次级淋巴组织趋化因子（SLC）基因克隆及在大肠杆菌中的表达

Cloning and Expression in Escherichia coli of Secondary Lymphoid-tissue Chemokine(SLC) Gene

摘要:

次级淋巴组织趋化因子（SLC）是通过搜索表达序列标签（EST）数据库克隆出来的一CC类趋化因子。以人SLC序列为蓝本，利用重叠PCR（SOE-PCR）的方法获得了适宜在大肠杆菌中表达的SLC基因，将此序列分别克隆至表达载体pTMF和pALM中，转化大肠杆菌，诱导表达。Western Blotting鉴定结果表明目的蛋白以可溶蛋白和包涵体两种形式表达，两种形式的蛋白所占比例依培养和诱导条件的不同而变化。对两种形式的表达产物分别用Ni-NTA金属亲和层析和包涵体复性方法纯化，在实验中还对纯化条件进行了探索。对纯化蛋白的电泳结果显示：纯化样品的分子量比预期的分子量要大。

Abstract:

Secondary lymphoid-tissue chemokine (SLC) is a type of CC chemokine identified by searching the Expressed Sequence Tag (EST) database. The full-length SLC gene was synthesized based on human SLCsequence using SOE-PCR. The sequenced SLC gene was cloned into expression vector pTMF and pALM, which used to transform Escherichia coli. Then the E.coli was cultured and induced according to protocol. The expressed target protein was identified by Western blotting. The target protein was expressed as soluble protein as well as inclusion bodies, the ratio of these two forms target protein varied with the difference conditions of culture and induction. The target protein was purified with the methods of nickel-nitrilotriacetic acid(Ni-NTA) metal-affinity chromatography. The results of electrophoresis of the purified target protein showed that the molecular weight was larger than the predicted molecular weight.