Expression And Purification Of Recombinant Human Zona Pellucida 3 Protein In Pichia Pastoris

The zona pellucida (ZP) is a special noncellular shell that surrounds all mammalian oocytes and is also the site at which gamete recognization occurs and the spermatozoa are induced to undergo the acrosome reaction. Among the three immunologi-cally distinct zona pellucida's glycoproteins, ZP3, human's for example, serves as the primary receptor of sperm, and thus is chosed as a potenional target antigen in developing pre-fertilization immunocontraceptive vaccines. Autoantibodies to the zona pellucida are likely to cause ovarian dysfunction, sterility and abortion. The acrosome reaction (AR) induced by the zona pellucida can be used to estimate the function of sperm. With gene-engineering developing, and in order to achieve mass high-purity zona pellucida protein for the study of controceptive vaccine and diagnosis of sterility, researchers cloned some mammal zona pellucida's cDNAs including human ZP3 to produce zona pellucida proteins. Taking gene-recombination technology to express human zona pellucida 3 protein in vitro can offer abundant and low-cost ZP material and accelerate the reseach in this domain.In this study, According to the sequence of hZP3 cDNA coding for amido acid 23-408, two primers were designed which include EcoR I and Not I sites respectively and a (His)e codon cassette was added to 5 ' -terminal. The amplified fragment of hZP3 by PCR excluded the N-terminal leader and reserved most of the C-terminal transmembrane-like domain, and then was incorporated into expression vector pPIC9K. The obtained recombinant yeast expression vector was designated pPIC9K-rhZP3, which after being linearized was then transformed into Pichia pastoris by electroporation. From increasing gradient of G418-YPD plates the Pichia transfomants including multicopy inserts were screened and then cultured and induced according the manufacturer's protocol. The rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blotting analyses showed the Pichia transformant that possesses the highest yield of rhZP3 protein. Simultaneously, the immunogencity of rhZP3 was also comfirmed. Then, the culture condition was optimized which would be used for culture transformant in high-density fermentation, purified with Ni-chelating affinity chromatography, the interesting protein was attained.The results show that the amplified hZP3 fragment can be inserted into vector plasmid pPIC9K, and the recombinant plasmid is also successfully transformed into competent DH5. by PCR identification. Furthermore, the analysis of map of the sequence shows that the interest gene was correctly cloned into vector. Multicopy integrant transformants were screened and then cultured and induced to secrete interesting protein, and the culture supernate was analysed by SDS-PAGE and Western blotting. A reactive band, whose apparent molecular weight was about 32~43KD,can be recognized in Western blotting by the polyclonal antibodies against porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. Besides, the interest protein can combine with Ni ion in affinity chromatography. These results indicate that the recombinant human Zona Pellucida 3 protein was successfullyExpressed in Pichia Pastoris.