Cloning And Expression Of ScFv Gene Anti-Bull Sperm Membrane Proteins

It was difficult to confirm the role of sperm membrane protein during mammalian fertilization and embryogenesis for the multiple configuration and lack of purified protein of sperm membrane proteins. In our previous studies, the primary role of sp18 family proteins, located on the post head of bull sperm plasma was described. In the present study, the express library of monoclonal anti-sp18 ScFv (Single Chain Fragment Variable) gene is constructed and selected for further study of sp18 antigen on mammalian fertilization and embryogenesis.Total RNA were firstly isolated from these growing hybridoma cells which secretes monoclonal anti-sp18 antibodies. After obtained using RPAS system, VH and VL genes were used to assemble ScFv gene fragment with a Linker primer. The ScFv fragment was then amplified with RS primer, followed by Sfi I and Not I degistion together and ligation into pCANTAB5E vector. ScFv antibody library was constructed after transform of pCANTAB5E into E.coli TGI. The library was rescued with phage M13K07 in order to display ScFv on the surface of the phage and to form the recombinant phage antibody library. One of positive ScFv clones, named pCSAl, was selected with phage-ELISA after panning and screening by bull sperm three times. ScFv fragment, amplified from pCSA1, was ligated to pMD18-T vector for sequencing analysis. Analysis on sequence indicated that the ScFv shown more than 80% homogolous sequence with anti-phosphorylcholine mAbs. These results suggested that ScFv CDR of pCSAl, and sp18 antigens, might be phosphorylcholine. The recombinant protein of pET30a/ScFv was expressed by E.coli BL21 (DE3) after ligation ScFv fragment into pET-30a (+) vector followed by transform and induction of IPTG. The patterns of SDS-PAGE indicated more than 30 % of recombinant proteins could be obtained from the extract of E.coli BL21.Furthermore, library of recombinant phage antibodies was constructed from total RNA isolatedfrom spleen of the female BALB/c mice immunized by bull sperm. PCR detection found there were1.2x104 TFU in the library. One of colonies of ScFv antibody, which binds to bull sperm, was selectedand sequenced. Sequence analysis indicates that the ScFv colony resembles the mouse antibody.