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Method for modifying bird flu virus through interferon inducible protein ESAT6

An avian influenza virus, interferon-induced technology, applied in the field of medicine and biology, can solve the problem of in vitro modification is not easy, and achieve the effect of small molecular weight

Inactive Publication Date: 2010-08-04
GUANGDONG LAB ANIMALS MONITORING INST +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in this decade of attempts, it was found that it is not easy to modify in vitro using the current 12-plasmid system or 8-plasmid system

Method used

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  • Method for modifying bird flu virus through interferon inducible protein ESAT6
  • Method for modifying bird flu virus through interferon inducible protein ESAT6
  • Method for modifying bird flu virus through interferon inducible protein ESAT6

Examples

Experimental program
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Embodiment Construction

[0016] 1. Cloning of ESAT6 gene

[0017] ① Extraction of the genome of the standard strain of Mycobacterium tuberculosis H37Rv: operate in a P3 laboratory biosafety cabinet, take 500ul culture of the standard strain of Mycobacterium tuberculosis H37Rv into a 1.5ml tube, add 1ml of absolute ethanol, and seal it. Take it out, put it at -20°C overnight, centrifuge at 12000rpm at 4°C, remove the supernatant, air-dry, dissolve the precipitate in 100ul sterilized water, boil for 10min, and extract the bacterial genomic DNA according to the usual operation.

[0018] ② Take bacterial genomic DNA for PCR

[0019] In a 25uL system,

[0020] 10×buffer 2.5uL

[0021] Mg 2+ 1.5uL (25mM),

[0022] dNTP 1uL (25mM)

[0023] Modified DNA1 1uL (10pmol / mL)

[0024] Modified DNA2 1uL (10pmol / mL)

[0025] Taq DNA polymerase 1u (5u / uL)

[0026] Add 25ng of the prepared DNA template

[0027] Finally, make up to 25uL with sterile deionized water.

[0028] 95°C for 3min, 50°C for 30s,...

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Abstract

The invention relates to a method for modifying a bird flu virus through interferon inducible protein ESAT6, which adopts an early secretory antigen target ESAT-6 gene of mycobacterium tuberculosis to modify the bird flu virus and is a method for splicing into an exogenous DNA on the basis of establishing a bird flu virus reverse genetic operating system. The selected ESAT-6 protein is secretory protein with a T cell multifunctional epiposition and has the main function of stimulating an organism to generate IFN, which has uniqueness in researching a virus anti-infective immune mechanism path. After a bird flu virus gene is modified through the DNA, when the modified virus is rescued, not only the transcription, the translation and the virus packaging of bird flu virus protein are not influenced, but also the ESAT-6 protein can be simultaneously generated.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and in particular relates to a method for modifying and processing avian influenza virus in vitro by using reverse genetic manipulation technology to obtain a new modified virus, which is used in the study of anti-infection immune mechanism and immune prevention the use of. Background technique [0002] Avian influenza virus not only brings huge losses to the poultry industry, but also poses a huge threat to human public health. Understanding the mechanism of interaction between avian influenza virus and the body is of great significance for the prevention of avian influenza. Avian influenza virus belongs to Orthomyxoviridae, and influenza virus belongs to type A virus. It is an enveloped and polymorphic single-stranded negative-sense RNA virus. Its viral genome consists of 8 segments and encodes 11 proteins. Among them, the hemagglutinin protein HA, neuraminidase NA, and nonstructural prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12R1/93
Inventor 罗琴芳郭霄峰黄韧
Owner GUANGDONG LAB ANIMALS MONITORING INST
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