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
View PDF3 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap