Cascade Connection And Prokaryotic Expression Of ChIL-2 Gene And Mycoplasma Gallisepticum H3 Strain TM-1 Gene

In order to further research DNA vaccine of MG and improve its efficiency in immune response,we cloned chicken IL-2 gene by means of the gene manipulation technology and constructed recombinant plasmid containing TM-1-IL-2 fusion gene.These genes were well expressed in prokaryotic expression cells.Firstly,according to the ChIL-2 cDNA sequence registered in GenBank and multi-clone restriction enzyme sites of prokaryotic expression plasmid pET-30a(+),we designed one pairs of special primers for the ChIL-2 gene.To manupulate the cloning and expression of the ChIL-2 genes,corresponding endonuclesae sites,protective sites,linkers,inititation and termination codons were designed at the 5'terminal and 3'terminal of the PCR primers.By reverse transcriptase polymerase chain reaction (RT-PCR),the DNA fragment about 460bp was amplified from chicken spleen cells that were stimulated with ConA for 6 hours,furthermore,the fragment was inserted into pMD19-T vector and identified with PCR and restriction enzyme digestion,the positive recombinant clones was sequenced and analysed.it was the ChIL-2 cDNA sequence registered in GenBank.The results suggested that ChIL-2 gene was cloned successfully.Secondly,by the technology of DNA recombination,we linked TM-1 gene fragment with IL-2 fragment by the linker as a fusion gene and inserted it into EcoRI/HindIII sites of pET-30a(+) plasmid.The further results of PCR, restriction enzyme digestion and sequencing showed that a recombinant plasmid pET30-a-TM-1-IL-2 was constructed.Finally,we transformed the recombinant plasmid into E.coli.BL21(DE3).Under the induction of IPTG at 37℃for 4 hours,a 43000 fusion protein highly expressed in E.coli.BL21(DE3) and could be detected by SDS-PAGE.The expressed fusion protein was localized in inclusion bodies,which could be solubilized by supersonication with urea.The fusion protein was further purified.All should provide effective experimental foundation to further research bio-activity of ChIL-2 and MG.In conclusion,we successfully cloned MG TM-1 gene of H3 strain and chicken IL-2 gene and constructed the recombinant palsmids containing TM-1-IL-2 fusion gene,which were expressed in prokaryotic expression cells.All should provide effective experimental materials to further research MG DNA vaccine.