人NY-ESO-1基因的原核表达、纯化和初步应用

目的：克隆人NY-ESO-1基因，进行原核表达和分离纯化，并初步应用于肝癌患者血清NY-ESO-1抗体的检测。方法：通过RT-PCR技术从人肝癌组织中扩增NY-ESO-1基因片段，插入原核表达载体pGEX4T-1( )中，构建重组表达质粒pGEx／ESO1，导入大肠杆菌BL21(DE3)，诱导目的蛋白表达，经包涵体透析复性和GSTrap亲和层析纯化目的蛋白，Westem blot鉴定。应用间接ELISA方法初步检测50例肝癌患者血清和20例正常人血清NY-ESO-1抗体的表达。结果：扩增得到NY-ESO-1基因3′端276bp片段，与GeneBank公布序列一致，构建的pGEX／ESO1原核表达质粒经IPTG诱导，在大肠杆菌中表达分子量约36kD的GST-ESO1融合蛋白，纯化后蛋白纯度为90％。50例肝细胞癌患者血清中4例检测到NY-ESO-1抗体(8％)，正常人血清均为阴性。结论：成功构建pGEX／ESO1重组表达质粒，得到有活性的NY-ESO-1融合蛋白，对肝癌患者血清NY-ESO-1抗体的检测有-定的应用价值。

The Prokaryotic Expression, Purification and Preliminary Application of Human NY-ESO-1 Gene

Objective: To clone human NY-ESO-1 gene, induce its prokaryotic expression and purify the protein for preliminary use of detecting NY-ESO-1 of sera in HCC patients. Methods: NY-ESO-1 segment was amplified by RT-PCR and cloned into the expression vector pGEX4T-1(+) to construct the expression plasmid pGEX/ESO1. The recombinant vector was transfected to BL21(DE3) and GST fusion protein was expressed by IPTG. The protein was purified by GST affinity chromatography and inclusion bodies renaturation and was identified by Western blot. NY-ESO-1 of sera from 50 cases of HCC and 20 cases of normal controls was detected by indirect ELISA. Results: The sequence of 3' terminal 276bp NY-ESO-1 segment was amplified segment and identical with that published in GenBank. The BL21(DE3) containing the pGEX/ESO1 expressed a Mr 36kD fusion protein after being induced by IPTG. The purity of the protein was 90%. NY-ESO-1 in 4 of 50 cases HCC patients was positive, whereas that in the normal controls was negative. Conclusion: The prokaryotic expression vector of NY-ESO-1 has been constructed and the fusion protein is successfully expressed. It can be used in detecting NY-ESO-1 of sera in the HCC patients.