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Recombinant TM4 bacteriophage library for constructing NADH dehydrogenase gene family deleted mycobacterium tuberculosis and application thereof

A Mycobacterium tuberculosis and phage library technology, applied in the field of tuberculosis, can solve the problems of affecting autophagic cell apoptosis, affecting the growth and virulence of Mycobacterium tuberculosis, and achieve the effect of good knockout specificity and high positive rate

Inactive Publication Date: 2019-02-26
上海晶诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the 14 proteins NuoA-NuoN, the NuoG protein has been studied more. It has been found that NuoG can affect the autophagy, release of inflammatory factors and apoptosis of macrophages infected by Mycobacterium tuberculosis, and affect the growth of Mycobacterium tuberculosis. , virulence, etc., while the other 13 proteins are relatively less studied, and the specific functions need to be further studied

Method used

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  • Recombinant TM4 bacteriophage library for constructing NADH dehydrogenase gene family deleted mycobacterium tuberculosis and application thereof
  • Recombinant TM4 bacteriophage library for constructing NADH dehydrogenase gene family deleted mycobacterium tuberculosis and application thereof
  • Recombinant TM4 bacteriophage library for constructing NADH dehydrogenase gene family deleted mycobacterium tuberculosis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] The design of embodiment 1 knockout primers

[0073] According to Mycobacterium tuberculosis NADH dehydrogenase family genes (Rv0392c, Rv1854c, Rv3145, Rv3146, Rv3147, Rv3148, Rv3149, Rv3150, Rv3151, Rv3152, Rv3153, Rv3154, Rv3155, Rv3156, Rv3158) gene group in Mycobacterium tuberculosis Select the appropriate fragment length on the selected target sequence as the position of the left and right homology arms to design primers (about 600-1000bp) and synthesize them, and further screen the following knockout primers according to the amplification and knockout effects.

[0074] Rv0392c:

[0075] Left arm upstream primer Rv0392cLF: GTCGGTGGCATACAACTGGG (SEQ ID NO: 1),

[0076] Left arm downstream primer Rv0392cLR: GGTGGGTCGTTGTCTTGGAG (SEQ ID NO: 2),

[0077] Right arm upstream primer Rv0392cRL: CCTGGTCTACCTGGTCGGCTAT (SEQ ID NO: 3),

[0078] Right arm downstream primer Rv0392cRR: AAGTGGTGCTGGACAACG (SEQ ID NO: 4);

[0079] Rv1854c:

[0080] Left arm upstream primer Rv...

Embodiment 2

[0171] Example 2 Construction of the recombinant shuttle vector library required for knocking out the Mycobacterium tuberculosis NADH dehydrogenase gene family phage library

[0172] Using the primer combination in Example 1, a recombinant shuttle vector library (the recombinant shuttle vector library required for knocking out the Mycobacterium tuberculosis NADH dehydrogenase gene family phage library) was constructed.

[0173] 1. Using Mycobacterium tuberculosis H37Rv as a template, using the primer combination in Example 1 as a primer and adding a corresponding restriction site at the 5' end, using KOD high-fidelity enzyme for PCR, and over-amplifying the corresponding fragments respectively.

[0174] Among them, the amplification of the left and right arms is carried out according to the following system:

[0175]

[0176] KOD PCR reaction conditions are:

[0177]

[0178] After the completion of PCR, 1% agarose gel electrophoresis was performed, and gel recovery was p...

Embodiment 3

[0182] Example 3 Construction of phage library for knocking out Mycobacterium tuberculosis NADH dehydrogenase gene family

[0183] The shuttle vector obtained in Example 2 was electrotransfected into Mycobacterium smegmatis to obtain a recombinant phage integrated with a homology arm, and a high-titer recombinant phage was obtained through in vitro amplification.

[0184] The specific operation is:

[0185] 1. Prepare Mycobacterium smegmatis competent cells;

[0186] 2. The shuttle vector obtained in Example 2 was electrotransferred into Mycobacterium smegmatis competent cells;

[0187] 3. The Mycobacterium smegmatis after electroporation is spread on the resistance selection medium containing hygromycin;

[0188] 4. Pick phage plaques, infect freshly cultured Mycobacterium smegmatis, and collect phage lysates.

[0189] Results: Phage plaques were grown successfully. After picking the plaques and infecting Mycobacterium smegmatis, high-titer phages were obtained from the co...

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Abstract

The invention discloses a recombinant TM4 bacteriophage library for constructing NADH dehydrogenase gene family deleted mycobacterium tuberculosis and an application thereof. The invention firstly provides a recombinant shuttle vector library required for constructing the recombinant TM4 bacteriophage library. The recombinant shuttle vector library comprises 16 recombinant shuttle vectors which insert homologous arm sequences at both sides of 16 member genes of the NADH dehydrogenase gene family of mycobacterium tuberculosis. For facilitating the function research and vaccine development of the NADH dehydrogenase gene, the homologous recombinant bacteriophage library is constructed. The library can be used for knocking out the NADH dehydrogenase gene of mycobacterium tuberculosis, verifying whether the genes are necessary genes and constructing the NADH dehydrogenase knockout strain, and is used for optimizing and improving BCG vaccines. When the phage library is used for knockout, theknockout specificity is good and the positive rate is high.

Description

technical field [0001] The invention relates to the technical field of tuberculosis, and more specifically relates to a recombinant TM4 phage library for constructing NADH dehydrogenase gene family-deleted mycobacterium tuberculosis and its application. Background technique [0002] Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, is the world's leading infectious disease killer, claiming the lives of more than 4,500 people every day. WHO has a vision for TB control: 95% reduction in TB deaths by 2035 compared to 2015 levels, 90% reduction in TB incidence by 2035 compared to 2015 levels, and no catastrophic costs due to TB by 2035 of households affected by tuberculosis. However, the treatment of tuberculosis is facing the severe challenge of drug resistance of Mycobacterium tuberculosis. It is urgent to study the pathogenic mechanism and drug resistance mechanism of Mycobacterium tuberculosis and develop new anti-tuberculosis drugs. But rese...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/90C12N15/53C12N15/11C40B40/02
CPCC12N9/0036C12N15/74C12N15/902C12Y106/99003C40B40/02
Inventor 周亚凤王绪德高磊鑫刘雪宾李茜茜李晓恬潘健昌李青申兆兴
Owner 上海晶诺生物科技有限公司