【作者】 黄天明；

【导师】 罗国容；

【作者基本信息】 广西医科大学 ， 人体解剖与组织胚胎学， 2006， 硕士

【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/L时,诱导效率最高;IPTG在工程菌37℃振荡培养2个小时后加入,可获得较优诱导效果。过柱纯化后的MBP-Kinectin融合蛋白电泳后可见一些分子量介于MBP和MBP-Kinectin之间的蛋白条带,降低破菌及过柱温度,并抑制蛋白酶的活性,可减少蛋白杂带的出现。结论:pMAL-C2载体可成功用于重组表达kinectin片断蛋白;在培养基中加入0.2%葡萄糖,37℃振荡培养两个小时后加入IPTG(终浓度为1mmol/L),34℃诱导3个小时,在冰浴中破菌并加入蛋白酶抑制剂PMSF更多还原

【Abstract】 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 transfected into E.coli DH5.The blue-white selection assay, PCR, incision enzyme assay and DNA sequencing were performed to find out the right recombinant clone. The right clone was transfected into the expression host (E.coli TB1), and the engineering bacterias were induced by IPTG in different conditions,such as different induction time,different induction temperature,etc.The products was analyzed throught the SDS-PAGE methods to find out the best induction conditions under which the engineering bacterias can obtain the highest expression efficiency.After the induction bacterias broken by ultrasound, purifing the supernatant throught the Amylose-resin column, and find out the better purification conditions to obtain the more purity MBP-kinectin.Result:The gene segment of kinectin have been successfully inserted into the vector pMAL-C2 at the multiple clone site, and the interest protein was expressed when the engineering bacterias induced by IPTG. When the engineering bacterias induced in 34℃,the production of the fusion protein is highter and form less inclusion body than in 37℃. After the IPTG is added,the production of the fusion protein increase in the first 3 hours,but drop after it. The更多还原