[Construction eukaryotic plasmids of the ureI and ctB-ureI and profile its' expression in cell].
OBJECTIVE
To construct the ureI and ctB-ureI eukaryotic expression plasmid with pIRES-RD2 and study the expressing characters of those plasmids in HEK 293.


METHODS
We got the ureI and ctB-ureI gene from the procaryotic expression plasmid pET32a(+)-ureI and pET32a(+)-ctB-ureI by restriction enzyme BglII and XhoI, respectively.The pET32a(+)-ctB-ureI had been designed that ureI fused with Cholerae toxin B gene (ctB) at the 5'terminal of ureI to increase the immunogenicity of ureI, and then inserted those gens to eukaryotic expression plasmid pIRES-RD2 by ligase. After sequenced, the pIRES-RD2-ureI and pIRES-RD2-ctB-ureI were identical with the targets. These plasmids were transfected HEK293T cell strain by Lipofetmiane 2000. Then studied the transfection ratio of those cells by Fluorescence microscope, studied the expression features and the immunoreactivity of those cells by Immunofluorescence and Immunohistochemisty with the special antibodies.


RESULTS
Fluorescence microscope showed about 80% cells had been transfected the target plasmids, and Immunofluorescence and Immunohistochemisty demonstrated that the transfected cells expressed the recombinant proteins which located at the cytoplasm of HEK 293 strain after 48-72 hours. And the expressed products could react with the special antibodies, respectively.


CONCLUSIONS
We successfully constructed the ureI and ctB-ureI eukaryotic expression plasmid with pIRES-RD2, which were pIRES2-DsRed2-ureI and pIRES2-DsRed2-ctB/ureI. Both these two plasmids could express ureI or ctB-ureI in HEK 293 cell, and the expressed products had immunoreactivity, respectively.
Wang
Bao-ning
BN
Department of Microbiology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Yang
Yuan
Y
Kuang
Yu
Y
Cao
Kang
K
Li
Ming-yuan
MY
Li
Wan-yi
WY
D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D002772:Cholera Toxin / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000276:immunology; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C114527:UreI protein, Helicobacter pylori; D002772:Cholera Toxin