摘要
哺乳动物授精是一个多步骤的复杂过程,包括很多独特的行为,有很多特异性蛋白或脂质等分子的参与。其中蛋白二硫键异构酶(PDI)家族成员Pdia3 (ERp57)和CRISP (cysteine-rich secretory proteins,CRISPs)家族蛋白参与了精卵融合,但是对于其介导精卵融合的分子机制尚不清楚。本实验克隆了绒山羊CRISPs家族各成员蛋白的cDNA序列,制备了小鼠抗羊CRISP2多克隆抗体,用免疫组织化学和免疫细胞化学技术研究了CRISP2蛋白在睾丸中的的表达定位,并用免疫共沉淀和酵母双杂交技术检测了绒山羊CRISP2和Pdia3在细胞内能否直接相互结合,以图解析CRISP2和Pdia3在精卵融合中的分子机制。
实验首先设计保守性引物,采用RT-PCR方法成功克隆了绒山羊的CRISP1、CRISP2、CRISP3 cDNA(同时也克隆到了绵羊的CRISP2、CRISP3 cDNA)。克隆到的绒山羊CRISPs蛋白长度均在240 aa左右,均有20 aa左右长度的信号肽。将所得到的绒山羊和绵羊CRISPs氨基酸序列同GenBank和Ensembl数据库中公布的其它物种的CRISPs氨基酸序列进行比对,发现不同物种的CRISPs蛋白同源性较高,且有16个高度保守的半胱氨酸残基,成为该家族的特征。将克隆得到的绒山羊的CRISP1、CRISP2、CRISP3 cDNA去信号肽序列后,分别构建了pGEX-CRISP1、pGEX-CRISP2、pET44a-CRISP3原核表达载体,诱导表达出融合蛋白GST-CRISP1、GST-CRISP2、Nus-CRISP3。用纯化的GST-CRISP2融合蛋白作为免疫原免疫昆明(KM)小鼠,最后腹腔注射S180细胞,制备了小鼠抗绒山羊CRISP2的腹水多克隆抗体,并采用戊二醛交联法纯化了自制的腹水多抗体。Western blotting分析显示所制抗体能特异性识别组织中的CRISP2蛋白。免疫组织化学检测绒山羊睾丸组织发现CRISP2蛋白大量出现于睾丸曲精细管内侧中央区,即成熟精子出现的区域。免疫细胞化学检测精子细胞显示CRISP2蛋白位于精子头部的赤道区至顶体部位。
本实验还构建了pcDNA-CRISP2和pcDNA-Pdia3真核表达载体,共转染Hela细胞,进行了免疫共沉淀实验分析。同时也构建了酵母双杂交载体pGAD-CRISP2与pGBK-Pdia3,进行了酵母双杂交实验。两实验结果均发现CRISP2与Pdia3在细胞内能够相互结合。
Mammalian fertilization is a complicated and orchestrated process, involving interactions of numerous specific proteins and lipide molecules. CRISPs and PDIA3 of PDI (protein disulfide isomerases) family have been shown to be implicated in gamete fusion, but the underlying molecular mechanisms by which they mediate gamete fusion remain unknown. This experiment cloned cDNAs of CRISPs family members of cashmere goat, generated anti-CRISP2 ascitic polyclonal antibody and displaied its localization in testis and sperms with immunohistochemistry and immunocytochemistry, and finally identified the potential interaction between CRISP2 and PDIA3 in vitro with co-immunoprecipitation (Co-IP) and yeast two hybridization (Y2H) to gain further insight into the mechanism of sperm-egg fusion.
With the conservative primers designed by comparing CRISPs mRNA sequences of various species released in GenBank and Ensembl databases we amplified the cDNA sequences of three CRISPs family members of cashmere by RT-PCR (including cDNA sequences of sheep CRISP2 and CRISP3, too). Analysis of the ORFs of these CRISP cDNAs showed that nearly all CRISPs proteins of cashmere goat consist of about 240 aa with a signal peptide of about 20 aa at their N-terminus. Alignement of amino acid sequences of our cloned cashmere goat and sheep CRISPs with those of the orthologues from other 25 species released in GeneBank and Ensembl databases indicated that the CRISPs family members share high homology among different species, all members have 16 cysteine residues which are highly conserved at their positions, thus is a hallmark of this family. To generate antibodies against cashmere goat CRISPs proteins, we then constructed the recombination vectors of pGEX-CRISP1, pGEX-CRISP2 and pET44a-CRISP3, and expressed fusion protein GST-CRISP1、GST-CRISP2 and Nus-CCRISP3 in E. coli BL21(DE3). The mouse anti-CRISP2 ascites polyclonal antibody was generated and prueified, and could recognize endogenous CRISP2 protein specifically. CRISP2 was detected to be expressed abundantly in the center region of seminiferous tubules in testis by immunohistochemistry, where the maturation of round sperms takes place, and it was also found in the head of mature sperm, from the equatorial segment to acrosome.
Finally, to clarify the binding ability of CRISP2 to Pdia3 in vivo, we constructed eukaryotic expression vectors pcDNA-CRISP2 and pcDNA-Pdia3 and transiently co-transfected them into HeLa cells to perform co-immunoprecipitation (Co-IP) experiment, and constructed pGAD-CRISP2 and pGBK-Pdia3 to carry out yeast two-hybrid (Y2H) screening. The results of Co-IP and Y2H proved the bingding events between CRISP2 and Pdia3 proteins in eukaryotic cells.
引文
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