[Biosynthesis of a single peptide chain containing human chorionic gonadotropin beta and C3D of complement].
In view of the strong immunity-enhancing function of HEL-C3d3 designed by Dr. Paul W. Dempsey, we made our efforts to produce a similar recombinant protein of hCG beta. With polymerase chain reaction, we introduced a Bam HI restriction site into the 3' terminal of hCG beta cDNA. The new cDNA and its terminal's correctness has been confirmed by sequencing. Then we have it covalently attached to the C3d3 cDNA at the pre-designed Bam HI/Bgl II site. Having the chimeric DNA correctly cloned into the protein nuclear polyhedrosis virus (AcNPV) expression vector pVL1393, we constructed the expression vector pVL1393-(hCG beta-C3d3). The insect cells were co-transfected with the expression vector and linearized nuclear polyhedrosis virus DNA, and recombinant viruses AcNPV-(hCG beta-C3d3) were screened out. Through anti-hCG beta immunoaffnity chromatography, the recombinant hCG beta-C3d3 chimera polypeptide was purified from culture supernatant of insect cells infected by the recombinant viruses. In RIA test, the expressed product competitively inhibits the binding of 125I-hCG beta to hCG beta-antibody. On SDS-PAGE and Western blot, the recombinant peptide hCG beta-C3d3 obviously appears to be with a molecular weight of 116KD. Therefore, we arrive at a conclusion that it has a normal immunogenic ability.
Yu
W K
WK
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031.
Shen
Q X
QX
Li
D J
DJ
Zhou
Q P
QP
Shen
W Y
WY
Wang
J
J
D000818:Animals; D002478:Cells, Cultured; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics*; D015933:Complement C3d / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D022642:Vaccines, Contraceptive
D018997:Chorionic Gonadotropin, beta Subunit, Human; D011993:Recombinant Fusion Proteins; D022642:Vaccines, Contraceptive; D015933:Complement C3d