Cloning, Identification And Analysis Of The Full-length CDNA Coding For Lagurus Lagurus Zona Pellucida Glycoprotein ZP3

Lagurus lagurus is a serious rodent pest not just because it can destroy local pasture in Xinjiang, but more importantly because its overpopulation changes the overall structure and composition of native grassland and other ecosystems. As one of the most promising population control tools in the world today, immuno- contraception using zona pellucida glycoprotein antigens, specifically ZP3, may be used to control Lagurus population.In this study, by combining RT-PCR and RACE, a full-length cDNA encoding the Lagurus lagurus ZP3 (lZP3) was isolated from the lagurus's ovary and cloned into a prokaryotic expression vector and a eukaryotic expression vector for further immunocontraceptive investigations.With a pair of degenerate primers designed according to the conserved region among the known cDNA sequences of five rodents, a cDNA fragment was generated by RT-PCR from total RNA isolated from ovaries of Lagurus lagurus. The cDNA fragment was cloned into a cloning vector, pUCm-T. Positive clones were analyzed with restriction enzyme digestions and further identified with sequence analysis. The sequencing results show that the cDNA fragment was homologous identical with the published sequences derived from five rodents.With a set of primers designed according to the sequence of the cloned cDNA fragment, the 3' and 5' ends of the cDNAs were obtained by 5' and 3' rapid amplification of cDNA ends (RACE), respectively. Combining the sequences of the 3' ends, 5' ends and cloned partial cDNA, the full-length sequence of the Lagurus lagurus ZP3 (lZP3) cDNA was assembled. To obtain full-length lZP3 cDNA, a pair of primers was designed according to the assembled lZP3 cDNA sequence. After RT-PCR of total RNA isolated from the ovaries of Lagurus lagurus, the lZP3 cDNA was cloned into pUCm-T and further identified with sequence analysis. The results of sequencing analysis show that the open reading frame (ORF) of the cDNA encodes a protein of 418 amino acids (45 kDa) that is significantly different from other published mammal ZP3 sequences at a few sites. The result may have important implications for the elucidation of mechanisms of fertilization and the development of contraceptive vaccine based on lZP3.For construction of lZP3 prokaryotic expression plasmid, pGEX-lZP3cf, a lZP3 cDNA core fragment (lZP3cf) excluding the regions encoding the leader sequence and the C-terminal transmembrane domain was obtained by RT-PCR and cloned into prokaryotic expression vector, pGEX-4T-1. In addition, for construction of prototype DNA vaccine, pCMV4-lZP3, the cloned lZP3 full-length cDNA was subcloned into a eukaryotic expression vector, pCMV4.These preliminary results could lead to further investigation for the development of immunocontraceptive vaccine based on lZP3 for Lagurus population control.