[Screening and analysis of in vivo induced genes of Mycobacterium tuberculosis].
OBJECTIVE
To screen in vivo induced genes of Mycobacterium tuberculosis and search possible molecular targets of new drugs, vaccines, and early diagnostic methods.


METHODS
In vivo induced antigen technology (IVIAT) was used in this study. Genomic DNA from M. tuberculosis of the strain H37Rv was extracted. The DNA was partially digested with Sau3A I and the purified fragments were inserted into the pET30a (+), pET30b (+) and pET30c (+) expression vectors to construct a genomic library. The library was induced with IPTG and then was screened with pooled tuberculosis patient sera preabsorbed with in vitro grown M. tuberculosis of the strain H37Rv and Escherichia coli of the strain BL21 (DE3). The inserts of positive clones were sequenced with primer T7 promoter. The sequences were aligned in the genomic database of M. tuberculosis strain H37Rv (http://genolist.pasteur.fr/Tuberculose) to identify the open reading frame (ORF).


RESULTS
The genomic expression library included 4.3 x 10(4) clones, and more than eighty percent were recombinant plasmids. The library reached the theoretic requirement. The successive adsorptions significantly decreased the anti-M. tuberculosis antibody titer of sera, and no significant difference was found between the last two adsorption groups, suggesting that the antibodies reactive against the M. tuberculosis H37Rv antigens expressed in vitro were removed. After screening of the genomic expression library and searching in the genome database, 51 ORFs were identified and they were classified into 8 categories according to the classification criterion on the website, including 1 virulence gene, 13 cell wall and cell processes genes, 11 intermediary metabolism and respiration genes, 7 lipid metabolism genes, 2 information pathways genes, 3 PE/PPE genes, 12 conserved hypotheticals, and 2 conserved hypotheticals with an orthologue in M. bovis.


CONCLUSIONS
Genes expressed specially during human M. tuberculosis infections can be identified with in vivo induced antigen technology. Analysis of these genes identified using IVIAT shows that some genes are related to virulence, some are essential genes for M. tuberculosis, and some encoded proteins have strong immunogenicity, suggesting that some of them can be used as molecular targets of anti-tuberculosis drugs, vaccines, and tuberculosis early diagnosis.
Zhang
Zong-de
ZD
Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
Li
Zi-Hui
ZH
DU
Bo-Ping
BP
Jia
Hong-Yan
HY
Liu
Zhong-Quan
ZQ
Chen
Xi
X
Huang
Hai-Rong
HR
Xing
Ai-Ying
AY
Gu
Shu-Xiang
SX
Ma
Yu
Y
D000942:Antigens, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D015698:Genomic Library; D006801:Humans; D008403:Mass Screening; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D017422:Sequence Analysis, DNA; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000175:diagnosis / Q000382:microbiology*
D000942:Antigens, Bacterial; D004269:DNA, Bacterial