Cloning, Expression and Purification of Chicken MHC I α-BirA Substrate Peptide (BSP) Sequence in Escherichia coli
LIU Guang-liang;TONG Tie-gang;WANG Qun;XIAO Yi-hong;MENG Qing-wen;WU Dong-lai
Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China; 2. Graduate School,Chinese Academy of Agricultural Sciences, Beijing 100081, China
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.
