Prokaryotic Expression, Purification And Antibody Production Of Sp56-lost Of Rat Sperm ZP3 Binding Protein Sp56

Mammal zona pellucida 3 (ZP3) has been characterized to be responsible for theprimary interactions between sperm and egg and inducement of acrosome reaction. Manydifferent sperm proteins have been identified as ZP3 binding proteins, among which mousesperm protein 56 (sp56) is considered as one of the candidates for mouse zona pellucida 3(mZP3)receptor. Up to date , its homology has only been cloned from guinea pig testis. Base onthe former result of study in our lab there are two sp56 transcripts( sp56l and sp56s) in ratsperm. Rat sp56l show very high homology to mouse sp56 and guinea-pig sp56 both ofwhich have seven Sushi domains. However, rat sp56s completely lacks the third one of theseven Sushi domains of mouse sp56.Here, with a pair of primers designed according to the third Sushi domain sequence ofrat sp56l, the sp56-lost sequence was generated by PCR from cDNA of sp56l. Forconstruction of prokaryotic expression plasmid, pGEX-4T-sp56-lost 2 , two sp56-lostsequences were contacted and cloned orientatively into the vector, pGEX-4T-3, by restrictionsites of pGEX-4T-3. Then pGEX-4T-sp56-lost 2 was induced to express by use of IPTG andwas selected. No soluble GST-(sp56-lost)2 fusion protein can be acquired through affinitypurify of Glutathione Sepharose 4B resin .For under denature conditions the recombinant Trx-(sp56-lost)2-His fusion protein wasaffinity purified. The vector, pGEX-4T-3, was replaced with the vector, PET-32a. Then, theprokaryotic expression plasmid,PET-32a-sp56-lost 2 was constructed, induced by IPTG andwas selected. Finally, the recombinant Trx-(sp56-lost)2-His fusion protein was affinitypurified using Ni-NTA His-Bind resin. After that, the affinity purified fusion protein wasundergone by SDS-PAGE. Target protein band was cutted, freezed out and pulverized. Malerabbits were immunized intraperitoneally with the mixture of the purified fusion proteinpower and Freund's Complete Adjuvant and boosted three times with the purified fusionprotein power in Freund's Incomplete Adjuvant. ELISA was done using the antisera raisedfrom ear vein of rabbits after three boosts. Then, blood of rabbits was released from carotids...