GCF2基因片段原核表达载体的构建与鉴定

Construction and Identification of Prokaryotic Expression Vector Containing GCF2 Fragment

目的构建肝癌相关抗原GCF2基因片断(323～1 234 bp)原核表达重组质粒。方法提取人肝癌组织总RNA,通过反转录酶合成cDNA;根据GenBank公布的GCF2基因cDNA序列设计引物,利用RT-PCR法扩增出人GCF2基因片断(编号2-5a),将此片段连接在克隆载体pGEM-T上进行测序,通过酶切、连接将目的片断重组到原核表达载体pET-30a上。结果酶切电泳鉴定结果显示GCF2基因同源片段克隆入载体中,测序结果证实克隆的基因序列与GenBank中GCF2基因相应序列完全相符。结论获得了含人GCF2基因同源片段重组原核表达质粒pET-30a/GCF2-5a,为进一步表达和纯化GCF2基因片段重组蛋白奠定了基础。

Objective To construct a prokaryotic expression vector of recombinant plasmid pET30a/GCF2-5a.Methods The total RNA of human hepatocellular carcinoma tissues was extracted for synthesizing cDNA by reverse transcriptase.The specific primers were designed according to the encoding sequence of GCF2 published by GenBank.Then the DNA segment of GCF2（2-5a）was amplified by reverse transcriptase polymerase chain reaction（RT-PCR）.This fragment was inserted into the cloning vector pGEM-T and sequenced.Finally,the cDNA fragment was cloned into pET30a expression vector.Results The results of electrophoresis showed that GCF2 gene fragment had been cloned into pET30a vector,and the sequence analysis results showed that sequence of GCF2 gene homolog fragment in recombinant vector was completely idential with GenBank data.Conclusion The prokaryotic expression vector of GCF2 gene fragment was successfully constructed,which lay a basis for purification of GCF2 homolog protein.