[Genetic engineering production of H37Rv M. tuberculosis proteins].
A complete library of M. tuberculosis H37Rv genes was produced by incorporating the DNA fragments of M. tuberculosis H37Rv into the lambda pSI phasmid. For this, DNA isolated from the mycobacteria was treated by EcoRI restrictases and the fragments of 8-17 thousand nucleotide pairs were crosslinked with the phasmid DNA. Hybrid DNA molecules were packed into the caspids from the proteins of E. coli BHB2688 and BHB2690 strains. By estimates, this library contained 98% of M. tuberculosis H37Rv genome so that any required gene can be found at 0.99 probability. The needed genes were sought by monoclonal antibodies against a protein with a molecular mass of 17-19 kDa (IT-12, IT-51, IT-54) obtained from the WHO. The protein gene was also produced by the method for raising the end sequences using synthesis of two oligonucleotides SP30 complementary to segments that limit this gene in the presence of Taq-DNA-polymerase and DNA of M. tuberculosis H37Rv. Copies of a gene (MT-1) were produced by denaturation, firing and raising. This gene was incorporated in the puC119 plasmid and expressed in E. coli cells, the direction of reading being checked up. A protein with a molecular mass of 19 kDa was detected in E. coli extracts with the expressed pMT-2 plasmid using monoclonal antibodies IT-12 and IT-54 in enzyme-linked immunoassay and immunoblotting.
Perel'man
B V
BV
Avdienko
V G
VG
Litvinov
V I
VI
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005818:Genetic Engineering; D015698:Genomic Library; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010957:Plasmids
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase
Genno-inzhenernoe poluchenie belkov M. tuberculosis H37Rv.