Expression Of Recombinant Human Zona Pellucida 3 In Both Prokaryotic And Eukaryotic System And Its Reproductive Immunogenicity

The zona pellucida (ZP), an extracellular glycoprotein coat surroundingmammalian eggs, plays a vital role in initial recognition and binding events ofsperm to the oocyte in a relatively species-specific manner. The human zonapellucida is composed of three glycoproteins, ZP1, ZP2, and ZP3. Considerableevidence suggests that ZP3 serves as the putative primary sperm receptor. SinceZP antibodies can prevent sperm from recognizing oocyte and reduce fertility,therefore, detection of ZP antibodies is very important in clinical diagnosis ofinfertility. The experimental studies show that the availability of large numbersof pig ovaries from abbatoire has made pig ZP glycoproteins promisingcandidates in the development of a contraceptive vaccine. However, theavailability of purified ZP glycoproteins without any other protein contaminantfrom ovaries will be limited and may not a feasible proposition as animmunogen for a contraceptive vaccine. The expression of ZP3 by recombinantDNA technology could help in overcoming this problem. In this experimentalstudy, we have undertaken to express the fusion protein ZP3 in E. coli.Furthermore, the ability of recombinant ZP3 to generate antibodies against themwas also investigated.As mention above, mammalian oocyte is enveloped by a unique, cellular,translucent matrix termed zona pellucida (ZP). Because ZP plays a vital role infertilization, it makes ZP potential candidate antigens for immunocontraception.In 1970's WHO proposed to develop immunocontraception vaccine that elicit animmune response capable of inhibiting more efficiently the human sperm-egginteraction. Based on previous studies, a modified ZP3 DNA fragment shouldelicit B cell responses only which exclude N-terminal peptide and C-terminaltrans-membrane domain. Thus a eukaryotic expression vector (pIRES1 neo) wasselected for construction of huZP3 expression vector, which acts as DNAvaccine (pIRESl-ZP3). The transformation of pIRESl-ZP3 into CHO cell wascarried out by liposome. ZP3 mRNA could be detected by RT-PCR. Afterimmunization of Balb/C mice using pIRESl-ZP3, antibodies direct againsthuZP3 in serum of mice could be examined by ELISA. In conclusion, theconstruction of huZP3 expression vectors in both prokaryotic and eukaryoticsystem make it possible that anti-fertility effect of active immunization withrecombinant huZP3 will come into reality in the future. In this study, theprotocol given describes the production of immunologically active recombinanthuZP3 using E. coli and CHO cell line as host cell, respectively.