肝细胞癌候选相关基因IDD01原核表达载体的构建与表达

目的克隆及表达肝细胞癌(hepatocellular carcinoma, HCC)候选相关基因IDD01 (Institute of Digestive Disease.01),研究其功能.方法用RT-PCR方法从HCC标本中扩增出IDD01的开放读码框(open reading frame, ORF),将目的基因插入表达载体pMAL-c2X中,用重组质粒转化大肠杆菌(E. coli TBl),并在E. coli TBl中进行表达.用SDS-PAGE方法对表达产物进行分析.结果扩增的ORF长度约为770 bp;经酶切和测序分析,插入片段序列和阅读框架正确;SDS-PAGE结果显示,目的基因表达产物的相对分子质量为28×103,融合蛋白的量占全菌总蛋白的30.4%.结论成功构建IDD01的原核表达载体,重组质粒在E. coli TBl中能够高效表达目的基因,该重组子的构建为IDD01基因的功能研究奠定了重要基础.

Construction and expression of prokaryotic vector of a novel candidate gene IDD01 associated with hepatocellular carcinoma

Objective To construct the prokaryotic vector and to express a novel candidate gene IDD01 associated with hepatocellular carcinoma (HCC) for its functional study. Methods The open reading frame (ORF) of IDD01, amplified from HCC tissue by RT PCR, was inserted into the expression vector pMAL c2X. The recombinant vector was transformed into E. coli TB1, and the expression products were analyzed by SDS PAGE. Results The length of PCR product was about 770 bp. The restriction enzyme digestion and sequencing conformed that the gene segment was correctly inserted into the vector. SDS PAGE and density scanning indicated that the protein was expressed as a fusion protein with 28 KD of molecular weight and the fusion protein possessed 30.4% of the total bacterial protein. Conclusion The recombinant vector is constructed successfully and IDD01 can be expressed at a high level, which lays a foundation for the further research on the functions of IDD01 gene.