Cloning, Sequence Analysis And Prokaryotic Expression Of Zona Pellucida Glycoprotein3from Chinese Zokor (Myospalax Fontanierii)

The Chinese zokor (Myospalax fontanierii) is a species of rodents in the Spalacidaefamily, endemic to China. Chinese zokor are major pests across the loess plateau area ofnorthwestern china which are highly specialized subterranean herbivores broadly distributedin farm, alpine prairie and meadow habitats. They are active year-round and excavatecaches in their burrow system to store food which is tenfold more than they need. Accordingto the national agricultural technology extension service center’s statistics (2012), thenumber is on average12.6per hectare. From2012，The number of rodent pests will growrapidly through the10-years conservation period. Usually, the traditional prevention methodis totally depending on artificial capture which is time-consuming, inefficient and quicklyrecovery of population density. Now there are successful cases which use the immuneinfertility technology to control the population of rodent pests. At its core, we can controlthe key factor of sperm-egg recognition or binding. So it’s crucial for us to obtain theefficient autoantigen. Scientists have improved the excellent performance about ZP3as anautoantigen. Our work is to obtain the Chinese zokor ZP3gene, using the rapidamplification of cDNA ends-polymerase chain reaction (RACE-PCR). And we study thestructural functions of ZP3and express it in E.coli.Specific conserved primers were designed according to the ZP3cDNA sequences ofrodents from GeneBank. The conserved sequence encoding Chinese zokor ZP3wereamplified by RT-PCR. After sequencing and homology analysis, we conformed that weobtained the conserved sequence of zokor’s ZP3. With the primers designed by thesequence of the cloned cDNA fragment, the3’and5’ ends were obtained by3’ and5’RACE, respectively. Combining the sequences of3’ends,5’ends and cloned conservedcDNA, the full-length sequence of the Chinese zokor ZP3(mZP3) cDNA was assembled.After the molecular biological analysis, we designed the specific primers. Forconstruction of mZP3prokaryotic expression plasmid, pET-mZP3, a core fragment of zokorZP3(excluding the regions encoding the signal peptide and the C-terminal transmembranedomain) was obtained and cloned into prokaryotic expression vector, pET-28a. Restrictionenzyme digestion verified that prokaryotic expression vector was successfully constructed. Recombinant pET-zokorZP3was expressed as a poly-histidine fusion protein in E.colistrain BL21. And then the expression vector was induced by IPTG. SDS-PAGE andWestern-blot revealed that we successfully expressed the Chinese zokor ZP3gene.We first cloned the full-length ZP3cDNA gene from Chinese zokor, and we hadsubmitted to GeneBank with the accession number: KF360058. This work protects thegenetic resources of national specific species. The successful recombined vector can be usedin subsequent antiserum immune infertility experiment.