| In recent years,a number of researchers pay attention to study on bacteria in vivo -expressed gene.Several studies have been reported that identified many genes of bacteria in vivo,including in vivo expression technology,signature-tagged mutagenesis(STM), and wholegenome expression profling,but little is reported on Pasteurella multocida of swine.In this study,we based on the reported some in vivo -expressed genes of Pasteurella multocida in chicken,identified two genes,pm0221 and pm1069,which were expressed in vivo in a septicemic mouse model by RT-PCR.The two prokaryotic expression vectors,pET-32a(+)-pm1069 and pET-32a(+)-pm221 were constructed.The prokaryotic expression of pm1069 recombinant and the immunological study on the expressed protein have been studied in the last..According to the 14 Pasteurella multocida genes of avian required for survival and expression in vivo,14 pairs of primers were designed,and also the infected swine Pasteu rella multocida models in septicemic mice were established.RNA was isolated from bacteria which were grew in vivo and in vitro by using Trizol reagent.Purified RNA was treated with Dnase,and then by using RT-PCR.Successfully,two genes,pm1069 and pm0221 were screened and identified which were expressed in vivo because of analysising of the results about agarose gel electrophoresis,that is,the electrophoresis purpose specific PCR bands could be detected if they can express in vivo,and could not be detected if they can not able to express in vitro.In this study,based on the results of identifing in vivo -expressed gene,genomic DNA as a template,the pm1069 and pm0221 amplified fragments were inserted into pMD18-T vector;their sequences compared with the pm1069 and pm0221 gene of pm70 which reported on the GenBank,analysis of the homology of 94.8%and 98.8%.After the relevant bioinformatics software analysis,its strong antigenic region was cloned and constructed recombinant prokaryotic expression vector pET-32a(+)-pm1069 and pET-32a(+)-pm0221. Recombinant plasmid,pET-32a(+)-pm1069,was transformed into E.coli strain Rosetta.In brief,E.coli strain Rosetta harboring the recombinant plasmid was cultured in TB medium at 30℃until absorbance at 600 nm reached 0.6.IPTG was added to a final concentration of 0.4mg/ml,and the culture was grown for another 4h.Successfully expressed recombinant pm1069 protein,63.0kDa,the protein mainly in the form of inclusion bodies.The recombinant pm1069 protein had a concentration ratio of the analysis,approximately 3.0 mg/ml,before were purified and ultrafiltered.Immuned health rabbit in accordance with the design of immunization procedures,preparation of antiserum.By using indirect ELISA method and titration side for the antiserum,selected the best coating concentration of antigen,at1:400(about 7.5ug/ml target protein) and the best anti-serum dilution,at1:1600.Western-Blot analysed for the purpose of protein, showed that the protein has antigenicity.The purified recombinant pm1069 protein,as envelope antigen,to test 20 negative serum diluted 1:1600 of rabbit by indirect ELISA.The average OD450 result of negative serum X is 0.072,standard deviation S is 0.006;X add to 3S is 0.090,as the positive deter -minant standard,to construct a method of indirect ELISA.The two groups of antiserum, which got from the immunized mice with live bacteria or dead bacteria respectively,tested by indirect ELISA.The results of OD450 all beyond 0.090 of group of live bacteria, oppositively,the OD450 of group of dead bacteria all under 0.090.The identifion of infection or immunity on porcine Pasteurella multocida can be determined by this indirect ELISA.At present,it has also not been reported that screened to identify in vivo -expressed gene of porcine Pasteurella multocida,this study was the fast reported in china that screened to identify porcine Pasteurella multocida in vivo -expressed gene by using RT-PCR technology in the level of nucleic acid,and good for building the way to identifying the infection or immunity on porcine Pasteurella multocida.
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