BIOSYNTHESIS OF A SINGLE PEPTIDE CHAIN CONTAINING HUMAN CHORIONIC GONADOTRGPIN β AND OVINE COMMON α SUBUNITS TANDEM
XIE Li LI Chang Lin SHEN Hui SHEN Qing Xiang (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031) WANG Jian (Shanghai Institute of Planned Parenthood Research , Shanghai 200032)
hCGβ-oLHα chimeric cDNA was constructed by using overlapping PCR to contact the codons of C-terminal end of hCGβ with the codons of N-terminal end of oLHα, then it was subcloned into nuclear polyhedrosis virus (AcNPV) expression vector pVL1393 to construct expression vector pVL1393-hCGβ-oLHα. The insect cells ( Sf9 ) were cotransfected by the expression vector pVL1393-hCGβ-oLHα and BaculoGoldTM AcNPV linearized genomic DNA, and recombinant viruses AcNPV-hCGβ-oLHα were screened out by plaque assay. Further the insect cells were infected by the recombinant viruses, the recombinant hCGβ-oLHα was purified by immunoaffinity chromatography column coupling anti-hCGβ monoclonal antibody from the conditioned media of infected cells. The results of SDS-PAGE silver staining and western blotting showed that hCGβ-oLHα single peptide chain had apparent molecular weights of 40. 5kD and 38.0kD under non-reducing and reducing conditions respectively, indicating the occurrence of disulfide bonds and significant tertiary structure in the single peptide chain. From the results of competitive inhibition of125I-hCGβ binding we can conclude that the anti-hCGβ antibody-binding activity of hCGβ-oLHα chimera is lower than that of native hCG, but higher than that of native hCGβ. Therefore, we assume that the hCGβ-oLHα chimera should have potential application as a target antigen of anti-hCG fertility regulatory vaccine.