Screening method of drug for resisting retention-state mycobacterium tuberculosis

A technology of mycobacterium tuberculosis and drugs, applied in the direction of antibacterial drugs, biochemical equipment and methods, and pharmaceutical formulations, which can solve problems such as easy recurrence and difficult to cure tuberculosis completely

Inactive Publication Date: 2010-02-10
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

In addition, tuberculosis is difficult to cure completely and is easy to relapse, which is also closely related to the long-term existence of Mycobacterium tuberculosis in a "persistent" state

Method used

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  • Screening method of drug for resisting retention-state mycobacterium tuberculosis
  • Screening method of drug for resisting retention-state mycobacterium tuberculosis
  • Screening method of drug for resisting retention-state mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The construction of embodiment 1 prokaryotic expression plasmid

[0083] Using Mycobacterium tuberculosis H37Rv genomic DNA as a template, the full sequence of pknG gene was amplified by PCR. The primer sequence is: upstream primer 5'TGGA CATATG GCCAAAGCGTCAGAG 3', downstream primer 5' TCG CTCGAG ATAGAACGTGCTGG 3' (the restriction sites of NdeI and XhoI are indicated by the underline).

[0084] The PCR reaction system is as follows:

[0085] 5×PrimeSTAR Buffer (Mg 2+ Plus) 10μl

[0086] dNTP Mixture (2.5mM each) 4μl

[0087] Forward Primer (20pM) 1μl

[0088] Reverse Primer (20pM) 1μl

[0089] Template (70ng / μl) 1μl

[0090] PrimeSTAR HS DNA Polymerase (2.5U / μl) 0.5μl

[0091] sterile ddH 2 O 32.5 μl

[0092] Total volume 50μl

[0093] The PCR reaction procedure is as follows:

[0094]

[0095] After the amplified product was verified, it was inserted into the multiple cloning site (NdeI and XhoI) of the prokaryotic expression vector pET30a (Novagen) t...

Embodiment 2

[0096] Induced expression of embodiment 2pknG gene

[0097] The recombinant plasmid pET-pknG constructed in Example 1 was transformed into Escherichia coli Bl21 (λDE3) competent cells. Escherichia coli Bl21 containing pET-pknG was inoculated in LB medium containing 50 μg / ml Kan, cultured with shaking at 200 rpm at 37 °C until the culture medium became turbid, and then the above culture was transferred to a medium containing 50 μg / ml Kan at a ratio of 1:100. ml Kan's fresh LB medium, 37 ° C 200 rpm shaking culture OD 600 = about 0.6. Add IPTG to the culture at a final concentration of 1 mM, transfer to 28° C. at 200 rpm to continue shaking culture for 3-5 hours, and harvest the cells after induction of expression by centrifugation.

Embodiment 3

[0098] The separation and purification of embodiment 3PknG protein

[0099] The bacterial cells harvested in Example 2 were resuspended by shaking with the lysis buffer, the bacterial cells were lysed by ultrasonic waves with a power of 400W, and centrifuged at 15000g for 30min to remove impurities such as cell debris. Since the induced expression of recombinant PknG has a His-tag, use HisTrap TM HP nickel ion affinity chromatography column is used as purification medium, through The explorer system separates and purifies PknG protein. The purified protein was desalted and stored at -80°C.

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Abstract

The invention discloses a screening method of a drug for resisting retention-state mycobacterium tuberculosis, which utilizes the change of the light absorption value of an enzyme-labeling instrumentdetection reaction system to judge the inhibition function to the activity of protein kinase G in a sample by adding the sample to be measured into an enzyme activity measuring system, thereby screening a drug for resisting the retention-state mycobacterium tuberculosis. The screening method selects the protein kinase G as a target point and is different from the pervious antitubercular drug whichmainly aims at bacteria with active growth and propagation. The invention aims at solving the difficult problem of long-term incubative infection of mycobacterium tuberculosis by aiming at bacteria which exists in host cells for a long time in a retention state.

Description

technical field [0001] The invention relates to a screening method for drugs, in particular to a screening method for drugs against persistent Mycobacterium tuberculosis. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that seriously endangers human health. For a long time, the main means for people to fight against tuberculosis is to use antibiotics for chemical treatment, which once effectively controlled the spread and spread of tuberculosis in the world. However, in recent years, tuberculosis, which has been effectively controlled, has made a comeback, and its morbidity and mortality have risen significantly, and it has become an infectious disease alongside AIDS and malaria. There are many reasons for the resurgence of tuberculosis, including increasing population mobility, increasing HIV infection and the emergence of multi-drug resistant bacteria. In addition, tuberculosis is difficult to cure completel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/32G01N21/33A61K45/00A61P31/06
Inventor 余利岩邵天舒岑山黄彬张玉琴
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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