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目的:利用原核表达系统构建Tec激酶区作用蛋白RAI16的融合蛋白表达载体,并进行表达条件的优化和初步纯化.方法:设计基因拼接引物,合成RAI16cDNA序列,将其连接于克隆载体pMD18-T中;将酶切、纯化的RAI16基因与pGEX4T-2载体相连接、转化、筛选.将鉴定阳性的重组子质粒转人大肠杆菌BL-21表达菌中,采用SDS-PAGE电泳分析不同浓度诱导剂异丙基硫代-D-半乳糖苷(IPTG)、不同诱导温度、不同诱导时间下目的蛋白的表达.结果:成功构建RAI16蛋白原核表达载体.采用0.4mmol/LIPTG、30℃诱导4h,获得较高表达目的蛋白.融合蛋白主要以包涵体形式表达,将包涵体进行尿素法变性复性和亲和层析柱处理后,获得可溶的高纯度GST-多肽融合蛋白.结论:Tec激酶区作用蛋白RAI16融合蛋白表达的载体和纯化,是制备RAI16多抗以及进一步验证与Tec体外结合作用实验的基础.
OBJECTIVE: To construct a fusion protein expression vector for the function of Tec kinase domain RAI16 by using prokaryotic expression system, and to optimize and purify the expression conditions.Methods: RAI16 cDNA was synthesized and ligated into cloning vector pMD18-T The digested and purified RAI16 gene was ligated into pGEX4T-2 vector, transformed and screened.The positive recombinant plasmid was transformed into E.coli BL-21 and analyzed by SDS-PAGE electrophoresis (IPTG), different induction temperature and different induction time.Results: The prokaryotic expression vector RAI16 was successfully constructed.Using 0.4mmol / L IPTG and induction at 30 ℃ for 4h, High expression of the target protein.The fusion protein was mainly expressed in the form of inclusion bodies, the inclusion body after urea denaturation renaturation and affinity chromatography column to obtain soluble high-purity GST-peptide fusion protein.Conclusion: Tec kinase domain-interacting protein The vector and purification of RAI16 fusion protein expression is the basis for the experiment of preparing RAI16 polyclonal antibody and further verifying the in vitro binding with Tec.