P-130: Designing and Construction of An Appropriate Eukaryotic Expression Vector to Generate Soluble Form of Human Hyaluronidase Type PH20 in Cell Culture Feasible for Application in IVF and ICSI

Background: The hyaluronidases are the enzymes hydrolyze β-1, 4 glycosidic linkage of hyaluronan. Hyaluronan is a polymer consisting of a repeating disaccharide unit found in cumulus ovuforus complex, semen liquid and other tissue. Addition to hydrolyzing the hyaluronan, hyaluronidase can penetrate through the cumulus cells layer that surrounds the oocyte, thus it terms spreading factor. Moreover, it is used to increase the absorption and dispersion of injected drugs. This enzyme used to denude oocytes from surrounding cumulus cells in IVF and ICSI.Materials and Methods: At first step, total mRNA was extracted from testis tissue and cDNA was synthesized. Ph20 coding sequence deleted GPI anchor was amplified by means of specific primers designed based on ph20 special CDS and also contained additional regions encoding His tag and distinctive sequence recognized by entrokinase enzyme. Then, an amplified fragment was inserted into pTZ57R and treated by appropriate restriction enzyme to sub clone into pBudCE4.1. In this recombinant expression vector, attB region was added for insertion of this construct into genome by phiC31 integrase produced by another vector termed as pCMV-Int. Results: The constructed expression vector was confirmed successfully as verified by sequence analysis. After transfection, culture media was extracted and tested on Granulosa cells. The cell mass was separated effectively that indicate this protein is active. Conclusion: In this study, we produced an appropriate vehicle encoding recombinant hyaluronidase for therapeutic approach and recombinant protein for MS and infertility therapy.