心肌特异表达基因p93的原核表达及鸡卵黄抗体的制备  

Preparation of an egg yolk antibody against cardiac-specific gene p93 expressed in Escherichia coli

作　　者：尤昕[1] 时娜[1] 赵秀文[1] 柳明洙[2] 刘冬青[1] 曹慧青[1] 孟宪敏[1] 

机构地区：[1]中国医学科学院中国协和医科大学阜外心血管病医院分子医学中心 [2]延边大学医学院生物化学与分子生物学教研室

出　　处：《中华检验医学杂志》2005年第10期1060-1063,共4页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(30270557);北京市重大科技资助项目(H020220020310)

摘　　要：目的在大肠杆菌中高效表达心肌特异表达基因p93,以此作为抗原免疫产蛋母鸡,制备抗p93蛋白鸡卵黄抗体(IgY)。方法将p93基因分别插入原核表达载体pGEX-5X-1、pBV220、pET28a(+)中,选取表达量最高的载体进行表达纯化,其产物免疫产蛋母鸡。结果插入pET28a(+)中N端带有His标签的p93表达水平最高,且以包涵体形式存在,从含有pET28a-p93质粒的1L培养的菌液中可获得3mg纯化的p93蛋白。免疫产蛋母鸡,末次免疫后其卵黄抗体最高滴度达到1∶64000,Westernblot结果显示该抗体具有高度特异性。结论此抗体的制备为新基因p93的检测提供了良好的工具。Objective To express cardiac-specific gene p93 in Escherichia coli, and prepare egg yolk immunoglobulin（IgY） against p93 recombinant protein. Methods The p93 gene was inserted into expression vectors pGEX-SX-1, pBV220 and pET28a （ ＋ ） to construct recombinant expression plasmids, respectively. One of the highest-yield expressions was purified with Ni^2＋ metal chelate affinity chromatography columns. Egg-laying hens were immnunorized with purified p93 protein that had been expressed in Escherichia coli. Results The highest-yield expression was achieved using pET28a-p93 recombinant plasmid as inclusion body, 3mg of p93 was obtained from 1 liter of E. coli. The highest titers of the IgY reached 1 ： 64 000 after the third immunization, Western blot analysis showed the antibody has good specificity. Conclusion The preparation of the egg yolk antibody provides a useful reagent for the detection of novel gene p93.

关 键 词：基因表达 大肠杆菌 蛋黄 抗体 

分 类 号：R392[医药卫生—免疫学]