鳞状细胞癌抗原SCCA基因的构建、表达及鉴定  

Construction and Expression of SCCA Fusion Protein

作　　者：于华实[1] 孙晓琳[1] 张天竹[1] 李梅[1] 李淑珍[1] 唐晓波[1] 

机构地区：[1]哈尔滨医科大学药学院生物制药学教研室,黑龙江哈尔滨150081

出　　处：《现代生物医学进展》2013年第18期3420-3423,共4页Progress in Modern Biomedicine

基　　金：哈尔滨市科技创新人才研究专项资金项目(2008RFXXS018)

摘　　要：目的:重组人鳞状细胞癌抗原(SCCA)的基因及表达融合蛋白,为下一步建立新的肝癌诊断方法奠定基础。方法:提取子宫颈癌细胞株(hela)中的总RNA,应用RT-PCR、PCR等技术扩增出SCCA基因,将其分别与PET-32a及PGEX-4T-1载体连接,转化入DH5α菌中进行克隆,并测序鉴定。异丙基β-D-硫代半乳糖苷(IPTG)诱导转入工程质粒的BL21菌中表达SCCA融合蛋白(分别含有HIS,GST-Tag),并进行SDS-PAGE及Western blotting鉴定。结果:经RT-PCR、PCR扩增后成功获得一条450 bp的DNA片段,经测序鉴定与预期序列一致;并成功在大肠杆菌中实现了高表达,经Western blotting鉴定为SCCA融合蛋白。结论:成功获得SCCA目的基因并获得高纯度的SCCA融合蛋白,为进一步开发针对肝癌的SCCA诊断试剂打下基础,开辟诊断肝癌新途径。Objective：To clone human squamous cell carcinoma antige（SCCA） gene and express fusion protein,and to establish an early diagnostic method of hepatocellular carcinoma（HCC）.Methods：The gene encoding SCCA was cloned by RT-PCR and PCR techniques from total RNA of Hela cell,and the amplified gene was inserted into vectors PET-32a and PGEX-4T-1 and was subcloned into DH5α.Transforming the recombinant plasmids which was confirmed into E.coli BL21 cells,and inducing protein expression（including His-Tag and GST-Tag,respectively） by IPTG and identified by SDS-PAGE and Western blotting.Results：We gained a positive band of DNA about 450 bp by RT-PCR and PCR techniques,and sequence result was agreement with expected sequence.The expression system was constructed and soluble expression of SCCA fusion protein was achieved successfully,which was identified by Western blotting.Conclusion：Fusion protein SCCA with high purity could be expressed in prokaryotic system.The study lays a foundation for opening up new ways for early diagnosis of liver cancer.

关 键 词：肝癌 SCCA基因 克隆 表达 

分 类 号：Q78[生物学—分子生物学] R735.7[医药卫生—肿瘤]