[Construction and identification of the E.coli-BCG shuttle vector expressing lipoprotein Der p2 on cell wall of mycobacterium vaccae].
OBJECTIVE
To construct the E.coli-BCG shuttle vector carrying and expressing dust mite antigen gene Der p2 on cell wall of mycobacterium vaccae.


METHODS
The gene fragment encoding 19 kDa antigen and the upstream control element (19-ss) was amplified by PCR from the mycobacteria tuberculosis H37Rv.Subsequently, the 19-ss gene was cloned into the E.coli-BCG shuttle vector pOLYG, which can schlepped and expressed exogenous antigen gene on cell wall of mycobacteria and containing the Der p2 gene. The expression of Der p2 gene in mycobacterium vaccae determined by indirect immunofluorescence staining.


RESULTS
Sequencing proved that the cloned sequence of 19-ss gene was correct. The constructed E.coli-BCG shuttle vector (pCW) containing 19-ss gene had function of shuttle between E.coli and mycobacteria, and mediated the expression of antibiotic resistance gene. Indirect immunofluorescence staining indicated that the Der p2 gene was expressed in the form of lipoprotein on surface of the mycobacterium vaccae.


CONCLUSIONS
E.coli-BCG shuttle vector has been constructed successfully, which can express exogenous antigen gene as a chimeric protein on cell wall.
Shi
Jie-Ran
JR
Department of Respiratory Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an 710032,China.
Li
Yuan
Y
Qi
Hao-Wen
HW
Li
Bie-Hu
BH
Fan
Xiong-Lin
XL
D002473:Cell Wall; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008074:Lipoproteins / Q000235:genetics; D009163:Mycobacterium bovis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics
D008074:Lipoproteins