绵羊Izumo2的cDNA克

发布时间：2018-02-10 08:56

本文关键词： 绵羊 IZUMO2 基因克隆免疫组织化学酵母双杂交　出处：《内蒙古大学》2015年硕士论文　论文类型：学位论文

【摘要】：受精作用是将上一代的基因组传递给下一代,并开始一个新生命体生长的细胞机制的总和。在哺乳动物中,精卵融合是通过精子和卵子表面多种分子的相互作用完成的,但目前对这些分子的相互作用机制还不清楚。到目前为止鉴定出若干参与精卵融合的蛋白质,其中精子蛋白有IZUMO蛋白家族、CRISPs家族蛋白、SLLP1、PDIA3、TSSK6、SPESP1和EQUATORIN等,卵子上有JUNO等蛋白。现在的研究多集中在这些蛋白质之间的相互作用模式上。本研究克隆了绵羊Izumo2 cDNA序列,原核表达并纯化GST-IZUMO2融合蛋白,用纯化后的融合蛋白免疫小鼠,制备小鼠抗绵羊IZUMO2腹水多克隆抗体,研究IZUMO2蛋白在睾丸和在精子上的定位。本研究还使用酵母双杂交技术初步探究了IZUMO2 与 IZUMO1、IZUMO3、IZUMO4、PDIA3、TSSK6、SPESP1、SLLP1、 CRISP1、MN9之间的相互作用。以绵羊睾丸组织总cDNA为模板,根据GenBank序列(XM 004015399.1)设计引物克隆绵羊Izumo2 cDNA,全长为813 bp,其中编码区为666 bp, BLAST结果显示其与GenBank数据库中的绵羊预测序列相似度为98.6%。克隆得到的绵羊Izumo2 cDNA序列编码222个氨基酸,其中从N端开始前15个氨基酸为信号肽,第186-206个氨基酸构成跨膜结构域。构建原核表达质粒pGEX-Izumo2,转化E. coli BL21(DE3)感受态菌,用IPTG诱导表达了分子量约为50 kDa的GST-IZUMO2融合蛋白,并采用SDS-PAGE切胶纯化法纯化GST-IZUM02融合蛋白。用纯化后的融合蛋白免疫小鼠,制备并纯化小鼠抗绵羊IZUM02腹水多克隆抗体。用纯化后的抗体进行免疫组织化学研究IZUM02蛋白在睾丸和精子上的定位,结果显示IZUMO2蛋白在绵羊睾丸组织的各期细胞中均会表达；在精子发生顶体反应前IZUMO2蛋白位于精子的头部,当精子发生顶体反应后IZUM02蛋白消失。为了探寻IZUM02蛋白与精子上的其他精卵融合相关蛋白的关系,本研究利用酵母双杂交技术初步探究了IZUMO2与IZUMO1、IZUMO3、IZUMO4、 PDIA3、TSSK6、SPESP1、SLLP1、CRISP1、MN9之间的相互作用,结果未发现相互作用。
[Abstract]:Fertilization is the sum of the cellular mechanisms that transmit the genomes of the previous generation to the next generation and begin the growth of a new organism. In mammals, sperm and egg fusion is accomplished by the interaction of multiple molecules on the surface of the sperm and egg. However, the interaction mechanism of these molecules is not clear. Up to now, a number of proteins involved in spermatozoa fusion have been identified. Among them, sperm proteins include IZUMO protein family, CRISPs family protein, SLLP1PDIA3, TSSK6, SPESP1 and EQUATORIN, etc. There are JUNO and other proteins on the egg. The current research focuses on the interaction pattern of these proteins. We cloned the sheep Izumo2 cDNA sequence, expressed and purified the GST-IZUMO2 fusion protein in prokaryotic, and immunized mice with the purified fusion protein. Mouse anti-sheep IZUMO2 ascites polyclonal antibody was prepared to study the localization of IZUMO2 protein in testis and sperm. The interaction between IZUMO2 and IZUMO1, IZUMO3, IZUMO3, TSSK6, SPESP1, SLLP1, CRISP1MN9 was studied using yeast two-hybrid technique. According to the GenBank sequence XM004015399.1), a primer was designed to clone sheep Izumo2 cDNA. the length of Izumo2 was 813 BP, and the coding region was 666bp.The BLAST result showed that the similarity between Izumo2 cDNA and the predicted sheep sequence in GenBank database was 98.6.The cloned sheep Izumo2 cDNA sequence encoded 222 amino acids. The first 15 amino acids were signal peptides from the N-terminal, and the 186-206 amino acids formed transmembrane domain. The prokaryotic expression plasmid pGEX-Izumo2 was constructed and transformed into E. coli BL21DE3) -receptive bacteria. The GST-IZUMO2 fusion protein with molecular weight of about 50 kDa was induced and expressed by IPTG. GST-IZUM02 fusion protein was purified by SDS-PAGE gel purification. Mice were immunized with the purified fusion protein. Mouse anti-sheep IZUM02 ascites polyclonal antibody was prepared and purified. The localization of IZUM02 protein on testis and spermatozoa was studied by immunohistochemistry with purified antibody. The results showed that IZUMO2 protein was expressed in all stages of sheep testicular cells. IZUMO2 protein is located in the head of sperm before acrosome reaction, and IZUM02 protein disappears after acrosome reaction. In order to explore the relationship between IZUM02 protein and other spermatozoa fusion related proteins, The interaction between IZUMO2 and IZUMO3 IZUMO4, PDIA3TSSK6 SPESP1 and SLLP1CRISP1MN9 was preliminarily investigated by yeast two-hybrid technique. No interaction was found.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826;Q78

【参考文献】