Cloning of Human IgG Fc cDNA and
Expression of Whole Human Anti-HBsAg Antibody in CHO Cells
TONG Yi-Gang, XU Jing, LIU Guo-Qi, ZHANG Yong-Guo,CHENG
Wan-Rong, LIU Shu-Ling, WANG Hai-Tao*
( Institute of Microbiology and Epidemiology, Academy of Millitary Medical
Science, Beijing 100071, China )
Abstract Messenger
RNA was extracted from human peripheral lymphocytes and first strand cDNA was
prepared by reverse-transciption. The cDNA of Fc fragment of human
IgG1 was then obtained by PCR and was cloned into the pGEM T-vector. The DNA
sequences encoding signal peptides of both light and heavy chains were
synthesized and cloned respectively. For construction of the light chain
expression plasmid, the light chain signal sequence was linked with the light
chain variable and constant regions (VL-CL) which had been cloned
previously by screening of phage display libraries with HBsAg. The resulting
full-lenth light chain sequence was then inserted into pcDNA3.1, a mammalian
expression vector. For construction of the heavy chain expression plasmid, the
heavy chain signal sequence, the variable region, the first constant region (VH-CH1,
cloned previously by screening of phage display libraries with HBsAg) and Fc
fragment sequence were ligated to form a full-length heavy chain ORF, which was
then cloned into another mammalian expression vector, pCI-DHFR1. CHO(dhfr–)
cells were cotransfected with the above light and heavy chain expression
plasmids, and cell clones expressing human anti-HBsAg antibodies were selected
by G418 and methotrexate (MTX). The recombinant human antibodies were purified
with protein L affinity chromatography from the cell culture medium. As human
serum IgG, the recombinant IgG exhibited only one band with a molecular weight
of more than 100 kD in non-reducing SDS-PAGE； in reducing SDS-PAGE,
however, it turned out to be two bands of approximately 50 kD and 25 kD
respectively. Western-blot analysis demonstrated that the whole IgG in the non-reducing
SDS-PAGE, and the heavy chain in the reducing SDS-PAGE both reacted with goat
anti-human Fc antiserum.
Key words antibody engineering； whole immunoglobulin； mammalian cell； gene expression
*Correspondent author：Tel，86-10-66948563；Fax，86-10-63869835；e-mail：[email protected]