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目的化学合成寡核苷酸片段克隆入pIVEX2.3质粒中,构建GCIP-27的原核表达载体并进行原核表达。方法化学合成GCIP-27的全基因序列,定向克隆到pIVEX2.3质粒中构建含有T7启动子的pIVEX2.3-GCIP27原核表达载体并酶切及测序鉴定;pIVEX2.3-GCIP27质粒转染E coli BL21(DE3)pLysE细菌,用IPTG诱导GCIP-27多肽的表达。结果成功构建了pIVEX2.3-GCIP27表达质粒,经测序鉴定结果与设计的完全相符;成功用BL21细菌表达了GCIP-27多肽,GCIP-27占细菌总蛋白的4.96%。结论本实验构建并表达了GCIP-27的重组质粒,为GCIP-27的大量原核表达奠定了基础。
The target chemical synthetic oligonucleotide fragment was cloned into pIVEX2.3 plasmid to construct the prokaryotic expression vector of GCIP-27 and prokaryotic expression. Methods The full-length GCIP-27 gene was chemically synthesized and cloned into pIVEX2.3 plasmid to construct the prokaryotic expression vector pIVEX2.3-GCIP27 containing the T7 promoter. The recombinant plasmid was digested with restriction endonucleases and sequenced. Plasmid pIVEX2.3-GCIP27 was transfected into E. coli BL21 (DE3) pLysE bacteria, the expression of GCIP-27 polypeptide was induced with IPTG. Results The pIVEX2.3-GCIP27 expression plasmid was successfully constructed. The sequencing result was in good agreement with the design. The GCIP-27 polypeptide was successfully expressed in BL21 and accounted for 4.96% of total bacterial protein. Conclusion This experiment constructed and expressed the recombinant plasmid of GCIP-27, which laid the foundation for the large number of prokaryotic expression of GCIP-27.