摘要
用两种不同的表达载体构建、表达人肝癌肿瘤抗啄——衰老标记蛋白-30(SMP—30),并在体外对其表达产物进行纯化和鉴定。PCR方法扩增经SEREX技术筛选出的人肝细胞癌抗原SMP—30羧基末端165个AA的DNA序列,分别与pMAL—C2和pET16b两种表达载体重组后转入相应的宿主菌中诱导表达,并对表达产物进行分离纯化和鉴定。结果克隆的SMP—30基因经DNA测序与已公布的序列相同。pMAL—C2和pET16b两种重组载体分别表达出带麦芽糖结合蛋白和带10个组氨酸的SMP—30融合蛋白,麦芽糖结合蛋白——SMP—30是以可溶性蛋白形式表达,而带组氨酸的SMP—30则以包涵体的形式存在,对表达蛋白质N-末端15个氨基酸进行测序,结果与预期的完全相同。结果说明pMAL—C2表达载体更为适合于SMP—30蛋白质的表达;实验为肿瘤抗原免疫研究打下了基础。
To expressed hepatocellular carcinoma antigen SMP—30 fusion protein by two different vectors and to isolated and purify aim protein, the cDNA that represents 165 amino acids of SMP—30 in its C-end was amplified and then recombined in pMAL—C2 pET16b expressing plasmid respectively, and then the expressing plasmid was transferred into corresponding host cells of TB1 and BL21 (DE3) plys to induce expressing aim protein. The expressed SMP—30 fusion protein was purified by affinity chromatography. The Result showed that the choning DNA sequence of SMP—30 is the same as the sequence of being reported. The two fusion proteins of maltose binding protein-SMP—30 and 10His-SMP—30 were expressed by pMAL—C2 and pET16b expressing plasmid respectively. The maltose binding protein-SMP—30 was expressed in soluble form but the 10His-SMP—30 was expressed as inclusion body. 15 amino acids of SMP—30 in the N-end were correct by sequence analysis. It is concluded that pMAL—C2 expressing plasmid is better for the expression of SMP—30 protein.
出处
《科学技术与工程》
2003年第6期553-556,共4页
Science Technology and Engineering
基金
国家自然科学基金(39960022)
国家教委高校骨干教师基金
