[Molecular cloning and expression of hypothetical proteins Rv1494 and Rv1495 of M.tuberculosis H37Rv strain].
OBJECTIVE
The mechanism by which M.tuberculosis persists and survives in host macrophage is not fully understood, however, the M. tuberculosis chromosome-encoded TA loci perform functions possibly of signaling to these processes. To explore the biological functions of M. tuberculosis chromosome-encoded TA loci, the Rv1494 and Rv1495 genes of M.tuberculosis H37Rv strain were cloned and expressed.


METHODS
The hypothetical proteins Rv1494 and Rv1495 were bioinformatically analyzed by means of Bioedit software, Dnaman software and Pfam database. The complete open-reading frame sequences of Rv1494 and Rv1495 genes were amplified by PCR using M.tuberculosis H37Rv genomic DNA as the template, and the PCR products were cloned into prokaryotic expression vector pET32a(+), respectively. After induction of expressions in E.coli host strain BL21 (DE3), the recombinant proteins were purified and detected by Western blotting.


RESULTS
According to bioinformatic analysis, the hypothetical proteins of Rv1494 and Rv1495 genes shared some homologies with mazEF family, one of E. coli chromosomal TA loci (homology at 26% and 29.5%). Sequence analysis showed that the inserted target genes and its reading frames were completely correct. The recombinant plasmids were induced with IPTG to effectively express the fusion proteins with relative molecular mass coincident with prediction. The specific positive signals were identified from the immunoblots.


CONCLUSIONS
For the first time, the Rv1494 and Rv1495 genes of M.tuberculosis H37Rv strain were cloned and its prokaryotic expression vectors were constructed successfully in this experiment, which may facilitate further functional study of this mazEF-like gene pair.
Shang
Zheng-Ling
ZL
Department of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. shangzhengling@126.com.
Bao
Lang
L
Yao
Su-Xia
SX
Zhang
Hui-Dong
HD
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins