附睾蛋白激酶抑制剂Eppin和精囊蛋白Semenogelin 1相互关系研究

发布时间：2018-03-13 20:13

本文选题：附睾辛蛋白激酶抑制剂Eppin　切入点：精囊凝固蛋白Semenogelin　出处：《南京医科大学》2006年博士论文　论文类型：学位论文

【摘要】：EPPIN(SPINLW1；Gene ID：57119)基因是一种在附睾和睾丸组织中特异性双表达并编码含胱氨酸残基丰富的蛋白质的单拷贝基因。其基因结构中含有KUNITZE和WAP两种特点的基因类型，有含四个二硫腱为核心的类似于蛋白激酶抑制剂样结构为特点的基因序列。本研究用六种不同的实验方法表明排精后的精浆中和在精子表面，精子表面的EPPIN结合来自于精囊的凝胶蛋白SEMENOGELIN1：1)用抗EPPIN的多克隆抗体从提取出的人类精子表面蛋白和精浆蛋白中行免疫沉淀法可沉淀出精囊蛋白Semenogelinl；2)用荧光标记的抗Eppin和抗Semenogelinl的抗体行免疫定位法显示Eppin和Semenogelin两者在精子表面共同定位；3)体外重组EPPIN及重组Semenogelinl蛋白质共同孵育，用抗Eppin或抗Semenogelinl的抗体行免疫共沉淀法，可沉降相应的结合蛋白；4)用重组Eppin和重组Semenogelinl进行Far-western印迹法证实两者相互作用；5)放射性碘125标记的重组Semenogelinl能饱和性结合重组Eppin，无放射性标记的Semenogelinl可与之竞争性地结合，反之亦然；6)放射性碘125标记的重组Semenogelin能直接与印迹膜上的重组Eppin结合的放射自显影法。所有这些方法从体内和体外都表明Eppin结合Semenogelinl。为了进一步研究Eppin和Semenogelinl结合的特异性，，制作重组Eppin和重组Semenogelinl的不同氨基酸片段，发现C端Eppin75-133氨基酸片段与Sg164-283氨基酸片段结合，这段Semenogelinl片段中含有人类Semenogelinl中唯一的胱氨酸残基(Cys239)。还原化或羧甲基化后的胱氨酸残基失去与放射性碘125标记的重组Eppin的结合能力，表明
[Abstract]:EPPING SPINLW1 / Gene IDW: 57119) is a single copy gene that is specifically expressed in epididymis and testis and encodes a protein rich in cysteine residues. Its gene structure contains two types of genes characterized by KUNITZE and WAP. There is a gene sequence with four disulfide tendons at the core, which is similar to the structure of protein kinase inhibitor. In this study, six different experimental methods were used to show that the spermatozoa after spermatogenesis was neutralized and neutralized on the surface of spermatozoa. Seminal vesicle protein Semenogelin 1: 1) seminal vesicle protein Semenogelin 2 was precipitated by immunoprecipitation from extracted human sperm surface proteins and seminal plasma proteins using polyclonal antibodies against EPPIN. Immunolocalization of Eppin and Semenogelin on spermatozoa surface showed that Eppin and Semenogelin were co-incubated with recombinant EPPIN and recombinant Semenogelinl protein in vitro. Immunoprecipitation was performed with antibodies against Eppin or Semenogelinl. Quantifiable binding protein 4) Far-western blotting with recombinant Eppin and recombinant Semenogelinl confirmed that the recombinant Semenogelinl labeled with radioactive iodine 125 could saturate and bind to recombinant Eppin, and non-radioactive labeled Semenogelinl could bind to it competitively. Radioiodine-125 labeled recombinant Semenogelin can be directly combined with recombinant Eppin on the imprinted membrane by autoradiography. All of these methods indicate that Eppin binds Semenogelinl. in order to further study the specificity of Eppin and Semenogelinl binding, Different amino acid fragments of recombinant Eppin and recombinant Semenogelinl were prepared. It was found that C-terminal Eppin75-133 amino acid fragment combined with Sg164-283 amino acid fragment. This Semenogelinl fragment contains the only cystine residue in human Semenogelinl Cys2390.The reduced or carboxymethylated cystine residues lose the ability to bind to the radioactive iodine-125-labeled recombinant Eppin.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R321.2

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