摘要
目的:构建人肿瘤-睾丸抗原LAGE-1的原核表达质粒,表达、纯化LAGE-1融合蛋白并制备GST-LAGE-1多克隆抗体。方法:逆转录聚合酶链反应(RT-PCR)方法扩增人肝癌组织LAGE-1基因3′端片段,连入原核表达载体pGEX-4T-1(+),构建原核表达质粒pGEX-LAGE-1并测序。将该质粒转化大肠埃希菌BL21(DE3)进行诱导表达,经包涵体透析复性和谷胱甘肽巯基转移酶纯化系统分离纯化目的蛋白。用纯化的融合蛋白GST-LAGE-1免疫新西兰大白兔,间接ELISA法测定抗体效价,饱和硫酸铵沉淀法纯化抗体,并采用Western blot检测GST-LAGE-1多克隆抗体特异性。结果:RT-PCR扩增得到LAGE-1基因3′端264bp片段,测序结果与GenBank公布序列一致;成功构建pGEX-LAGE-1原核表达质粒,含有该质粒的大肠埃希菌经IPTG诱导后表达相对分子质量约为36×103的蛋白,经包涵体复性和GST融合蛋白纯化系统纯化,得到重组蛋白GST-LAGE-1,纯度达90%;制备兔抗GST-LAGE-1抗体,纯化的抗体经Western blot证实可与目的蛋白特异性结合。结论:获得了纯化的肿瘤-睾丸抗原LAGE-1重组蛋白及其特异性多克隆抗体,为进一步研究LAGE-1抗原在肿瘤免疫治疗中的作用奠定基础。
OBJECTIVE： To construct human LAGE- 1 pro- karyotic expression plasmid, purify the recombinant protein, and prepare polyclonal antibody against LAGE- 1. METHODS.. LAGE-1 gene segment was amplified by RT-PCR. After sequencing, the gene was inserted into pGEX-4T-1 to construct LAGE- 1 expression plasmid pGEX-LAGE-1. The pGEX-LAGE-1 was transfected into BL21 （DE3） and expressed GST-LAGE-1 fusion protein induced by IPTG. The protein was purified through GST affinity chromatography and inclusion bodies renaturation. New Zealand white rabbits were immunized with the immunogen GST- LAGE-1 protein. The titer of polyclonal antibody was measured by indirect ELISA. RESULTS： The sequence of 3＇ terminal 264 bp LAGE-1 segment was amplified which was as the same as what published in GenBank. The expression plasmid pGEX- LAGE- 1 was successfully constructed. The BL21 （DE3） containing the pGEX-LAGE-1 expressed a Mr 36KD protein after induced by IPTG and the recombinant protein was purified. The anti-GST-LAGE-1 polyclonal antibody could specifically recognize LAGE-1 protein. CONCLUSIONS： The purifid recombinant LAGE-1 protein and the anti-GST. LAGE-1 polycolonal antibody are successfully comstructed and prepared. It lays the foundation for further research of the LAGE-1 protein as a promising candidate in tumor immunotherapy.
出处
《中华肿瘤防治杂志》
CAS
2006年第15期1154-1157,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
广州市科技计划项目基金(2003J1-C0221)
关键词
肝肿瘤/遗传学
抗原
肿瘤/生物合成
睾丸/免疫学
基因
抗体
逆转录聚合酶链反应
liver neoplasms/genetics
antigens, neoplasms/biosynthesis
testis/immunology
genes
antibodies
reverse transcriptase polymerase chain reaction
