Cloning Of Murine Zona Pellucida 3 CDNA And Its Expressing In Prokaryotic Cell

Population control has become a major problem in many wildlife species. Fertility control through immunocontraception has been proposed as a method for reducing population size. We selected the murine zona pellucida 3(mZP3) as the main role. Ovarian mZP3 is a major glycoprotein of murine zona pellucida and the primary sperm receptor. Because antibodies raised against mZP3 block sperm-egg interaction, ZP3 has been considered a candidate immunogen in the development of a contraceptive vaccine. In this study, we had cloned the mZP3 cDNA, expressed the fusion protein and produced the rabbit-anti-mZP3 antisera. Firstly, the sequence of murine zona pellucida 3 cDNA was obtained from the Gene Bank. According to the sequence , the special primers were designed. Meanwhile, the ovaries were gotten by dissected the non-pregnant Kunmingbai mouse. The total RNA was abstracted from ovaries and mRNA was purified by agarose linked with OligodT. Then the cDNA of mZP3 was cloned and joined into pUCm-T. The correction of the recombinant vector pUCm-ZP3 was tested by blue and white spot screen and restrictive digestion. Finally the sequence of the cloned mZP3 eDNA was defined by PE 377 DNA Sequencer (the primers were T7 and reverse SP6 ). The results showed that the sequence of the cloned mZP3 was the same with the Gene Bank and its length was 131 7bp. And then the ORF and the common restrictive digestion sites were judged on the basis of the results of sequencing and the restriction map and multiple cloning site of pGEX-4T- 1. The mZP3 gene was cut from the pUCm-ZP3 by XhoI and EcoRI and the same restrictive enzyme was used to cut pGEX-4T-1. Then the mZP3 gene was cloned into the pGEX-4T- 1. The correction of the recombinant vector GST-ZP3 was verified by the digestion of HindJII&BglI and PCR in which primers were special. Then GST-ZP3 was transformed into E. co/i BL2 1. After induced by IPTG, the cells were lysised and the fusion expression was measured by SDS-PAGE. And a 72kDa band was found. It suggested that the fusion protein be expressed in E.coli BL21. 3 ~I~h~i~c The E. coli BL2I which expressed the fusion protein was cultivated in big bulk and induced by IPTG. After the cell was lysised, the fusion protein was purified by agarose linked with GST. The big white rabbit was injected by the purified fusion protein with Feufund complete adjnvant. After boosted in two times, the rabbit-anti-mZP3 antisera was obtained. Because the result of Western blot showed a band in 72kDa., the immunocompetence of the fusion protein was verified. The titer of rabbit-anti-mZP3 antisera was measured by ELISA. The result was 1:1000. The base that mZP3 would be deeply studied as a candidate immunogen in the development of a contraception in mouse was founded by cloning mZP3 eDNA and expressed the active fusion protein which was coded by the cDNA of mZP3.