Cloning And Prokaryotic Expression Interferon Gamma (IFN-γ)gene Of Rongchang And Neijiang Porcine

Based on porcine interferon-gamma gene(PoIFN-γ) sequence(GenBank accessionnumber:S63967), a pair of specific primers was designed and synthesized. Meanwhile,total RNAs were isolated respectively from Rongchang and Neijiang porcine peripheralblood lymphocytes stimulated with ConA. A fragment of about 600bp was amplified byreverse transcription polymerase chain reaction. The amplified fragment was cloned intovector PMD18-T and then sequenced. The result indicates that the cloned gene was acomplete PoIFN-γgene including ORF of 501bp coding 166 amino acids. Sequencecomparison between acquired IFN-γgene sequences of rongchang and neijiang porcineand that of other breeds in NCBI indicates that homology are more than 99％. TheGenBank accession number of IFN-γgene sequence of Rongchang and Neijiang porcineare DQ 902588 and DQ913893.According to Rongchang porcine IFN-γgene sequence, specific primers weredesigned and synthesized, templated by pMD-R-IFN-γrecombinant plasmid. CompleteORFs of IFN-γgene sequence (IFN-γ_s) uncontainning signal peptide gene sequence(IFN-γ_m) were respectively subcloned, and inserted into prokaryotic expression vectorpET32(a)+. pET32(a)+R-IFN-γ_s,pET32(a)-R-IFN-γ_m prokaryotic expression plasmid weresuccessfully constructed. The expression plamids were transformed into E.coli BL21 andinduced for 4h with o.5mmol/L IPTG. SDS-PAGE and Western-Blotting analysisconfirmed that pET32(a)+R-IFN-γ_m expressed confluent protein was 36kd,with the yield accounting for 48.2％of the total bacterial protein. After dissolved into 0.3％SKL anddialyzed for renaturation, the confluent protein had bioactivity of promoting porcineperipheral blood lymphoblast proliferation.In addition, IFN-γ_s,IFN-γ_m was respectively inserted into prokaryotic expressionvector pBV220 constructing pBV220-R-IFN-γ_s, pBV220-R-IFN-γ_m prokaryotic expressionplasmid. Those expression plasmid were respectively transformed into E.coli DH5a, thenthe expression strains were induced for 5h under 42℃, SDS-PAGE confirmed that interestprotein was expressed by pBV220-R- IFN-γ_m strain and was 16kd in size. Its contentaccounted for 38.6％of the thalline protein.