[Construction and expression of eucaryotic recombinant plasmid of Legionella pneumophila mip gene].
OBJECTIVE
To construct recombinant plasmid of Legionella pneumophila mip gene and detect its expression in NIH3T3 cells.


METHODS
mip gene of Legionella pneumophila was amplified by PCR. The amplified DNA was ligated to pcDNA3.1(+) vector. The recombinant plasmid was named pcDNA3.1-mip. NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-mip with Lipofection strategy. Transient and stable products of mip gene were detected by immunofluorescence and Western-blot.


RESULTS
It was found that there was high green fluorescence on the cell membrane and inside the cell. It showed that NIH3T3 cell was transfected by pcDNA3.1-mip successfully. Rabbit serum antibody of Legionella pneumophila detected the NIH3T3 cell transfected with pcDNA3.1-mip. There was the protein in relative molecular weight 24 X 10(3), whereas no evidence for the protein in NIH3T3 cell transfected with pcDNA3.1(+) was seen. The protein expression of mip gene was shown.


CONCLUSIONS
We have successfully constructed the recombinant plasmid of Legionella pneumophila mip gene and detected the relative molecular weight 24 X 10(3) Mip protein in NIH3T3 cells.
Wang
Tao
T
Department of Parasitology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China.
Chen
Jian-Ping
JP
Li
Hong
H
Lei
Zhang
Z
Tao
Da-Chang
DC
Yang
Chun-Lei
CL
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D020104:Immunophilins / Q000096:biosynthesis* / Q000235:genetics; D016952:Legionella pneumophila / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D019696:Peptidylprolyl Isomerase / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D020104:Immunophilins; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase