Construction and expression of single-chain antibody derived from a new clone of monoclonal antibody against human CD14 in CHO cells.
Our aim was to construct the eukaryotic expressional system of single-chain antibody (ScFv(2F9)) derived from a new clone of monoclonal antibody named ZCH-7-2F9 against human CD14 antigen, and to obtain the ScFv(2F9) protein with a high biological activity for further studies on ScFv(2F9) immunotoxin and other kinds of genetically engineered antibodies. Primers were synthesized according to the gene sequence of ScFv(2F9), 4 tandem glysines and 1 serine (Gly4Ser)3 linker and multiple cloning site(MCS) of pSectag2A vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. ScFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pSectag2A vectors. Positive recombinants (pSectag2A/ScFv(2F9)) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells for expression. The relative molecular weight (MW) and the binding activity of the interesting protein were determined by SDS-PAGE, Western-blot and flow cytometry (FCM), respectively. The scFv(2F9) V(H) is a member of the IgH gene family V(1) subgroup and the scFv(2F9) V(L) gene belongs to the Ig(kappa) gene family V(10) subgroup. The recombined ScFv(2F9) gene was identified to be functional by sequencing and expressing. The interesting protein could be detected in the culture supernatant of transformed CHO cells with a MW of 31 kDa. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.3%and 63.38%, respectively after blockage of ScFv(2F9). The ScFv(2F9) gene was cloned and the interest protein against human CD14 antigen has been successfully expressed in eukaryotic cells with a high biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.
Tang
Yong-Min
YM
Department of Hematology and Oncology, the Children's Hospital of Zhejiang University School of Medicine, People's Republic of China. Y_M_Tang@zju.edu.cn
Ning
Bo-Tao
BT
Cao
Jiang
J
Shen
Hong-Qiang
HQ
Qian
Bai-Qin
BQ
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents; D018950:Lipopolysaccharide Receptors / Q000276:immunology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007202:Indicators and Reagents; D018950:Lipopolysaccharide Receptors