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Composition, screening model and screening method for screening anti-tuberculosis drugs

A technology of culture and aspartic acid, applied in the field of medicine, can solve problems such as difficult to break through MTBASD, and achieve the effect of high-throughput screening

Active Publication Date: 2020-11-13
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The possible reason is also that it is difficult to break through the bottleneck of MTBASD expression

Method used

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  • Composition, screening model and screening method for screening anti-tuberculosis drugs
  • Composition, screening model and screening method for screening anti-tuberculosis drugs
  • Composition, screening model and screening method for screening anti-tuberculosis drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Cloning and expression of embodiment 1 recombinant aspartate semialdehyde dehydrogenase ASD

[0064] (1) Cloning of asd gene and construction of expression vector

[0065] Primers were designed according to the ASD gene sequence published by NCBI (GenBank No. 9 885118): asdh F (5'-TTTT GAATTC ATGGGCCTGTCAATAGGGATC-3') and asdh R (5'AAAA AAGCTT TCACAAGTCGGCGGTCAGC) was used to amplify the ASD gene using the MTB genome as a template. Underlined parts are EcoR I and Hind III restriction enzyme sites. The reaction system is:

[0066]

[0067] PCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and primers were synthesized by Beijing Huada. The reaction procedure is:

[0068]

[0069] The PCR product was purified using a gel recovery kit (Beijing Quanshijin Biotechnology Co., Ltd.), the purified product and plasmid pET28a(+) were digested with EcoR I and Hind III (Dalian TaKaRa Company) respectively, and the digested fragments were heat-s...

Embodiment 2

[0074] Embodiment 2 Purification of recombinant aspartate semialdehyde dehydrogenase

[0075] Cell disruption: Suspend the collected cells with lysis buffer (20mM Tris-HCl, 500mM NaCl, 10mM imidazole, 1mM DTT, pH 8.0) into a 50mg / mL bacterial solution; use a pressure breaker pre-cooled to -15°C The cells were broken three times under the pressure of 800 bar; the broken cell solution was centrifuged at 10000 g for 20 min to remove insoluble matter, and then filtered with a 0.45 μm filter.

[0076] Protein purification: using The explorer system was used for protein purification, using 1mL prepacked column HiTrapChelating HP as the purification medium.

[0077] Elute with a buffer containing 20mM Tris-HCl, 500mM NaCl, 400mM imidazole and 1mM DTT, pH 8.0 according to the following procedure:

[0078] The eluent concentration was 0-100%, the elution time was 10 min, the collection volume was set at 1 mL, and the purification effect was detected by SDS-PAGE.

[0079] Protein de...

Embodiment 3

[0093] Example 3 Expression and Purification of Recombinant Escherichia coli Type III ASK (Lys C)

[0094] (1) Cloning of LysC gene and construction of expression vector

[0095] Primers were designed according to the LysC gene sequence published by NCBI (GenBank No. 948531): lysC F(5'-TTTT GAATTC ATGTCTGAAATTGTTGTCT-3') and lysC R(5'-AAAA AAGCTT TTACTCAAACAAATTACTA-3') was used to amplify the LysC gene using the Escherichia coli genome as a template. Underlined parts are EcoR I and Hind III restriction enzyme sites. The reaction system is:

[0096]

[0097] PCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and primers were synthesized by Beijing Huada. The reaction procedure is:

[0098]

[0099] The PCR product was purified using a gel recovery kit (Beijing Quanshijin Biotechnology Co., Ltd.), the purified product and plasmid pET28a(+) were digested with EcoR I and Hind III (Dalian TaKaRa Company) respectively, and the digested fragment...

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Abstract

The invention provides a composition for screening anti-tuberculosis drugs, and a screening model and a screening method and particularly provides a method for heterogeneously expressing aspartate semi-aldehyde dehydrogenase (ASD), which includes the following steps: 1) transferring a bacterial strain, containing a pET28a (+) :: asd expression plasmid, to a culture medium containing Km and a reagent for improving the osmotic pressure of cells, and culturing the strain to prepare a transferred culture, wherein the concentration of the reagent is 500-1000 mM; 2) adding IPTG to the transferred culture and culturing the culture at 20-23 DEG C to obtain the bacterial cells for expressing the ASD, wherein the final concentration of the IPTG is 5-20 [mu]M, preferably 10-15[mu]M; 3) collecting thebacterial cells in the step 2), and performing lysis to obtain the ASD. The invention also provides the composition containing the ASD and used for screening anti-tuberculosis drugs, and the screening model and the screening method, by which the anti-tuberculosis drugs can be screened out quickly and economically at high throughput.

Description

technical field [0001] The invention relates to the field of medicine, in particular, the invention relates to a composition for screening anti-tuberculosis drugs, a screening model and a screening method. Background technique [0002] The widespread application of anti-tuberculosis drugs such as rifampin, isoniazid and ethambutol has greatly reduced the mortality rate of tuberculosis. However, with the long-term use and irrational or irregular use of these anti-TB drugs, single drug resistance (SDR), multidrug resistance (MDR) and even extensive drug resistance (XDR) have emerged. ) Mycobacterium tuberculosis (MTB). Statistics show that 3.7% of newly infected tuberculosis patients are MDR-TB infected, the MDR-TB carrier rate of re-treated patients is as high as 20%, and XDR-TB patients among MDR-TB patients is as high as 9.0%. The continuous generation and spread of drug-resistant MTB has reduced the cure rate of tuberculosis, and the mortality rate of tuberculosis patien...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12Q1/48C12Q1/32C12R1/19
CPCC12N9/0008C12Q1/32C12Q1/485C12Y102/01011G01N2333/91225
Inventor 肖春玲蒙建州刘忆霜邓琪关艳王冕韩江雪李东升
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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