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Method for preparing isoniazid nicotinamide adenine dinucleotide by synthetic biology

A technology of hydrazine nicotinamide adenine and synthetic biology, which is applied in the field of bioengineering and can solve problems such as high toxicity

Pending Publication Date: 2018-10-02
FOSHAN UNIVERSITY
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  • Application Information

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Problems solved by technology

Second-line drugs are more toxic and less effective than first-line drugs, and are mainly used for the treatment of MDR-TB and prolonging treatment time
The anti-tuberculosis activity of isoniazid must be its activated compound (INH-NAD). The InhA protein in clinical isolates of drug-resistant tuberculosis usually mutates, resulting in the inability of INH-NAD to bind to the InhA protein and resulting in tuberculosis resistance. Influenced by INH

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  • Method for preparing isoniazid nicotinamide adenine dinucleotide by synthetic biology
  • Method for preparing isoniazid nicotinamide adenine dinucleotide by synthetic biology
  • Method for preparing isoniazid nicotinamide adenine dinucleotide by synthetic biology

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Embodiment 1

[0030] Example 1: Preparation of isoniazid nicotinamide adenine dinucleotide by synthetic biology.

[0031] PCR amplification of DNA sequence fragments of katG and inhA: PCR amplification of DNA sequence fragments of katG and inhA was amplified using primer pairs ethANFP / ethAKRP and inhABFP / inhAHRP, respectively, and the primer sequences were as follows:

[0032] katGBglIIFP: 5'-ACCAAGATCTATGCCCGAGCAACACCCACC-3' (SEQ ID No. 1);

[0033] katGEcoRIRP: 5'-CCCCGAATTCTCAGCGCACGTCGAACCT-3' (SEQ ID No. 2);

[0034] InhANcoIFP: 5'-ACAACCATGGGAATGACAGGACTGCTGGACGGC-3' (SEQ ID No. 3);

[0035] InhAHindШRP: 5'-ATGCAAGCTTGAGCAATTGGGTGTGCGCGCCG-3' (SEQ ID No. 4).

[0036] After the target DNA fragment amplified by PCR undergoes nucleic acid gel electrophoresis, the target DNA fragment is recovered using the gel recovery kit from Omega Company, and the recovered DNA fragment is recovered. Store in -20°C refrigerator for later use.

[0037] Construction of the pTrcHis2c-katG plasmid:

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Abstract

The invention provides a method for preparing isoniazid nicotinamide adenine dinucleotide by synthetic biology, wherein the method includes the steps: 1) PCR amplification of katG and inhA DNA sequence fragments; 2) construction of a pTrcHis2c-katG plasmid; 3) construction of a pET28a-inhA plasmid; 4) purification of an InhA protein; and 5) purification of an INH-NAD compound. The method has the beneficial effects of directly synthesizing the INH-NAD in escherichia coli cells and realizing purification and separation of the INH-NAD through ultrafiltration tube concentration and purification, short-time heating, centrifugation, filtration and other steps.

Description

technical field [0001] The invention relates to the technical field of bioengineering. Background technique [0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (TB) infection. Tuberculosis is one of the infectious diseases that have plagued mankind for the longest time and are the most harmful. In 1882, Mycobacterium tuberculosis, the pathogenic bacterium of tuberculosis, was isolated and identified by German scientist Koch. Prior to this, tuberculosis had occurred many times in large-scale epidemics around the world, and it was called the "white plague". Since the 1950s, humans have continuously discovered effective anti-tuberculosis drugs, which has brought the prevalence of TB under control. Anti-tuberculosis drugs are a double-edged sword. They can not only treat TB, but they can also lead to the emergence of corresponding anti-tuberculosis drug-resistant strains of Mycobacterium tuberculosis. [0003] Today, the prevention and co...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N9/02C12P19/36
CPCC12N9/001C12N15/66C12N15/70C12P19/36
Inventor 王绪德周亚凤毕利军周盈张晓丽
Owner FOSHAN UNIVERSITY
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