The Study Of Oviductin Of Mouse Cervix Expression, Recombinational Expression In Pichia Pastoris And Its Function

Oviductin used to be limited in oviduct mucosa epithelial microenvironment until we have found two components from mouse cervix is a nearly 60kDa and 30kDa by Western blotting. The 400bp oviductin cDNA was got by mouse cervix homogenization. Sequence comparison were performed using BLAST alignment, and This region is 100% homologous with the region from +310 to +714 of mouse MUC-9 by RT-PCR and western blot. Western blot showed 55KD and 30 KD from mouse cervix as from mouse oviduct mucosa epithelial. Semi-quantitative RT-PCR Analysis from immature and mature estrous, non-estrous show that the expression of these components were different, suggest that the mRNA level of mouse cervix oviductin was related to estrous ,implied with estrogen — dependent.The function of oviductin on viability of human sperm was studied using conditioned medium derived from the rabbit oviduct mucosa epithelial cells. "Correlative of function" experiment and "Gain-of-function" experiment showed no difference between their effects on the viability of human sperm anti-CTP DPF-1 antiserum. These indicated that DPF-1 could not affect the viability of human sperm.The plasmid pBluescript DPF cDNA as a template was used as a template to amplify the 1.3kb DPF c DNA, , was subcloned into EcoRI-NotI site of pPIC9K for removing its leading sequence and the sequence encoding signal peptide, the 1.3kb DPF-1 cDNA was obtained. The recombinant expressing plasmid pPIC9K/DPF-l cDNA was constructed by ligating 1.3kb DPF-1 cDNA with expressing vector pPIC9K. The recombinant pPIC9K/DPF-l cDNA was transformed into competent TOP 10 bacteria and the TOP 10 containing the recombinant plasmid was identified by proliferating, extracting the plasmids and digesting them with EcoRI and NotI, linearized with Bgl II and electrotransformed into Pichia pastoris GS115.A Mut~s recombinant was selected from YEPD medium with 2mg/ml Genectin,G418. The recombination of DPF-1 has been inducedly expressed in Pichia pastoris and has been purified from ferment supernant. The fusion proteins showed expectedmolecular weigh through SDS electrophoresis and was recognized in Western-blot by anti-rabbit—DPF-GST polyclonal antibody.Total RNA ws extracted from five hundred superovulated mouse oocytes, was prepared to cDNA with primer-randomly designed by SMART technology. The cDNA was transformed into Saccharomyces cerevisiae AH109 together with yeast expressing plasmid pGADT7-Rec in the theory of. From the counting of colonies growing on the nutrition-selecting SD-L, the titer of library was estimated about 3.4×10~6,and the ratio of recombinants to non-recombinant was 60%. In order to study the target protein of the rabbit DPF-1 and identify the components in the pathway of signal transduction involved, a cDNA library for yeast two hybrid of mouse oocyte has been constructed relied on interchanging parts of the plasmid pGADT7-Rec by homologous recombination.Also templated by The plasmid pBluescript DPF cDNA, 1.3kb DPF c DNA and 0.27kb DPF c DNA with EcoRI and BamHI sites were amplified, was subcloned into pGBKT7 withing the removal of signal peptide. The recombinant expressing plasmid pGBKT7/1.3 kb DPF-1 cDNA and pGBKT7/0.27kb DPF-1 cDNA were ligased. The two bait plasmid:pGBKT7/1.3kbDPF and pGBKT7/0.27kbDPF were constructed, and show no evident self-activation.In all, particularly, the following questions remain to be answers : 1) Which pathway of signal transduction is involved in the activation of zygotic gene by oviductin? 2) Is the mode of OGP association with gametes through a protein backbone or is it through carbohydrate residues? 3) Is there a separate and distinct binding domain on OGP that interacts with sperm and zona/eggs? and 4) What are the putative partners involved in binding OGP with sperm?...