Expression And Immunogenicity Assay Of Transferrin Binding Protein Of Pasteurella Multocida

Pasteurella multocida(Pm) is an antigen causing a range of infectious disease in wild and domesticated animals, such as cattle, pigs, poultry, and rabbits. The infection usually shows hemorrhagic septicemia and respiratory infection, which affects the growth of domestic animals and remains a significant threat to the farming industry. Currently,using vaccinations is the main method to prevent P. multocida prevalence, including inactivated vaccine, attenuated vaccine and subunit vaccine.Iron plays an important role in electron transfer of microbial metabolic processes.Many different ferroproteins can be found in animals, such as transferrin in blood,secretions of lactoferrin and intracellular ferritin, however, bacteria can not use the Iron from these ferroproteins directly, thus the majority bacteria generated a mechanism that can help them to absorb iron from their host, including Pasteurella multocida which encodeds several membrane protein. For example, bacterial transferrin binding protein can regulate bacterial to absorb iron from host which can be divided into two protein subsets: Tbp A and Tbp B. Recent studies have reported that Tbp A protein of bovine Pm can mediate cattle transferrin absorption effectively, while the Tbp B protein is not included in this process.In this study, the type A bovine Pasteurella multocide full-length Tbp A fragment were cloned and expressed, and protection of the Tbp A were explored by immunising mice. Afterwards, we would also like to study whether the Haemophilus parasuis and Actinobacillus pleuropheumoniae Tbp A protein have the cross-link protecitive immunity.So as to lay a foundation for the development of vaccine for the prevention of the subunit of the disease.The specific research contents are as follows:1. Clone expression of Pm-Tbp A, HPS-Tbp A, and APP-Tbp A proteinSpecific primer pairs of Pm-Tbp A, HPS-Tbp A and APP-Tbp A were designed according to the Pasteurella multocide HB01 genome, Haemophilus parasuis SH0165 genome and Actinobacillus pleuropheumoniae JL01 genome respectively. Tbp A fragment were amplified by PCR using HB01, SH0165 and APP4074 bacterial genome as templates, and recombinant the p ET-Pm-tbp A, p ET-HPS-tbp A, p ET-APP-tbp A vector were constructed. These successful vector were transformed into prokaryotic expressd strain BL21, which were induced at 37 ℃ by 0.8mmol/L IPTG for 3 hours, and these protein were confirmed expressed successfully by SDS-PAGE. Afterwards, the largenumber of protein were expressed and purified successfully, achieving the final concentration of Pm-Tbp A: 1.2mg/m L, HPS-Tbp A : 0.8mg/m L, APP-Tbp A :0.75mg/m L.2. Evaluation the immunogenicity of Pm-Tbp A protein in mice100μg aliquot of Pm-Tbp A was applied to immunise mice in experiment group and PBS was applied to immunise mice in control group mice, After the 14 day of the second immunization, we challenged the mice with 5LD50 HB01, and then tested the bacteria counts of the lung and blood after 15 hours. The results showed that bacteria counts in the immunized mice were 107 CFU/g in lunge and 2x107CFU/ml in blood, meanwhile, the control group showed that the bacteria concentration in lunge and blood were2x108CFU/g and 108CFU/ml, respectively. Compared with the control group, the bacteria numbers of immunized mice decreased dramatically by 20 times in lunge and 5 times in blood. The protective ability of Pm-Tbp A protein was determined, and the results showed that the protection was 70%, which proved that the Pm-Tbp A protein has a good protection3. Evaluation the cross-immunity protection of three kinds of bacteria after Tbp A protein immunization in mice Based on the good protective ability of Pm-Tbp A protein, the cross-immunity protection of Pm-Tbp A, HPS-Tbp A and APP-Tbp A proteins was also been tested. Pm-Tbp A,HPS-Tbp A and APP-Tbp A proteins were used to immunize mice separately, and A/B/C,G/H/I, D/E/F, The J/K/L group were named as the PBS control group. After twice immunized, A, D, G, J group with 5LD50 HB01; B, E, H, K group with 5LD50 SH0165 were Challenged; and C, F, I, L group with 5LD50 APP4074 were Challenged. The clinical symptoms, the number of death and lesions were recorded for 72 hours continuous observation used for pathological observation analysis. In the course of immunization in 0,14, 28 days, blood samples were collected from tail vein, and antibody level was detected.The results showed that the protection rate of three different protein from different strains were all 67%, HPS-Tbp A, APP-Tbp A protein group of mutual cross protection rate is50%, and the HB01 protection is weak, only 16.7% and 33%. The Lung tissue group appearance observation showed that bleeding phenomenon of survivors vaccinated mice pulmonary edema is lighter, while the control group showed serious edema and bleeding phenomenon; the pathological sections of control group displayed that the alveolar septum rupture and hemorrhage seriously, while alveolar of immune group damage less,and which only slightly congestion is observed. These results showed that the threeproteins had certain protective effect,tough had different degrees of cross protection.