鸡neurexophilin 1基因在毕赤酵母中的表达

Expression of Chicken Neurexophilin 1 Gene in Pichia pastoris

Neurexophilin 1是母鸡子宫阴道结合部位的差异表达基因。根据鸡nxph1的序列,结合毕赤酵母密码子的偏好性,合成nxph1基因,设计引物以合成基因为模板,扩增其去除信号肽的DNA序列△nxph1,将合成的nxph1基因及去除信号肽的nxph1基因片段△nxph1分别插入pPICZαA真核表达载体,构建重组表达质粒pPICZαA/nxph1和去除信号肽的重组表达质粒pPICZαA/△nxph1,电转入毕赤酵母GS115,阳性菌用终浓度为1%的甲醇诱导表达,SDS-PAGE电泳和Western blot检测表达产物,结果表明重组菌成功分泌鸡NXPH1蛋白和去除信号的△NXPH1蛋白,大小约29 kD,为探索NXPH1在母鸡生殖道内的功能奠定基础。

Neurexophilin 1 is the differentially expressed gene at the chicken utero-vaginal junction. According to the mRNA sequence of chicken nxph1 and codon bias of Pichia pastoris, the m odified DNA sequence for chicken nxph1 which encodes the same amino acid sequence as that of chicken nxph1 were synthesized. Specific primers were designed to amplify part of the synthesized DNA sequence whose expression product has no signal peptide. The two sequences were inserted into pPICZαA vector to construct the recombinant p PICZαA/nxph1 and pPICZαA/ △ nxph1（without signal peptide）, and then the recombinant plasmids were transfected into Pichia pastoris GS115 by electroporation. The positive recombinant strains were inducted to express protein by addition of 1 % methanol. The expression protein was displayed on the Tricine- SDS- PAGE electrophoresis and Western blot. The result showed that chicken NXPH1 protein and △NXPH1 protein were successfully expressed, and the molecular weight of the protein was approximately 29 kD, which provided foundations for analyzing func tions of NXPH1 in chicken reproductive tract.