[Biosynthesis of a single peptide chain containing human chorionic gonadotropin beta and ovine common alpha subunits tandem].
hCG beta-oLH alpha chimeric cDNA was constructed by using overlapping PCR to contact the codons of C-terminal end of hCG beta with the codons of N-terminal end of oLH alpha, then it was subcloned into nuclear polyhedrosis virus (AcNPV) expression vector pVL1393 to construct expression vector pVL1393-hCG beta-oLH alpha. The insect cells (Sf9) were cotransfected by the expression vector pVL1393-hCG beta-oLH alpha and BaculoGold AcNPV linearized genomic DNA, and recombinant viruses AcNPV-hCG beta-oLH alpha were screened out by plaque assay. Further the insect cells were infected by the recombinant viruses, the recombinant hCG beta-oLH alpha was purified by immunoaffinity chromatography column coupling anti-hCG beta monoclonal antibody from the conditioned media of infected cells. The results of SDS-PAGE silver staining and western blotting showed that hCG beta-oLH alpha single peptide chain had apparent molecular weights of 40.5 kD and 38.0 kD 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 of 125I-hCG beta binding we can conclude that the anti-hCG beta antibody-binding activity of hCG beta-oLH alpha chimera is lower than that of native hCG, but higher than that of native hCG beta. Therefore, we assume that the hCG beta-oLH alpha chimera should have potential application as a target antigen of anti-hCG fertility regulatory vaccine.
Xie
L
L
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031.
Li
C L
CL
Shen
H
H
Shen
Q X
QX
Wang
J
J
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics*; D003062:Codon; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D022642:Vaccines, Contraceptive
D018997:Chorionic Gonadotropin, beta Subunit, Human; D003062:Codon; D015292:Glycoprotein Hormones, alpha Subunit; D011993:Recombinant Fusion Proteins; D022642:Vaccines, Contraceptive