Mycobacterium tuberculosis inclusion body protein renaturation method and special renaturation buffer solution thereof

A Mycobacterium tuberculosis, protein renaturation technology, applied in the field of protein purification, can solve problems such as renaturation difficulties and affect product development, and achieve good immunogenicity effect

Pending Publication Date: 2022-04-22
中国人民解放军总医院第八医学中心
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Problems solved by technology

Inclusion body renaturation is a complex process, and its success largely depends on the various physical and chemical properties of the protein, especially the content of cysteine ​​in the protein sequence. The more cysteine ​​content, the greater t

Method used

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  • Mycobacterium tuberculosis inclusion body protein renaturation method and special renaturation buffer solution thereof
  • Mycobacterium tuberculosis inclusion body protein renaturation method and special renaturation buffer solution thereof
  • Mycobacterium tuberculosis inclusion body protein renaturation method and special renaturation buffer solution thereof

Examples

Experimental program
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Example Embodiment

[0032] Example, the establishment of the method for renaturation of Mycobacterium tuberculosis inclusion body protein (such as W544 protein)

[0033] First, the acquisition of Mycobacterium tuberculosis protein inclusion body engineering bacteria

[0034] 1. Artificially synthesize the double-stranded DNA molecule shown in SEQ ID NO: 2. In SEQ ID NO: 2, the 1st to 6th positions from the 5' end are the recognition sites of the restriction endonuclease NheI, the 7th to 2010th positions are the W544 gene, and the 2011th to 2016th positions are the restriction endonuclease EcoRI recognition site.

[0035] The W544 gene encodes the W544 protein whose amino acid sequence is shown in SEQ ID NO: 1. The W544 protein belongs to the Mycobacterium tuberculosis protein.

[0036] 2. The double-stranded DNA molecule shown in SEQ ID NO: 2 was digested with restriction endonucleases NheI and EcoRI, and the digested product was recovered.

[0037] 3. The pET28a vector was digested with rest...

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Abstract

The invention discloses a mycobacterium tuberculosis inclusion body protein renaturation method and a special renaturation buffer solution thereof. Experiments prove that the renaturation buffer solution provided by the invention can be used for renaturating mycobacterium tuberculosis protein W544, and the renaturated W544 protein can effectively stimulate splenic lymphocytes of a mouse infected by mycobacterium tuberculosis to generate immune response and does not stimulate splenic lymphocytes of a healthy mouse to generate immune response. Therefore, the renatured W544 protein has very good immunogenicity. The renaturation buffer solution and the mycobacterium tuberculosis inclusion body protein renaturation method established according to the renaturation buffer solution have important application value.

Description

technical field [0001] The invention belongs to the technical field of protein purification, and in particular relates to a method for renaturation of mycobacterium tuberculosis inclusion body protein and a special renaturation buffer thereof. Background technique [0002] Strengthening the research on the diagnosis, prevention and treatment of tuberculosis is of great significance to the control of the tuberculosis epidemic. Mycobacterium tuberculosis protein is widely used in the basic research and development of tuberculosis. Because the natural source of Mycobacterium tuberculosis protein is greatly limited, the use of genetic engineering technology has almost become the only effective means to obtain Mycobacterium tuberculosis protein. [0003] The expression of proteins in the E. coli system has the characteristics of easy operation, clear genetic background, and high expression levels. However, when E. coli is used to express Mycobacterium tuberculosis proteins, the e...

Claims

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Application Information

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IPC IPC(8): C07K14/35C07K1/34C12N15/31C12N15/70C12N1/21C12R1/19
CPCC07K14/35C12N15/70
Inventor 阳幼荣吴雪琼梁艳薛勇贾再兴张俊仙
Owner 中国人民解放军总医院第八医学中心
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