Antifertility Effects Of Zona Pellucida 3 DNA Vaccine In Lagurus Lagurus

Lagurus lagurus is a serious rodent pest in Xinjiang. It can destroy pasture and more importantly its overpop -ulation changes the overall structure and compostion of native grassland and other ecosystems. So, how to control it' s overpopulation becomes an important public problem for the husbandry development in Xinjiang. With the development of biotech, zona pellucida(ZP) has been found to be an extracellular matrix surrounding the developing and mature ovum. ZP is composed of three major glycoproteins ZP1, ZP2 and ZP3 in mouse. ZP3 is a major glycoprotein of zona pellucida and the primary sperm receptor. Because antibodies raised agai -nst ZP3 can block sperm-egg interaction, ZP3 has been considered a condidate immunogen in the development of contraceptive vaccine. This study explored the possibility of using a DNA vaccine expressing Lagurus ZPa to elicit an antibody response and fertility control in Lagurus lagurus. In this study, an eukaryotic expression vector of I ZP3 has been constructed, expressed the fusion protein and the test of fertility control in Lagurus lagurus.According to the full-length sequence of lagurus ZP3, combining to pUCm-T and the sequencing eukaryotic expression vector pCMV4, two restrictive enzymes, Kpnl and HindIII,were used to digest its and to construct the recombinant plasmid pCMV4-LZP3. Then the purified pCMV4-LZP3 was transfected into Hela cells to test if the Hela cell containing pCMV4- LZP3 can produce the specific ZP3 protein. RT-PCR showed eukaryotic express plasmid pCMV4 LZP3 can produce IZP3 protein. It indicated that the recombinate pasmid DNA can immunize the Lagurus asa DNA vaccine.By same method , two restrictive enzymes, EcoRI and Sail, were used to digest the core sequences of Lagurus ZP3(IZP3cf), combining to pUCm-T and the sequence of prokaryotic expression vector pGEX-4T-l. then the IZPacfgene was cloned into pGEX-4T-l, the correction of th recombinant vector GST-ZP3cf was verified. GST-ZPsCf was transformed into E. coli BL21. After induced by IPTG, the cell were lysised and the fusion expression was confirmed by SDS-PAGE. It suggested that fusion protein be expressed in E. coli BL21.Female Lagurus were chosen and enlisted to 2 groups randomly, and were received intramuscular immunization with pCMV4-ZP3 or pCMV4. respectively. Each time, per Lagurus received 50ug/50ul of pCMV4-LZP3 or pCMV4 at hind leg. Collected sera from the postorbital vein after each immunization. Analyzing the sera by ELISA, the result showed that the recombinant pCMV4-LZP3 can elicited significant anti-ZP3 antibody, in contrast to the control group.To test whether immunization of lagurus with LZP3 DNA vaccine expressing ZP3 can induce infertility, 5 week later , nonimmunized male lagurus were introduced to immunized females and allowed to mate with them. The fertility trial demenstrated, in females immunized with pCMV4 LZP3, 2 females were not pregnant at the end of the trial. The other 7 female gave birth to litters of normal size, but gave an average litter size for the entire group of 2. 9 pups. In control, all female in the control group became pregnant, delivering litters with an average of 5. 4 pups per lagurus. There is a significant difference between the number of pups per litter in the LZP3 immunization group and in the control group.This study show LZPs DNA vaccine can reduce the reproductive rate of Lagurus. Therefore, this work may provide evidence to support the feasibility of gene immunization strategy in immunocontrceptive vaccine to control the lagurus' overpopulation and promote the husbandry development in Xinjiang.