Recombinant plasmid PEGFP-12 obtained by mutating mycobacterium tuberculosis ERP 12 sequence motif to PGLTS

A technology of PEGFP-12 and Mycobacterium tuberculosis, applied in the field of bioengineering, can solve the problems of unknown molecular mechanism, the relationship between conservation and function has not been studied, and achieve the effect of obvious inflammatory response

Inactive Publication Date: 2015-08-26
NINGXIA UNIVERSITY
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Problems solved by technology

The N-terminal, middle repeat sequence and C-terminal of Erp protein play a very important role in the function of the protein, but the specific molecular mechanism is still unknown, especially the relationship between the conservation of the PGLTS in the middle repeat sequence and the function has not yet been established. Research

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  • Recombinant plasmid PEGFP-12 obtained by mutating mycobacterium tuberculosis ERP 12 sequence motif to PGLTS
  • Recombinant plasmid PEGFP-12 obtained by mutating mycobacterium tuberculosis ERP 12 sequence motif to PGLTS
  • Recombinant plasmid PEGFP-12 obtained by mutating mycobacterium tuberculosis ERP 12 sequence motif to PGLTS

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Embodiment Construction

[0012] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0013] The complete sequence of ERP with 12 motifs completely mutated into PGLTS was synthesized by KingScript Biotechnology Co., Ltd., humanized and optimized, and constructed on the PEGFP-N1 eukaryotic expression vector. The cloning site is HindIII / PstI, and Named PEGFP-12. The mutated sequence is shown as SEQ:ID:NO:1.

[0014] Transform the vector into Escherichia coli to extract the plasmid, transfer the plasmid into A549 cells by lipofection, and then do protein chip detection to see the changes of cytokines after transfection compared with wild type, for research PGLTS motif mutations and functional relationships provide robust data.

[0015] PEGFP-12 and PEGFP-ERP recombinant plasmids were transfected into A549 cells as follows: figure 1 , figure 2 shown. The recombinant plasmid was transformed i...

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Abstract

The invention discloses a recombinant plasmid PEGFP-12 obtained by mutating mycobacterium tuberculosis ERP 12 sequence motif to PGLTS. According to a sequence of mycobacterium tuberculosis wild ERP, an ERP complete sequence of which the 12 sequence motif is fully mutated to PGLTS is synthesized, is optimized by way of humanization to be constructed to a PEGFP-N1 eukaryotic expression vector, and the obtained product is named PEGFP-12. The sequence after mutation is shown in SEQ ID: NO: 1. According to the invention, the recombinant plasmid PEGFP-12 of which mycobacterium tuberculosis ERP 12 sequence motif is mutated to PGLTS is constructed. The plasmid is transferred to the A549 cell by virtue of a lipofection transfection method, then protein chip detection is carried out and the sequence is compared with the wild ERP sequence, and after the sequence motif is fully mutated to 12 PGLTS, expression of cell inflammation related factors is obviously up-regulated. Therefore, a relatively obvious reaction can be induced after mutation, thereby providing powerful data for researching PGLTS sequence motif mutation and functional relationship.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to a recombinant plasmid PEGFP-12 in which the ERP 12 motif of Mycobacterium tuberculosis is mutated into PGLTS. Background technique [0002] Bovine tuberculosis is a chronic wasting infectious disease that affects the output of milk, meat and livestock products of animals, causing serious economic losses. Mycobacterium tuberculosis (Mycobacterium tuberculosis) is the main pathogenic bacterium that causes bovine tuberculosis. related to immune damage. [0003] Erp (exported repeated protein), also known as P36, Pirg or Rv3810, is an important virulence factor of Mycobacterium tuberculosis. Mycobacterium africanum, Mycobacterium africanum, BCG and Mycobacterium microti) also exists in the leprosy-causing bacterium Mycobacterium leprae, but subsequent studies have shown that in Mycobacterium smegmatis and the opportunistic pathogen Mycobacterium tuberculosis Expression of this...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
Inventor 王玉炯李敏郝秀静何玉龙马臣杰
Owner NINGXIA UNIVERSITY
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