Immunocontraceptive Vaccine For Overpopulation Control In Lagurus Lagurus

Immunocontraceptive Vaccine for OverpopuLation Control in Lagurus lagurusLagurus lagurus is a dominant species of wild pest mouse living in Xinjiang desert grassland, which contributes to a great loss of thousands upon thousands pasture because of its overpopulation every year. In order to prevent and control this damage, traditional methods are mainly based on physical methods or chemical poisons. However, these methods inevitably lead to some fatal disadvantages, such as short effective periods, non-specific targets and pollution of environment. Now a new strategy, immunocontraceptive technology begins to attract more attention for the control of wild mouse or other pest animals. We have designed an immunocontraceptive vaccine based on DNA vaccine for Lagurus zona pellucida 3 to prevent fertilization of Lagurus lagurus. The mammalian oocyte is surrounded by an extracellular translucent envelope termed the zona pellucida(ZP). The ZP mediates the initial recognition and binding events of sperm to the oocyte in a relatively species specific manner, thus playing a vital role in fertilization. In most species, ZP is composed of three biochemically and immunologically distinct glycoproteins, ZP1, ZP2, and ZP3, which can recognize and combine with sperm and induce acrosome reaction. It has been widely concentrated that fertilization could be successfully prevented when the combination of ZP3 and sperm was blocked by monoclonal antibodies both in vivo and in vitro, and so ZP3 glycoprotein has been considered a good candidate for developing an immunocontraceptive vaccine. In our studies, we mainly utilize ZP3 gene of Lagurus lagurus for a new strategy, involved the prokaryotic expression in E. coli, preparation of specific antiserum, constructing and inspecting immune effects for DNA vaccine and infertility control experiment.Firstly, to express and purify Lugurus zone pellucida 3 in prokaryotic system, a core fragment (1025 bp) corresponding to the lugurus zona pellucida glycoprotein-3 (LZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. After inserted and identified exactly into the appropriate sites of pGEX-4T-1 vector, the construct was induced by IPTG to express fusion protein with glutathione s-transferase(GST) in E.coli BL21(DE3). Optimum expression of recombinant LZP3 was observed at 0.08 mM IPTG, and maximum expression was obtained when cultures were induced during the mid-log phase of growth. Then GST-LZP3 fusion protein was induced largely by IPTG and further purified by Glutathione Sepharose 4B to obtain a specific fusion protein with molecular weight of 63kD. Localization studies revealed that the recombinant protein was mostly presented in a soluble intracellular fraction. To prepare antiserum specific to LZP3 for further studies, two New Zealand rabbits were immunized with GST-LZP3 fusion protein, and LZP3 antiserum was successfully prepared. Immunoblots with rabbit anti LZP3 polyclonal antibody revealed a major band of 63 kDa. Two female New Zealand rabbits immunized with GST -LZP3 fusion protein by Glutathione Sepharose 4B purification generated high antibody titers against rec-LZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with GST- LZP3 fusion protein was checked by a Western blot. The availability of GST-LZP3 fusion protein will help in evaluating its efficacy for fertility control in wild mouse living Xinjiang desert grassland. For infertility control experiment, female lagurus was also vaccinated with LZP3 DNA vaccine. Spleen was taken out for cytokine assay and T cell proliferation. When immunized mice mate with males, 13 of 18 females immunized with pCDNA3-LZP3 were infertile,and the infertility rate was 73%. In contrast, 2 of 11 females mice received pCDNA3 vector were infertile. All individuals in control group were fertile. The results of cytokine assay and T cell proliferation revealed that LZP3 DNA vaccine induced immune respo...