摘要
目的:构建表达重组人精子蛋白FSCB的重组质粒,获得其纯化重组蛋白并制备其多克隆抗体.方法:分别成功构建了表达人精子全长FSCB蛋白和截短FSCB(FSCBt)蛋白的重组菌株BL21,IPTG诱导表达,镍柱亲和层析法和分子筛层析法纯化目的蛋白FSCBt,免疫接种雌性家兔制备多克隆抗体.结果:重组菌成功诱导表达了重组蛋白FSCB和FSCBt,相对表观分子量分别为230 000和30 000,重组蛋白FSCBt经纯化后纯度达到95%,制备的多克隆抗体效价达到1:10~5,Western Blot显示多克隆抗体与重组蛋白FSCB和FSCBt均具有良好的反应性.结论:人精子蛋白FSCB在重组菌株中得到表达,成功制备高效价的多克隆抗体,为进一步研究该蛋白在人体组织中的表达、分布以及生物学功能奠定了实验基础.
Abstract
AIM:To express the recombinant protein and prepare the polyclonal antibody(PAb)of human sperm protein FSCB.METHODS:Two recombinant bacterium B121 stains expressing human full length FSCB and truncated FSCB(FSCBt)proteins were successfully constructed,and the FSCBt protein was purified by Ni~(2+) affinity chromatography and molecular sieve chromatography.The specific PAb of FSCB was prepared by immunizing female rabbits.RESULTS:The recombinant protein FSCB and FSCBt could be expressed stably by the recombinant bacterium and be recognized by the PAb,which relative molecular mass were about 230 000 and 30 000.The purity of purified recombinant protein FSCBt was about 95%.The titers of PAb were 1:10~5.CONCLUSION:Expression of the recombinant protein FSCB and preparation of its polyclonal antibody laid a foundation for further research of its functions in human sperm.
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NSTL
维普
万方数据