Prokaryotic Expression, Ascitic Polyclonal Antibody Preparation and Identification of Cashmere Goat Izumol

• Published in: Agricultural sciences in China 2010 (4), p.605-613
• Main Author: LIU Zhi-da XING Wan-jin WANG Lian-qing LV Li-xia
• Format: Article
• Language: English
• Subjects: 免疫球蛋白超家族; 原核表达; 多克隆抗体; 抗体制备; 白绒山羊; 聚丙烯酰胺凝胶
• ISSN: 1671-2927

Izumol is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumol was fractionated into 6 fragments （F0-F5） which were ligated with pGEX-4T1 to construct the prokaryotic expression vectors pGEX-Fn. The recombinant plasmids were transformed into Escherichia coli BL21 （DE3） and the GST-Fn fusion proteins were expressed successfully by induction with IPTG. GST-F0, a recombinant fusion protein of GST with the full length of extracellular region of mature cashmere goat Izumol, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumol ascetic polyclonal antibody with intraperitoneal injection of S 180 cells. Subsequently, the anti-GST-Izumol polyclonal antibody was purified with miscellaneous antigen by glutaraldehyde cross-linking method. Western blotting analysis showed that the purified ascetic polyclonal antibody had high affinity to all 6 GST-Izumol fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumol proteins were at the equatorial segment of sperm head surface. These results indicate that this polyclonal antibody has high specificity and lays the foundations for further study on the expression pattern of Izumol in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumol to the egg surface.