group Ⅰ recombinant proteins p36, p46 and p65, p36, p46, p65 and p97R1-Nrdf group Ⅱ, swine mycoplasma vaccine safety one hundred grams (M PAC) for the test group Ⅲ, PBS adjuvant blank control group immunization of BALB / c mice. Swine Mycoplasma pneumoniae antibody detection in serum, lungs, and by the protein stimulates the spleen lymphocytes, IFN-γ and IL-4. Test results: Mycoplasma hyopneumoniae antibody levels of the test group II was significantly higher than the test group Ⅰ, Ⅲ group (P lt; 0.01); the group Ⅱ IFN-y levels significantly higher than the test group Ⅲ and group Ⅱ Ⅰ group, the test group Ⅰ and Ⅲ group of IFN-y levels had no significant significant difference (P lt; 0.05); group Ⅰ, group Ⅱ, Ⅲ group of IL-4 level difference is not significant with (P lt; 0.05) but are very significantly higher (P lt; 0.01); Mycoplasma hyopneumoniae antibodies, IFN-y and IL-4 levels in the lung test results presented consistency for group Ⅱ gt; test group Ⅰ gt; trial Ⅲ group gt; control group; M. hyopneumoniae antibodies and IFN-y the highest level of the test group II test results compared with stimulated spleen lymphocytes, IL-4 levels for the group Ⅲ highest. Thus, the proteins expressed by E. coli having a good immunogenicity, mice can be induced to produce a better immune effects, the results show two subunit vaccines at the same time activate the humoral immune activation cell immune. Among them, the swine mycoplasma pneumonia group Ⅱ humoral and cellular immune pathways generated the highest antibody levels; test group Ⅰ able to achieve through these two routes of immunization with the the group Ⅲ same immune effect. 2 the Mycoplasma hyopneumoniae genetically engineered live vaccine based on the known Gene-Bank primers were designed using molecular cloning techniques will be able to prokaryotic expression of Mycoplasma hyopneumoniae p46 and p65 gene was cloned into the expression plasmid pYA3493 recombinant plasmid the pYA-46 and pYA-65. The recombinant plasmid pYA3493 electrically the transferred the asd gene deletion strains of Salmonella choleraesuis C500-preparation of the recombinant strain C46 (pYA-46) and C65 (pYA-65) and the empty vector control strain CpYA (pYA3493). Western-blot results showed that p46 and p65 gene expression in recombinant strains. Research results show that the recombinant strain C46 (pYA-46) and C65 (pYA-65) compared with the parental strain C500, biochemical and growth characteristics is not changed, the insertion of exogenous genes capable of secreting the expression also exist stably. The recombinant strain C36 the recombinant strains C46 and C65 and experimental smothering built combination preparation of genetically engineered live vaccine (pYA-36), and at the same time and then be prepared in conjunction with the C97R1-Nrdf (pYA-97R1-Nrdf) another expression immunogenicity protein gene engineering seedlings, and to mice as animal model to evaluate the recombinant strain in the immunogenicity of the oral, intramuscular, two different routes of immunization. The vaccine immunogenicity results showed that mice: oral C36 C46 C65 C97R1 Mycoplasma hyopneumoniae antibody-Nrdf group extremely significantly higher than C36 C46 C65 group oral and intramuscular merchandise vaccine group (P lt; 0.01), but with the intramuscular injection of C36 For C46 C65 group, no significant differences (P gt; 0.05); IFN-γ compared with intramuscular C36 C46 C65 group was significantly higher than the the intramuscular commodity vaccine group (P lt; 0.05), with the oral group C36 C46 C65 or C36 C46 C65 between C97R1-Nrdf group differences were not significant (P gt; 0.05); export clothing IL-4 levels are showing C36 C46 C65 group gt; oral C36 C46 C65 C97R1-Nrdf group gt; the intramuscular merchandise vaccine group gt; intramuscular injection of C36 C46 C65 group, but the differences between the groups are not significant (P gt; 0.05). Control group swine Mycoplasma pneumoniae antibody, IFN-y, and of IL-4 with the differences in the experimental group was significantly (P lt; 0.01). Thus, the constructed expression the Mycoplasma hyopneumoniae immunogenicity gene recombinant Salmonella, mice with better immunogenicity, and the use of intramuscular its better immunogenicity, is expected to develop into swine mycoplasma pneumonia genetic engineering vaccine. Anti-of M. hyopneumoniae p36 protein monoclonal antibody preparation and application of E. coli containing pGEX-36 plasmid constructed in our lab by IPTG induction, glutathione agarose gel purification of soluble proteins, fusion proteins GST-36. The recombinant protein to take conventional of subcutaneous immune and abdominal strengthening the immune combination immunized BALB / c mice. SP2 / 0 myeloma cells are fused with immune spleen cells of mice by ELISA screening, five times the limiting dilution method and cloning to obtain 19 of hybridoma anti-Mycoplasma hyopneumoniae p36 protein. Western-blot results showed that monoclonal antibodies react specifically with the p36 protein. The titer of the culture supernatant by the cells was detected by most 1:25600 to 1:51200 the mouse ascites titer 1:13107200 to 1:52428800; ISOTYPE were IgG2b and IgG1, light chain K type. Of monoclonal antibodies for accurate and specific immunohistochemical methods for diagnosis of Mycoplasma hyopneumoniae protection. The preparation of monoclonal antibodies against Mycoplasma hyopneumoniae p36 protein as the primary antibody, to establish monoclonal antibody immunohistochemical methods used to detect blank challenged group, negative control group, group, and clinically suspected cases of lung tissue. The test results show that the immunohistochemical method accurate positioning and lower costs, more than the PCR method accuracy. Further confirm the anti-Mycoplasma hyopneumoniae p36 protein monoclonal antibody obtained in this test with high specificity, so that the antigen-specific antibody response, and to reduce cross-reactivity, and the test results are more accurate and specific.

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