猪白细胞介素6和α干扰素基因的融合及其原核表达

Fusion and prokaryotic expression of interleukin-6 and interferon-α genes in swine

为了获得猪白细胞介素6(IL6)和α干扰素(IFNα)双重活性的融合蛋白,研究其作为免疫佐剂的可行性,利用IL6和IFNα基因的编码区序列以及原核表达载体pET32a序列,设计了带有限制性内切酶位点、Linker序列以及His标签的特异性引物扩增出猪IL6和IFNα的成熟肽基因序列,并分别与克隆载体pMD18连接后转化Esche-richia coli DH5α,提取质粒酶切并连接构建重组质粒pMD18-IL6-IFNα。将该重组质粒和表达载体pET32a同时酶切并连接构建pET32-IL6-IFNα重组质粒,依次转化E.coli DH5α和E.coli Rosetta(DE3)感受态细胞,并经不同终浓度的IPTG以及不同时间的诱导表达。结果成功构建了IL6和IFNα的融合表达载体,SDS-PAGE检测pET32-IL6-IFNα融合蛋白在E.coli Rosetta(DE3)中得到了较高表达,且经1.00 mmol/L IPTG诱导7.0 h后表达量最大,目的蛋白的相对分子量为44 960,与理论预期值一致。SDS-PAGE检测显示融合蛋白主要以不溶性的包涵体形式存在。

To achieve the fusion of interleukin-6/interferon-α （IL6/IFNα） genes and to investigate the feasibility of fusion protein as an immunoadjuvant, the coding sequences of IL6 and IFNα were cloned. Based on the coding sequences and the sequence of prokaryotic expression vector pE332a, the mature peptide sequences of IL6 and IFNcl were amplified using the specific primers designed with restriction enzyme sites, linker sequences and His tag and then transfected into Escherichia coli DI45α after linked with pMD18, respectively. The plasmids were extracted and digested by restriction enzyme respectively, and were linked together to construct the recombinant plasmid of pMD18-1L6-IFNcl. The plasmid was tranformed into E. coli DH5α and Rosetta（ DE3 ）, and then expressed by the induction of different concentrations of IPTG for different time. The results showed that the recombinant plasmid of pET32-1L6-1FNα was constructed successfully. SDS-PAGE revealed that recombinant fusion proteins were highly expressed in E. coli Rosetta （ DE3 ）, with the molecular weight of 44 960. The expression were made the greatest after induction by 1.00 mmol/L IPTG for 7.0 h. PET32a-IL6-IFNα existed in inclusion body form.