绵羊精子特异性蛋白TSSK6与IZUMO1相互作用的研究

发布时间：2018-04-22 22:27

本文选题：Tssk6 + Izumo1　；参考：《内蒙古大学》2017年硕士论文

【摘要】：哺乳动物的受精作用是一个精密且复杂的生理过程,主要包括精子获能、精子与卵子透明带结合引发顶体反应、精子和卵子膜融合三个主要过程。精子膜上的蛋白质之间的相互作用为之后的精卵融合创造了结构基础,近来研究表明,鼠精子特异性蛋白精卵融合蛋白IZUMO1在精子膜上的运动跟另一个精子特异性蛋白激酶TSSK6有关,但是两者之间是否有直接的相互作用还没有被证明。本文用酵母双杂交、免疫共沉淀和哺乳动物双杂交技术鉴定了 IZUMO1与TSSK6之间的相互作用。本实验选取绵羊的睾丸组织,克隆出长度为985 bp的Tssk6的cDNA全长,并上传至NCBI,登录号为KY914489。其中CDS区中的第144位和672位碱基与GenBank预测的序列存在差异,但没有发生氨基酸改变。将Tssk6和本实验室已有的绵羊Izumo1 cDNA分别插入pGEX-4T1构建pGEX-Tssk6和pGEX-Izumo1原核表达载体,在大肠杆菌中表达重组蛋白TSSK6-GST和IZUMO1-GST,提取纯化后分别免疫小鼠和家兔,得到兔抗的IZUMO1血清抗体和鼠抗的TSSK6血清抗体。酵母双杂交实验中,我们把转化了pGAD-Tssk6的Y2H酵母菌和转化了Pgbk-Izumo1的Y187酵母菌进行杂交,杂交后代可以在SD/-Trp-Leu二缺培养基和SD/-Ade-His-Trp-Leu培养基上分别长出蓝色菌落,实验结果说明TSSK6和IZUMO1在酵母细胞中发生了相互作用。在哺乳动物双杂交实验中,把pFN-10A-Izumo1/pFN-11A-Tssk6和报告基因载体PGL4.31共转染人293T细胞,然后进行双荧光素酶报告基因检测,并对结果进行差异显著性分析。其中阳性对照组p0.05,实验组p0.01,实验结果说明IZUMO1和TSSK6在人293T细胞中发生了较强的相互作用。在免疫共沉淀实验中,我们将pCMV-Flag-Tssk6和pCMV-HA-Izumo1载体共转染293T细胞,·然后使用抗HA标签的磁珠抗体对IZUMO1-HA进行沉淀,用抗Flag标签的抗体进行Western-blot检测,蛋白印迹结果显示IZUMO1-HA蛋白与TSSK6-Flag蛋白一起被沉淀下来,这说明TSSK6和IZUMO1在293T细胞中是结合在一起的。综合以上实验结果,我们可以确定IZUMO1和TSSK6之间发生了直接的相互作用。
[Abstract]:The fertilization of mammals is a precise and complex physiological process, mainly including sperm capacitation, the combination of sperm and the oval zona pellucida triggering acrosome reaction, and the fusion of sperm and egg membrane three main processes. The interaction between the proteins on the sperm membrane creates a structural basis for the subsequent fusion of sperm and eggs. Recent studies have shown that the rat The movement of sperm specific egg fusion protein IZUMO1 on the sperm membrane is related to another sperm specific protein kinase TSSK6. But whether there is a direct interaction between them has not been proved. In this paper, the interaction between IZUMO1 and TSSK6 was identified by yeast two hybrid, CO immunoprecipitation and mammalian two hybrid techniques. In this experiment, we selected the testis tissue of sheep and cloned the full length of Tssk6 cDNA length of 985 BP, and uploaded to NCBI. The login number was KY914489. in the CDS region and the 672 base bases were different from the GenBank predicted sequences, but there was no amino acid change. Tssk6 and the existing sheep Izumo1 cDNA in our laboratory were inserted into pG, respectively. EX-4T1 constructed the prokaryotic expression vector of pGEX-Tssk6 and pGEX-Izumo1, expressed recombinant protein TSSK6-GST and IZUMO1-GST in Escherichia coli, and immunized mice and rabbits after extraction and purified respectively. The Rabbit anti IZUMO1 serum antibody and mouse anti TSSK6 serum antibody were obtained. In the yeast two hybrid experiment, we transformed the Y2H yeast and transformation of pGAD-Tssk6. The Pgbk-Izumo1 Y187 yeast was hybridized. The hybrid progeny could grow blue colonies on the SD/-Trp-Leu two deficient medium and the SD/-Ade-His-Trp-Leu medium. The experimental results showed that the interaction between TSSK6 and IZUMO1 had occurred in the yeast cells. In the two hybrid experiment of mammalian, the pFN-10A-Izumo1/pFN-11A-Tssk6 and the report base were used. The carrier PGL4.31 was co transfected with human 293T cells, then the double luciferase reporter gene was detected and the results were statistically analyzed. The positive control group, P0.05, and the experimental group P0.01, showed that IZUMO1 and TSSK6 had a stronger interaction in human 293T cells. In the immunoprecipitation experiment, we will have pCMV-Flag-Tss The K6 and pCMV-HA-Izumo1 vectors co transfected 293T cells. Then the IZUMO1-HA was precipitated with the anti HA labeled magnetic bead antibody, and the Western-blot detection was carried out with the antibody labeled Flag. The Western blot results showed that the IZUMO1-HA protein was precipitated together with the TSSK6-Flag protein, which indicates that TSSK6 and IZUMO1 are combined in the 293T cells. Based on the above results, we can determine the direct interaction between IZUMO1 and TSSK6.

【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;S826

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