Cloning And Expression The PlpE Gene Of Cattle Pasteurella Multocida Strain CQ2and Study On The DNA Vaccine

Pasteurellosis, which pathogen is pasteurella mutocida, is a kind of infectious diseases in animals and humans. Cattel pasteurellosis has a short incubation period, a fast spreading and a high morality and often causes major economic loss in cattle industry. Inactivated vaccines and attenuated vaccines are used to prevent pasteurellosis at present and play a certain positive role. But these traditional vaccines usually only have protection to the same serotype strains and attenuated vaccines could pose a security risk. People want to work out a new vaccine which is safe and has a cross protection. Our laboratory study showed that the plpE gene of Multocida Pasteurella has a high conservative. Literature reported both at home and abroad that the PlpE protein may be a cross protective antigen. This study focused on cloning, expression the plpE gene of Pm CQ2and the DNA vaccine, which were summarized as follows.1. Cloning, Expression the plpE Gene of CQ2and Anslysis of Immunological Activity of the Expressing ProteinsThe plpE gene (signal peptide removal) of cattle A-Pm CQ2was amplified and named el. Gene sequencing found385-387of el was TAA (termination codon) prompting expression of the plpE gene may intercept. DNA fragment el was divided into first half (384bp) and second half (564bp) because of TAA. The second half was amplified and named e2. Fusion gene of first half and second half was amplified and named e3.Recombinant expression plasmids pET30a(+)/el, pET30a(+)/e2and pET30a(+)/e3 were constructed and transformed into E.coli BL21(DE3) competent cells. After induction by IPTG,24kD,28kD and44kD recombinant proteins were expressed, which were in line with the expected values and named E1, E2and E3. Western blot analysis revealed that E1and E3have immunological activity, but E2have no reaction with cattle antisera by the challenge of CQ2. Western blot analysis revealed that E2have immunological activity but have no reaction with cattle antisera by the challenge of CQ2. Therefore, expression of the plpE gene of CQ2is intercepted.Similarity analysis of the plpE gene of CQ2and other Pm strains revealed that only CQ1and CQ2have intercepted termination codon, which were isolated from the same specimen. Intercepted expression of the plpE gene of CQ2is a special case, not universality.2. Immunogenicity of the PlpE Protein in MiceSome mice were immunized with E1and E3. Detection of antibody by ELISA found that antibody levels of the two immunity groups are significantly higher than the control groups and the two immunity groups have no significant differences. Mice were challenged with injection of cattle A-Pm CQ2, CQ6, B-Pm and avian A-Pm. The reasults showed E1and E3can’t protect mice against attack of avian A-Pm, but are able to provide some protection for the other three types strains. The protection of E1to CQ2, CQ6and B-Pm were42.9%,57.1%and28.6%, and E3were57.1%,71.4%and14.3%.3. Study on the DNA VaccineEukaryotic expression plasmids pcDNA3.1(+)/el and pcDNA3.1(+)/e3were constructed. Some mice were immunized with pcDNA3.1(+)/el and. Detection of antibody by ELISA found that antibody levels of the two immunity groups are significantly higher than the control groups and the pcDNA3.1(+)/e3group is significantly higher than the pcDNA3.1(+)/el group and the two protein groups are significantly higher than the two DNA vaccine groups. Mice were challenged with injection of cattle A-Pm CQ2, CQ6, B-Pm and avian A-Pw.The reasults showed the two DNA vaccines can’t protect mice against attack of cattle B-Pm and avian A-Pm, but are able to provide some protection for CQ2and CQ6. The protection of pcDNA3.1(+)/el to CQ2and CQ6were28.6%and57.1%, and pcDNA3.1(+)/e3were28.6%and42.9%.Mice survival curves were drawn. Judging from the trends of the curves, four vaccine groups declined more slowly than the control groups. The results showed that four vaccines have some protective effect against four strains and from strongest to weakest in the following order:E3> E1> pcDNA3.1(+)/e1>pcDNA3.1(+)/e3.This order is basically the same as the antibody levels, describes the proteins have a higher protection than the DNA vaccines and the complete protein have a higher protection than the truncated protein.