Prokaryotic Expression And Immunological Characteristics Study Of The Lipoprotein LppA From Mycoplasma Mycoides Subsp.Capri Of PG₃

The Office International des Epizoofies classify Contagious Caprine Pleuropneumoni-a(CCPP) is the B list of communicable animal disease,and is an acute or chronic severe contagius respiratory disease of goats.In this experiment we cultivated the Mycoplasma mycoides subsp.Capri of PG3in abundance after resurrection,then we extracted its genome.And with it as a template, used a pair of specific primer (the sites of enzyme BamH I and Xho I were added to the two primers respectively) amplification LppA gene.LppA gene has a TGA codon which is encoded tryptophan in Mycoplasma but as terminator codon in prokayotic expression,so TGA was mutated to TGG by site-directed mutagenes,after we builded the cloned plasmid.Then constructed expression recombinant plasmid of pET-32a-LppA by estriction enzyme digestion of prokaryotic expression vector of pET-32a(+) and cloned plasmid which contained LppA gene. Then expression recombinant plasmid was transferred in to host bacterium and induced to expression,The recombinant protein were properly expressed just as expected, and SDS-PAGE showed that the target protein, s molecular weights is64kDa.Then purified the recombinant protein and was immunogenic by Western blot analysis.Ultimate, immunized some mice for three times with purified recombinant proteins and collected serum.The LppA antibody of serum of vaccinated mice were tested by the methods of ELISA,the results showed that LppA antibodies were detected in the serum of mice in every immunized three times. The serum antibody of purified recombinant proteins immunization group was highly significantly different from that of the blank control group(P<0.01).By opsonophagocytic assay to text whether the whole blood can enhance opsonophagocytic function,the results showed that the whole blood of purified recombinant protein has an extremely significant difference compared with the blank control group(P<0.01).The above results showed that,This study successfully mutated TGA to TGG and cloned of LppA gene from Mycoplasma mycoides subsp.Capri of PG3,and successfully constructed recombinant expression plasmid PET-32a-LppA, after successfully expressed and purified recombinant protein,using immunological analysis shows that the recombinant protein has a good reactogenicity and immunogenicity. This study provided the evidence for genetically-engineered subunit vaccine of prevention of CCPP.