[In vitro expression of Lgeionella pneumophila pilE gene and its immunogenicity].
OBJECTIVE
To construct a recombinant plasmid containing Lgeionella pneumophila pilE gene, detect its expression in NIH3T3 cells and evaluate its immunogenicity.


METHODS
PilE gene (LP) was amplified from Legionella pneumophila DNA by PCR and inserted into pcDNA3.1(+) vector to construct the recombinant plasmid pcDNA3.1-pilE, which as verified by restriction endonuclease digestion, PCR and DNA sequencing analysis. NIH3T3 cells were transfected with the recombinant plasmid with Lipofection strategy. Transient and stable pilE gene products were detected by immunofluorescence and Western blotting, respectively. To evaluate the immunogenicity of pcDNA3.1-pilE, the recombinant plasmid was used as a DNA vaccine to immunize female BALB/c mice intramuscularly and the specific antibodies, lymphocyte proliferation response, interferon (IFN)-gamma production and cytotoxic T-lymphocyte response of the immunized mice were detected and evaluated.


RESULTS
The pilE gene of 429 bp in length was amplified. After transfection of NIH3T3 cells with the recombinant plasmid, strong green fluorescence was observed on the cell membrane and inside the cell. A protein with relative molecular mass of 15.7 kD was detected in the transfected cells with Western blotting, suggesting successful protein expression of pilE gene. pcDNA3.1-pilE resulted in much stronger immune response in the immunized mice than pcDNA3.1(+) (P<0.01).


CONCLUSIONS
The recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 cells, and can induce specific humoral and cellular immune responses in mice.
Yang
Zhi-Wei
ZW
Department of Parasitology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. yangzhw0817@163.com
Chen
Jian-Ping
JP
Wang
Tao
T
Tian
Yu
Y
Liu
De-Song
DS
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D036781:Fimbriae Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010861:Fimbriae, Bacterial; D005455:Fluorescent Antibody Technique; D006801:Humans; D007114:Immunization / Q000379:methods; D007273:Injections, Intramuscular; D007371:Interferon-gamma / Q000378:metabolism; D016952:Legionella pneumophila / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology; D014162:Transfection; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D036781:Fimbriae Proteins; D007371:Interferon-gamma