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Antibody and encoding gene of mycobacterium tuberculosis Acr protein and application thereof

A gene and protein technology, applied to the antibody of Mycobacterium Acr protein and its encoding gene and application field, can solve problems such as latent infection

Inactive Publication Date: 2013-06-05
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The currently used anti-tuberculosis drugs are mainly obtained through traditional random screening from 1945 to 1966. In recent years, some new rifampicin drugs (rifabutin, rifapentine, KRM-1648, etc.) and New quinolones (levofloxacin, ofloxacin, sparfloxacin, moxi-floxacin, etc.), these drugs can only be used for active pulmonary tuberculosis, and latent infection (accounting for about 90% of all infected patients) %) does not work, and with the increasing severity of drug-resistant strains and multi-drug-resistant strains, it is urgent to develop new methods to prevent and control this disease. The fully human antibody drug screened against the dormancy protein Acr may Addressing two of the biggest challenges in the current landscape (i.e. latent infection and drug resistance)

Method used

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  • Antibody and encoding gene of mycobacterium tuberculosis Acr protein and application thereof
  • Antibody and encoding gene of mycobacterium tuberculosis Acr protein and application thereof
  • Antibody and encoding gene of mycobacterium tuberculosis Acr protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1, the acquisition of the antibody of Acr protein

[0033] 1. Prokaryotic expression of Mycobacterium tuberculosis Acr protein

[0034] ①Take the genome of MTB H37RV strain (Expression and purification of ACR protein of Mycobacterium tuberculosis in E.coli.Chinese Journal of Preventive Veterinary Medicine, 2009.Vol.31 No.7pp.528-531) as a template, and use the synthetic primers (sequence as follows :primer -f:

[0035] 5'-GGATCCAAAATGGCCACCACCCCTTCCCGTT-3', primer-r:

[0036] 5'-GGAAGCTTCAGTTGGTGGACCGGATCTGAATGTG-3) for PCR amplification, successfully amplified a PCR product of 435bp with the expected length of Mycobacterium tuberculosis Acr ( figure 1 , M: 2000bp DNA Marker, 1: PCR product).

[0037] The PCR product was connected to the peasy-blunt simple vector (Beijing Quanshijin Company, catalog number CB111-01) to obtain a recombinant vector, and the recombinant vector was digested with BamHI and Hind III to obtain a 435bp fragment as a positive recom...

Embodiment 2

[0062] Embodiment 2, the functional verification of the antibody of Acr protein

[0063] 1. Human antibody neutralization activity experiment

[0064] (1) Coating: the purified Acr protein obtained in Example 1, ovalbumin (OA) (purchased from sigma company product number: A5503-1G), SC protein (amino acid sequence is sequence 30), HIV-1 core protein P24 protein (The amino acid sequence is sequence 31) Antigens were diluted with coating solution to 2.5 μg / ml, 100 μl was added to each well of a polystyrene enzyme-linked detection plate, and coated overnight at 4°C.

[0065] (2) Blocking: add 200 μl per well or top up with blocking solution (5% skimmed milk powder), after 2 hours at 37°C, wash 3 times with PBST (0.05% Tween20), and pat dry.

[0066] (3) Add the sample to be tested: take the soluble single-chain antibody scFv1-scFv14 obtained in the third step of Example 1, add 100 μl / well to the coated microtiter plate, at the same time, incubate at 37°C for 30 minutes, and wash...

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Abstract

The invention discloses an antibody and an encoding gene of mycobacterium tuberculosis Acr protein and application of the antibody and the encoding gene of the mycobacterium tuberculosis Acr protein. The invention provides polypeptide which is the antibody of the mycobacterium tuberculosis Acr protein. The mycobacterium tuberculosis Acr protein can be any one of (1)-(15), including (1) polypeptide composed of amino acid sequences shown in a sequence 2 of a sequence table, (2) polypeptide composed of amino acid sequences shown in a sequence 4 of the sequence table, (3) polypeptide composed of amino acid sequences shown in a sequence 6 of the sequence table, and the like. According to experimental evidence, a humanized antibody bank is adopted to screen humanized antibodies of the mycobacterium tuberculosis Acr protein, a humanized antibody with high-neutralization activity is screened out according to an external neutralization test, and fourteen strains of humanized antibody sequences are obtained. Good foundation is provided for construction of humanized single-chain antibody preparations which are used for preventing mycobacterium tuberculosis and have good medical value, and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody to Acr protein of Mycobacterium tuberculosis and its coding gene and application. Background technique [0002] Tuberculosis has always been a global health problem that people are concerned about. Latent infection and drug resistance are two major problems in the treatment of tuberculosis. The development of new fully human antibody drugs can solve the two problems currently encountered, which is of great significance . [0003] Latent infection and drug resistance are two major problems in tuberculosis treatment: MTB latent infection (latent tuberculosis infection, LTBI) is a subclinical state, at which time the bacteria are dormant. About 1 / 3 of the general population is infected with Mycobacterium tuberculosis, and about 10% of those infected may develop TB, and most of them are in the form of latent infection. This latent infection state has a latency period of up ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/11C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21G01N33/569C12Q1/68C12Q1/04
Inventor 童贻刚周丽君冯福民安小平米志强王晓娜
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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