PTD融合蛋白的原核表达及其抗体制备  

Study on Prokaryotic Expression PTD-apoptin Fusion Protein and Produce Polyclonal Antiserum

作　　者：张慧瑛[1] 张玉静[1] 胡薇[2] 明宇[1] 张蕾[1] 郝军元[3] 

机构地区：[1]吉林大学农学部人兽共患病研究所,吉林长春130062 [2]吉林农业大学,吉林长春130118 [3]河西学院生物系,甘肃张掖734000

出　　处：《生物技术》2008年第4期20-22,共3页Biotechnology

基　　金：国家自然科学基金项目资助(30170694)

摘　　要：目的:为制备凋亡素重组蛋白抗体,首先获得凋亡素重组蛋白融合基因LTA,而且通过原核表达系统表达重组蛋白并制备其抗体,为进一步利用凋亡素重组蛋白导向治疗肿瘤的检测奠定基础。方法:应用重叠延伸的基因融合技术将LHRH(黄体生成激素释放激素)基因、TAT(HIV-1反式转录激活因子)基因和凋亡素基因重组,构建成凋亡素重组蛋白原核表达载体pET-28a-LTA,随后将表达质粒转入BL21菌株,经IPTG诱导表达重组融合蛋白,将已表达的重组蛋白通过Ni-NTA亲和色谱柱进行纯化,并制备LTA凋亡素重组蛋白抗体。结果:表达产物经聚丙烯酰胺凝胶电泳检测,LTA蛋白融合基因获得高效表达,凝胶薄层扫描分析表明表达蛋白占菌体蛋白12.6%。LTA蛋白经Ni-NTA亲和色谱柱柱纯化,以纯化蛋白为抗原免疫獭兔制备凋亡素重组蛋白抗血清。结果表明抗体的效价为1:12800。结论:应用重叠延伸的基因融合技术获得凋亡素重组蛋白融合基因LTA,通过原核表达系统表达重组蛋白并制备其抗体。Objective： In order tn produce polyelonal antiserum of apoptin recombination protein, the apoplin thsion gene was amplified by PCR and was ligated with an expression vector pET - 28a to express, After purified the recombination protein was used to produce polyelonal antiserum, Supporting that it would be a promising candidate for tumor targeted inununotherapy. Method： The genes encoding LHRH and TAT be integrated into the gene encoding apoptin. The apoptin fusion gene was ligated with an expression vector pET- 28a to constnuct the prokaryotie expression plasnfid PET - 28a - LT4, the pnsitive reeombinant plasmids were, transformed into host slrain E. colt BL21 and induced to express LTA recombination protein by IPTG .After purified the recombination prolein was used to produce polyclonal antiserum. Result： The. specific protein expressed was deter＇ted by SDS- PAGE , LTA fusion protein was expressed at high level amounting to 12.6% of the total baeterial protein confirmed by thin - layer .scanning. After purified the supernatant protein, Purified LTA protein was used as antigen to inuntme rabbits and induce the production of polyclonal antibody against LTA, of which tilers is 1：12800. Conclusion： The apoptin fusion gene was amplified by LTA PCR, Mter purified the recombination protein was used to produce polyclonal antiserum.

关 键 词：凋亡素融合蛋白 TAT 原核表达 抗体 

分 类 号：R730.54[医药卫生—肿瘤]