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Method for secretory expression of honeybee melittin signal peptide-mediated IBV (infectious bronchitis virus) N protein

A technology for bronchitis and chicken infectivity, applied in the direction of viruses, viral peptides, viruses/phages, etc., can solve the problems of easy loss of biological activity, target protein hydrolysis, and difficult purification.

Inactive Publication Date: 2016-05-25
GUANGXI UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, in the published articles on IBV structural proteins expressed by the insect baculovirus system, the expression level is low, and the target protein is easily hydrolyzed by the endogenous protease of the cell; Loss of biological activity; Difficult to purify, etc.

Method used

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  • Method for secretory expression of honeybee melittin signal peptide-mediated IBV (infectious bronchitis virus) N protein
  • Method for secretory expression of honeybee melittin signal peptide-mediated IBV (infectious bronchitis virus) N protein
  • Method for secretory expression of honeybee melittin signal peptide-mediated IBV (infectious bronchitis virus) N protein

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Embodiment Construction

[0028] The inventor designed a pair of primers based on the N gene sequence of the Chinese isolate of chicken infectious bronchitis virus GX-YL5 registered in GenBank, and at the same time designed a pair of primer sequences for amplifying the signal peptide of apitropin, and obtained the fusion apitropin by biological methods. IBVN gene HBM-N of the signal peptide, construct and identify the recombinant transfer vector pFast-HBM-N, construct and identify the recombinant bacmid rHBM-N, transfect the recombinant bacmid into Sf9 insect cells, use indirect immunofluorescence (IFA) and Western -blot identification showed that N protein was secreted and expressed. The specific operation is as follows:

[0029] 1.1 strain

[0030] IBV strain GX-YL5 (GenBankNO.HQ848267.1)

[0031] 1.2 Method

[0032] 1.2.1 Design of primers: According to the GX-YL5N gene sequence registered in NCBI GenBank (accession number: FJ548847.1), the specific primers N-F and N-R for amplifying the N gene w...

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Abstract

The invention discloses a method for secretory expression of littin signal peptide-mediated IBV N protein, that is firstly, IBV N gene HBM-N fusing honeybee melittin signal peptide is obtained, then a recombination transfer vector pFast-HBM-N and recombination bacmid rHBM-N are constructed, and finally, the recombination bacmid transfers Sf9 insect cells for secretory expression of IBV N protein. According to the method, the honeybee melittin signal peptide is introduced before IBV N protein, N protein efficient secretory expression is realized, the obtained IBV N protein has excellent biological activity, hydrolysis of endogenous protease to target protein is reduced with the method, purification is facilitated, the foundation is laid for further study of N protein biological functions, development of novel diagnostic antigen, development of novel vaccine and the like, and at the same time, a new thought is provided for expression of other structural protein of the IBV.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for mediating the secretion and expression of chicken infectious bronchitis virus N protein by a mespaline signal peptide. Background technique [0002] Infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens caused by infectious bronchitis virus (IBV), which mainly affects the respiratory system, digestive system and genitourinary system of chickens. Chickens of different ages and breeds are susceptible. Since the disease was discovered in North America in 1930, it has been found in other countries one after another. At present, it has become a worldwide epidemic, causing huge economic losses to the poultry industry. [0003] Among the four structural proteins of IBV, N protein has important biological functions. It not only combines with viral RNA to form nucleocapsid, participates in the synthesis, transcription and transl...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/62
CPCC07K14/005C07K2319/02C12N15/625C12N15/86C12N2710/14043C12N2770/20022C12N2800/105
Inventor 磨美兰张志鹏张丽华何怡宁孙新宽韦平韦天超
Owner GUANGXI UNIV
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