[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].
OBJECTIVE
To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line.


METHODS
The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA.


RESULTS
A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L.


CONCLUSIONS
A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.
Li
Jian-min
JM
Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
Chen
Wei
W
Jia
Xiu-jie
XJ
An
Xiao-ping
XP
Li
Bing
B
Fan
Ying-ru
YR
Tong
Yi-gang
YG
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002460:Cell Line; D002646:Chikungunya virus / Q000276:immunology*; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology*; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection
D000906:Antibodies; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins