Cloning And Prokaryotic Expression Interleiukin-2 Receptor Gamma Chain (Il-2γ) Gene Of Rongchang Porcine

Based on porcine interleiukin-2 receptor gamma chain gene(pIL-2Rγ) sequence(GenBank accession number:NM 214083),a pair of specific primers was designed and synthesized.Meanwhile,total RNAs were isolated from Rongchang porcine peripheral blood lymphocytes stimulated with ConA.A fragment of about 1100bp DNA was amplified by reverse transcription polymerase chain reaction.The amplified fragment was cloned into vector PMD18-T and then sequenced.The result indicated that the cloned gene was a complete pIL-2Rγgene including ORF of 1107bp encoding 368 amino acids.Sequence comparison between acquired IL-2Rγgene sequences of Rongchang porcine and that of other breeds in NCBI indicates that homology are all more than 99%.The GenBank accession number of IL-2Rγgene sequence of Rongchang porcine are EU026383.According to Rongchang porcine IL-2Rγgene sequence,specific primers were designed and synthesized.Non-signal peptide gene sequence(IL-2Rγ_m) was subcloned by PCR amplifing with pMD18-T-R-IL-2Rγrecombinant plasmid as template and was inserted into prokaryotic expression vector pET32(a)+. pET32(a)-R-IL-2Rγ_m prokaryotic expression plasmid was constructed successfully. The expression plamids were transformed into E.coli BL21 and induced for 4h with 1.0mmol/L IPTG.SDS-PAGE and Western-Blotting analysis confirmed that the confluent protein expressed by pET32(a)-R-IL-2Rγ_m was 47KD in size.The yield of confluent protein accounted for 41.8%of the total bacterial protein.IL-2Rγ_s was inserted into eukaryotic expression vector pcDNA3.1(+) to construct eukaryotic expression plasmid of pcDNA3.1(+) -R-IL-2Rγ_s.The plasmid was vertifed by PCR and restriction enzyme digestion.The mices were immunized with the plasmid for three times.The amount variation of T-cell subgroup of mice was detected with flow cytometer.The result indicated that IL-2Rγhad the capability of stimulating immunocell multiplication.In this research,IL-2Rγgene sequence of Rongchang porcine was cloned by RT-PCR for first time in domestic.Prokaryotic expression system for expressing maturation protein of IL-2Rγwas established.Eukaryotic expression vector of IL-2Rγprotein for bulk preparation of the vector was constructed.The biological activity of IL-2Rγwas detected by immunized mice with itself.This work supported the advanced research on the structure,biological activity and mechanism of action with the rationale foundation.