【中文摘要】 目的:研究肝癌相关抗原kinectin基因片断体外重组表达的方法,探讨高效表达及纯化的条件,为进一步开发应用该抗原打下基础。 方法:用PCR法扩增肝癌相关抗原kinectin的基因片断,将目的基因插入表达载体pMAL-C2的麦芽糖结合蛋白基因下游,转化DH5α菌,通过蓝白斑筛选、PCR鉴定、内切酶鉴定及DNA测序筛选出正确的重组子,并用其转化TB1菌。采用不同的诱导温度、诱导时间、诱导剂IPTG浓度及IPTG加入时机对工程菌进行诱导表达,SDS-PAGE电泳鉴定,并用Quantity one凝胶分析软件进行分析,以找出最适诱导条件。按优化条件诱导的工程菌超声破碎后,上清液过Amylose-resin亲和层析柱,对过柱纯化条件进行优化,以获取纯化的MBP-kinectin融合蛋白。 结果:目的基因正确插入载体质粒的多克隆位点,并成功诱导表达出MBP-kinectin融合蛋白。工程菌在34℃诱导时不仅可以获得较高的目的蛋白产量,且其可溶性部分所占的比例也较高;工程菌在诱导的最初3个小时内,目的蛋白表达量会随着诱导时间的延长而增加,但超过3个小时,则会明显下降;IPTG终浓度为1mmol/...

【英文摘要】 Objective: To study a method to recombinate and express the gene segment of HCC-associated antigen-kinectin in vitro. Find out the suitable conditions under which the fusion protein can be expressed most efficiently. Then provide a basis for the production and application of this antigen. Methods: The gene segment of Kinectin was amplified by PCR (Polymerase Chain Reaction), and then inserted into the vector pMAL-C2 at the downstream of the gene encoding the MBP protein. The recombinant vector was ...