Expression And Immunogenicity Analysis Of Outer Membrane Protein Of Actinobacillus Pleuropneumoniae

1 Development of indirect ELISA for detecting antibodies against Apxâ…¡of ActinobaciIlus pIeuropeumoniaeThe expression plasmid pET-Apxâ…¡was transformed into E.coli BL₂₁(DE3).Clones of higher expression were selected to induce expression.An indirect enzyme-linked immunosorbent assay(ELISA) detecting antibodies against Actinobacillus pleuropneumoniae(App) was developed.Purified fusion protein was used as coating antigen.The test conditions were optimized by reagent titration utilizing known positive and negative sera.The ELISA was used to detect clinical sera.The results showed that the ELISA had good reliability,speciality and sensitivity.2 Cloning and expression of one section of omlA gene from Actinobacillus pleuropneumoniaeOne section of omlA gene of App amplified by PCR was cloned into pMD18-T vector,and transformed into host strain E.coil DH5a.The plasmid of positive clone was enzymolysized and sequenced.The result of sequencing showed that the consistency was 100%compared with the sequence of AB007572,AB007573 and AB007579 from GenBank.Then the target DNA was cloned into pET-32a vector.The recombinant plasmid was transformed into E.coli BL₂₁ The target protein with about 56kDa was induced by IPTG and detected by western blotting.3 Cloning and expression of one section of aopA gene from Actinobacillus pleuropneumoniaeOne section of aopA gene of App amplified by PCR was cloned into pMD18-T vector,and transformed into host strain E.coli DH5a.The plasmid of positive clone was enzymolysized and sequenced,the result of sequencing showed that the consistency was 99%compared with the sequence of U24492 from GenBank.Then the target DNA was cloned into pET-32a vector.The recombinant plasmid was transformed into E.coli BL₂₁ The target protein with about 69kDa was induced by IPTG and detected by western blotting.4 Immunogenicity analysis of outer membrane protein of Actinobacillus pleuropneumoniaeThe recombinant proteins rAopA,rOmlA1,rOmlA2 and rOmlA7 against App were obtained from prokaryotic expression system.The ICR mice were vaccinated subcutaneouly with the fusion protein above on day 0,14 and 28,respectively.The antibody titers of mice were examined every week.All mice were challenged intraperitoneally with serotype 1(2×10⁷CFU) and serotype 7(4×10⁸CFU) of App 35 day post-vaccination,respectively.The protective rates of experimental group against App(serotype 1 and serotype 7) were significantly higher than the control group.Among the experimental group,the protective rates of groupâ… (positive control) show lower than experimental group although it's antibody titer(anti-Apxâ…¡) is higher;And the protective rates of groupâ…¡I and groupâ…¤seem better than others.The results revealed that these outer membrane proteins of App play an important role in protect from attacking of App.