Immunocontraception In Lepus Capensis (Brown Hare)-expression Of Zone Pellucida-3(ZP3) In Prokaryotic Expression System And Eukaryotic Expression System

Lepus capensis (brown hare), the commonly known as rabbit, Lepus, Leporidae,mammals Lagomorpha, is different with the Lepus europaeus and New Zealand White rabbits,which originated from Oryctolagus, belong to the homologous family but not belong to thesame genus. Despite many efforts, the control of reproduction in wildlife species (just asLepus capensis) populations is still a problem around the world for their high fecundity,remote and broad distribution regions. It’s a severe blow to the forestry, agriculture andanimal husbandry. But until now, control of wildlife pest populations has usually relied onconventional ways of thinking that increase mortality, such as disease, introduced predators,trapping, shooting, or poisoning. Many of these strategies are ineffective in the long term forpopulation rapid return, and there are new challenges, growing public opinions, that morenovel humane and environmental methods should be taken into account. Fertility controlthrough immunocontraception has been proposed as a new method for reducing the birth rateinstead of increasing mortality, especially for those species like Lepus capensis (brown hare)with high fecundity.Ovarian ZP3, the primary sperm receptor, that is a major glycoprotein of rabbit zonapellucida (ZP), for blocking sperm-egg interaction has been considered as a candidateimmunogen in the development of a contraceptive vaccine.Thus the present study on this potential use can be facilitated by the availability ofrecombinant proteins. Several different internal fragments corresponding to the Lepuscapensis (brown hare) Zona Pellucida-3(ZP3) were expressed in two different expressionsystems: two different ZP3segments, which one excluding the N-terminal signal sequenceand the C-terminus transmembrane-like domain (aa residues23~382) named ZP3b, while theother one excluding the N-terminal signal sequence only (aa residues23~419) named ZP3a,were cloned in pET-28a vector and expressed in Escherichia coli; and the full-length ofcomplete open reading frame (aa residues1~419) was cloned in pEGFP-N1vector andexpressed in RK-13cells and Chinese Hamster Ovary (CHO) cells. The recombinant proteins in prokaryotic system was engineered with a N-terminal six histidine (His6) segment that wasused to purify the proteins by metal affinity column chromatography and detect the objectiveprotein with western blot analysis. The ZP proteins produced in bacteria, formation ofinclusion bodies, could only be purified after being solubilized by strong denaturants.Optimum expression conditions of ZP3with respect to the IPTG concentration, time, andtemperature used for induction of ZP3were at1mM, at37℃, for4hours. While theeukaryotic system’s was engineered with a C-terminal GFP-Tag that was used to detect theobjective protein with western blot analysis and fluorescence microscopy. Green fluorescencewas found in cytoplasm of the experiment group while not in the control group.Approximately,65%~75%transfected RK-13cells and55%~60%transfected CHO cellswere positive for expression of ZP3. Westernbolt and RT-PCR further proved that thepEGFP-N1-ZP3is expressed in vitro. In this report the availability of ZP3will further help inevaluating its efficacy for fertility regulation and understanding the concrete mechanismassociated with ZP3immunization in wildlife species.