Molecular Cloning Of Crisps Family Of Cashmere Goat, And Localization Of CRISP2 And Identification Of Its Binding To PDIA3

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.