[Establishment of a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase gene].
BACKGROUND
To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.


METHODS
The full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T. The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits. The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established. Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro. The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay.


RESULTS
SDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000. The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells. Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method.


CONCLUSIONS
The established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities. Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene.
Gao
Chen
C
Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China.
Hou
Xingsheng
X
Zhang
Fuping
F
Zhou
Wei
W
Yuan
Yukang
Y
Dong
Xiaoping
X
D000818:Animals; D000906:Antibodies / Q000032:analysis; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D017930:Genes, Reporter; D008297:Male; D027383:Papillomaviridae / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase