[Expression and immunity of fused protein H1N1 M2e and cholera toxin B].
OBJECTIVE
To construct the eukaryotic recombinant plasmid pcDNA3.1a(+)-M2e/CtB which contains H1N1 M2e gene and cholera toxin B subunit gene (CtB) and to study the expression and immunity of recombinant protein M2e/CTB in NIH3T3 cells.


METHODS
M2e/CtB gene was cloned by PCR and digested with Hind III and Xho I. M2e/CtB was linked into pcDNA3.1a(+) to build eukaryotic expression plasmid pcDNA3.1a(+)-M2e/CtB. The pcDNA3.1a(+)-M2e/CtB was transfered into competent E.coli JM109. The transformed colonies were verified by Hind III and Xho I, PCR, and sequencing. The NIH3T3 cells were transfected with pcDNA3.1a(+)-M2e/CtB by positive ion polymer. The product of M2e/CtB gene with stable expression was analysed by immunofluorescence, RT-PCR sequencing, and Western blot.


RESULTS
The digested pcDNA3.1a (+)-M2e/CtB contains correct M2e and CtB gene. Blastn results showed the genetic homongenicity of M2e and CtB were 100%, respectively. The pcDNA3.1a(+)-M2e/CtB efficiently expressed M2e/CTB protein in the eukaryotic NIH3T3 cells. The cell lysis and supernant both contained the M2e/CTB protein, which had the immunity and reactivity to both anti-M2e and anti-CTB. The relative molecular mass of M2e/CTB protein was about 18 000 analysed by Western blot.


CONCLUSIONS
A new recombinant eukaryotic plasmid pcDNA3.1a(+)- M2e/CtB has been successfully constructed. The plasmid can express the fused protein of M2e with CTB. Our study will help further research into new and effective DNA vaccines against influenza virus A.
Yang
Xiao-fang
XF
School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. yxfang1981@sina.com
Jiang
Yan
Y
Li
Wan-yi
WY
Kuang
Yu
Y
Jiang
Zhong-hua
ZH
Wang
Feng-ping
FP
Li
Ming-yuan
MY
D000818:Animals; D015153:Blotting, Western; D002772:Cholera Toxin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D002772:Cholera Toxin