Construction method of complete mycobacterium tuberculosis genome ORF clone library and application thereof

A Mycobacterium tuberculosis, whole genome technology, applied in the field of constructing Mycobacterium tuberculosis whole genome ORF clone library, can solve problems such as inability to guarantee complete and correct expression of cloned genes, high randomness, and inconvenient screening work

Inactive Publication Date: 2009-09-23
HUAZHONG AGRI UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

The bacterial two-hybrid clone library used in their research is composed of randomly interrupted gene fragments of Mycobacterium tuberculosis H37Rv. The defect is that the complete and correct expression of the cloned gene cannot be guaranteed, and the randomness is high, which hinders the screening work. Brings great inconvenience, especially some important protein-protein interactions may be neglected

Method used

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  • Construction method of complete mycobacterium tuberculosis genome ORF clone library and application thereof
  • Construction method of complete mycobacterium tuberculosis genome ORF clone library and application thereof
  • Construction method of complete mycobacterium tuberculosis genome ORF clone library and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Transformation of bacterial two-hybrid pTRG vector

[0029] The XbaI restriction site at position 211 on the bacterial two-hybrid vector pTRG (accession number: GI: 13958039) purchased from Invitrogen was eliminated, and the XbaI restriction site at position 1076 was retained to obtain an intermediate vector pTRG-XbaIΔ. A gene fragment of the microchromosome maintenance gene MCM gene (accession number: GI: 15897676) of the extreme thermophilic archaea S. solfataricus was introduced into the multiple cloning site, with a length of 2061bp. The recombinant plasmid was named pTRG-MCM. Since there are two BamHI restriction endonuclease recognition sites inside the MCM, they affect the application of the vector in the construction of the cloning library, so the pTRG-MCM plasmid is fully digested with BamHI to form a linear deletion of 844 bases. Fragment, and then the fragment is further flattened and then self-connected to form pTRG-MCMΔ, the nucleotide sequen...

Embodiment 2

[0068] Example 2: Amplification of the whole genome ORF of Mycobacterium tuberculosis H37Rv

[0069] According to the genome sequence of Mycobacterium tuberculosis H37Rv provided by GenBank (GenBank accession number: AL123456), the restriction sites inside all coding genes were analyzed, and the restriction sites not inside all coding genes were selected as restriction enzymes for primer design site. When designing primers, all genes were divided into six categories. The first five categories of genes introduced EcoRI and XbaI, NotI and XbaI, EcoRI and XhoI, NotI and XhoI, BamHI and XhoI restriction sites in the upstream primer and downstream primer respectively. The six types use linkers to introduce an EcoRI restriction site into the linker and an XbaI restriction site into the downstream primer. By using the design method of the primers, the entire coding gene of Mycobacterium tuberculosis H37Rv can be amplified.

[0070] Examples are as follows:

[0071] Primers were de...

Embodiment 3

[0091] Example 3: Construction of the complete genome ORF bacterial two-hybrid clone library of Mycobacterium tuberculosis H37Rv

[0092] The six types of gene amplification products described in Example 2 were mixed and ligated with the pTRG-MCMΔ vector prepared by the corresponding combination of restriction enzymes, and ligated overnight at 16°C. After the ligation product was desalted, it was electrotransformed into Escherichia coli DH10B electroporation competent (transformation conditions: 1800V, 4-6mS), and more than 12,000 recombinant clones were obtained, that is, the gene coverage reached more than 3 times. The bacterial strain (Escherichia coli DH10B) containing the recombinant clone was mixed and cultured, the plasmid was extracted according to the routinely reported method, and the obtained glycerol bacteria and the plasmid were stored at -20°C. For the electropherogram of grouped mixed recombinant plasmids, see Image 6 .

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Abstract

The invention belongs to the technical field of microbial genetic engineering, specifically relates to a construction method of a complete mycobacterium tuberculosis H37Rv genome ORF bacterial two-hybrid clone library and application thereof. The construction method is characterized by comprising the following steps: preparing a bacterial two-hybrid carrier pTRG-MCM delta suitable for constructing the complete mycobacterium tuberculosis H37Rv genome ORF clone library; obtaining all encoded genes of the mycobacterium tuberculosis H37Rv by a PCR reaction; accurately cloning all encoded genes of the mycobacterium tuberculosis H37Rv into the constructed bacterial two-hybrid carrier pTRG-MCM delta; and transforming Escherichia coli DH10B to obtain the complete mycobacterium tuberculosis H37Rv genome ORF bacterial two-hybrid clone library.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering. In particular, it relates to a method and application for constructing a whole genome ORF clone library of Mycobacterium tuberculosis. Background technique [0002] Tuberculosis is a worldwide infectious disease caused by Mycobacterium tuberculosis, and it is also the disease with the highest mortality caused by a single bacterial infection. At present, there are 8 to 10 million new tuberculosis patients in the world every year, and the annual death toll is about 2 million (World Health Organization (2008) WHO Report 2008 Global Tuberculosis Control.), it is one of the infectious diseases that are controlled globally. [0003] The interaction between proteins and the protein complexes formed through the interaction are the main completion of the basic functions of cells. Almost all important life activities, including DNA replication and transcription, protein synthesis an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/74C40B50/06C12R1/19C12R1/32
Inventor 何正国王毅
Owner HUAZHONG AGRI UNIV
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