【摘要】 实验克隆了莱航鸡(Gallusgallus)MHCIα(BF2)(GenBank登陆号:AY989897)全基因,分析了其信号肽序列,构建了缺失信号肽且拼接了BirA底物肽序列(BSP)的融合蛋白重组表达载体pET-BF2-BSP,在大肠杆菌(Escherichiacoli)中得到表达,并优化了诱导剂浓度、起始诱导菌体浓度及诱导时间等表达条件。SDS-PAGE分析结果表明所得融合目的蛋白约为40kD,Western-blot结果显示该重组蛋白成功融合了6×His标签;pET-BF2-BSP最优化表达条件分别为:菌体起始诱导浓度OD600nm0.8￣1.2,IPTG诱导浓度1.1mmol/L,诱导时间2￣4h;建立了该重组蛋白变性状态下通过镍柱亲和层析纯化包涵体的方法。实验获得了高纯度的在体外构建鸡MHC-肽四聚体所需的重组蛋白BF2-BSP。更多还原

【Abstract】 The gene of chicken MHC I α (BF2 ) was amplified from total RNA of peripheral blood by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T. And then the PCR product which deleted the signal sequence at the 5’ end in the BF2 gene and included BirA substrate peptide(BSP ) sequence at the 3’ end was amplified from pMD18-T including BF2 gene by PCR amplification and subcloned into an expression vector pET-28a(+) named pET-BF2-BSP. The recombinant vector pET-BF2-BSP successfully expressed in the Escherichia coli strain BL21(DE3). The results of SDS-PAGE and Westernblot showed that the target protein was about 40 kD containing 6×His tag. The overexpressed recombinant protein could be gained when the bacteria was at OD600nm of 0.8 to 1.2 induced for 2 to 4 h and 1.1 mmol/L IPTG. Additionally, the recombinant target protein was purified with a Ni2+NTA column including His-Bind Resin and high quality of recombinant protein was recovered from the inclusion body. All these results indicated that high quality of recombinant protein of BF2-BSP was obtained, which will be contributed to the construction of chicken MHC peptide tetramer.更多还原