[Study on gene cloning of Chlamydial pneumonia CPn0308 and its endogenous localization].
OBJECTIVE
To clone CPn0308 gene from Clamyida pneumonia and express its fusion protein, to make antibodies to fusion protein GST-CPn0308, and to further localize endogenous protein preliminarily using antibodies raised with CPn0308 fusion protein.


METHODS
The open reading frame (ORF) coding for CPn0308 in the Chlamydia pneumonia AR 39 genome was cloned into the pGEX6p2 vector after it was cloned using PCR and digested by the restriction enzymes BamHI and NotI. The recombinant plasmid pGEX6p2-CPn0308 was transformed into XL1-blue bacteria and the gene CPn0308 was expressed as fusion proteins with the glutathione-s-transferase (GST) tagged to the N-terminus. The GST-CPn0308 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CPn0308 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA).


RESULTS
The CPn0308 gene, which was 366bp in length,was successfully cloned and the GST fusion protein with molecular weight of 39kD was expressed. It was found that the hypothetical protein CPn0308 was located in the inclusion membrane of Chlamydia pneumonia-infected cells using IFA of mouse anti-fusion protein antibodies.


CONCLUSIONS
Using antibodies raised with GST-CPn0308 fusion protein, the hypothetical protein CPn0308 was identified to be a Chlamydia pneumoniae inclusion membrane protein. It could be the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells.
Jia
Tianjun
T
Department of Epidemiology, Hebei Medical University, Shijiazhuang, Shijiazhuang 050017, China. jiatianjun@yahoo.com
Liu
Dianwu
D
Luo
Jianhua
J
Zhang
Shumin
S
et al
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D061387:Chlamydial Pneumonia / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D054884:Host-Pathogen Interactions; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; C094163:IncA protein, bacteria; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase