【关键词】 原核表达
Cloning of human sCD40L cDNA and construction of vector for expression in E. coli
【Abstract】 AIM: To clone human sCD40L(184-831) cDNA and construct a prokaryotic vector for expression in E. coli. METHODS: The human sCD40L(184-831 ) cDNA was amplified with the total RNA from the human tonsil by reverse transcriptase (RT)PCR, and was analyzed by enzymatic digestion and sequencing. Then, the insert of human sCD40L(184-831) cDNA was cloned into the vector pET28a+ for expression in BL21, and then the interest protein was identified by Western blot. RESULTS: The result of sequencing was accordant with the one reported. The prokaryotic expression vector was constructed successfully and the interest protein was expressed in BL21. CONCLUSION: The human sCD40L(184-831) cDNA has been successfully cloned and a prokaryotic system been constructed for expression. The interest protein was expressed successfully in BL21.
【Keywords】 CD40L; cloning; prokaryotic expression
【摘要】 目的: 利用基因技术克隆CD40L分子的胞外区184～831片断cDNA,构建其原核表达载体,从而建立原核表达体系. 方法: 采用RTPCR 方法从人扁桃体的总RNA中扩增CD40L分子184～831片断的cDNA,以限制性酶切和DNA 测序进行鉴定,并将目的片断克隆至原核表达载体pET28a+,让重组质粒在BL21中表达,利用Western blot鉴定目的蛋白. 结果: 克隆到人sCD40L分子184～831片断的cDNA,DNA测序结果与报道的完全一致;构建了该片断的原核表达载体, 且目的蛋白能在BL21中表达. 结论: 成功克隆人sCD40L分子184～831片断的cDNA并构建了其原核表达载体. 在BL21中成功表达了目的蛋白.
【关键词】 CD40L; 克隆; 原核表达
0引言
人CD40L由261个氨基酸残基组成,为典型的Ⅱ型跨膜蛋白, CD40L包括胞浆内区,跨膜疏水区及胞浆外区组成,其中胞膜外区214个氨基可形成可溶性蛋白,与其膜型分子一样可与B细胞表面的CD40分子
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