[Construction and expression of eukaryotic expression vector for intact IgG against human keratin].
OBJECTIVE
To construct an eukaryotic expression vector containing the gene of anti-keratin antibody and express it in CHO(dhfr(-)) cells.


METHODS
Both the V(L) and V(H) genes of an anti-keratin Fab from a phage display library were amplified by PCR. Using PCR product as the template, the V(kappa) and V(H) genes with the leader sequences (named L(kappa) and L(H) respectively) were amplified by overlapping PCR. After digested with endonuclease Xba I/BamH I and Xho I/Hind III, L(kappa) and L(H) genes were inserted into pWD digested with Xba I/BamH I and Xho I/Hind III, respectively to construct pWDkappaH. After PCR and sequencing, the expression plasmid pWDkappaH was transfected into CHO (dhfr(-)) cells. The culture supernatant of the transfected cells was collected and assayed for IgG activity.


RESULTS
The eukaryotic expression vector pWDkappaH was constructed successfully and expressed in CHO(dhfr(-)) cells. The expression of intact IgG against keratin was identified by ELISA and RT-PCR.


CONCLUSIONS
The successful expression of intact IgG against keratin lays the foundation for its clinical application.
Xia
Ru-shan
RS
Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Wang
Gang
G
Fan
Jian-yong
JY
Li
Wei
W
Liu
Yu-feng
YF
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism; D007633:Keratins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D012333:RNA, Messenger; D007633:Keratins