Cloning And Expression Of The TBPB Gene Of Actinobacillus Pleuropneumoniae And The Protection Of The Recombinant Protein In Mice

Actinobacillus pleuropneumoniae is a porcine respiratory tract pathogen which causes disease ranging from peracute to chronic,with infected pigs typically showing a hemorrhagic,necrotizing pneumoniae often associated with fibrinous pleuritis.The pathogen encountered worldwide, and existed in china. It become the most important disease threat the pig industry, transferrin binding protein B, one of the most important virulence factor and required for persistence and multiplication of APP,have good immunogenicity and is the valid component of vaccine.1 Amplification an expression of tbpB gene of APP1 and analyse the immunoginity of the rTbpB protein.this study, complementary DNA corresponding to the entire sequence of tbpB gene of the shope strain of APP were cloned into vector pMD19-T simple vector after being amplified by polymerase chain reaction (PCR) method. Then tbpB gene was subcloned into prokaryotic vector pET-32a(+) to express recombinant TbpB protein for the purpose of studing the protection of the rTbpB to APP in mice. It plays important role in prevention of the disease.According to the nucleotide sequence of tbpB of APP1, a pairs of primes P1\P2 for tbpB gene were designed, to amplify the corresponding target gene After purified, the gene fragments were cloned into a vector pMD19-T simple vector. Positive clones were screened and identified by PCR and enzyme-digestion. The homology of tbpB gene to the reference strain were 99.72%.The tbpB gene contained in pMD19-T simple-tbpB were subloned into prokaryotic vector pET-32a(+) to construct a recombinant plasmid pET-32a(+)-tbpB. After recombinant plasmid pET-32a(+)-tbpB being proved to be successfully constructed by PCR and enzyme-digestion, it was transformed into the Escherichia Coli BL21(DE3) competent cells. Recombinant protein was expressed highly after induced by IPTG in BL21 (DE3). Sodium dodecyl sulphate-polyacrylamide gel (12%) electrophoresis(SDS-PAGE) analysis showed that the molecular weight of the fusion protein was approximately 84KD in agreement with the expect. Furthermore, the optimizing results of induction condition indicated that recombinant protein could be expressed most efficiently after being induced at 37℃for 1～3 hours under 0.2～0.8mM IPTG condition The orientation analysis demonstrated that the fusion protein was highly expressed in insoluble forms.The insoluble fusion protein was mainly accumulated as inclusion bodies which could be solubilized in 8M carbamide. Fusion protein was purified by Ni2+-imetal chelation affinity chromatography under denaturing conditions.The results of western blotting analysis demonstrated that the fusion protein possesed antigenic activity and could be specifically recognized by antiserum against APP1.2 Protection of rTbpB to APP in miceIn this study,mice were divided into three groups: rTbpB+ inactivate bacterin; rTbpB;passive control group. The adjuvant is the SDS-PAGE gel.Each group has 10 mice.The mice were vaccinated subcutaneously in o day, 14 days.The antiserum was taken before the day of the first vaccine and 14 days after each vaccine. And the antibody of ELISA-rTbpB is determinded. After 14 days of the second vaccine,the seroconversion rate of the TbpB+ inactivate bacterin,rTbpB group is 100% and 48%.On the same day,the mice were challenged with 1×LD50 dose of APP1,2,6 by peritoneal injection. The survival rate of the rTbpB+ inactivate bacterin, rTbpB and passive control group is 90%,87.5%,70%, 100%, 40%,37.5% and 10%,20%,20 %. The results indicate that rTbp can partly protect mice from the challenge of APP1,2,6.