Prokaryotic Expression, Ascitic Polyclonal Antibody Preparation and Identification of Cashmere Goat Izumo1

Izumo1 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, Izumo1 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 Izumo1, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumo1 ascetic polyclonal antibody with intraperitoneal injection of S180 cells. Subsequently, the anti-GST-Izumo1 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-Izumo1 fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumo1 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 Izumo1 in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumo1 to the egg surface.