Method for expressing IBV-HN99 membrane protein gene in insect-rhabdovirus system

A baculovirus, membrane protein technology, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve problems such as systems that cannot be commonly used

Inactive Publication Date: 2009-10-21
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The green fluorescent protein gene egfp is a widely used reporter gene. Early studies have shown that it can be expressed normally in insect cells and has the ability to produce green fluorescence, so it has also been applied to the baculovirus expression system, but before

Method used

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  • Method for expressing IBV-HN99 membrane protein gene in insect-rhabdovirus system
  • Method for expressing IBV-HN99 membrane protein gene in insect-rhabdovirus system
  • Method for expressing IBV-HN99 membrane protein gene in insect-rhabdovirus system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction and Identification of Recombinant Expression Vectors

[0040] 1 material

[0041] 1.1 Primer design and synthesis

[0042] Referring to the IBV M gene sequence published in GenBank, a pair of primers was designed using the primer design software Prime Premier5.0:

[0043] Upstream primer: 5′-CG GAATTC AGTTTCCTAAGAACGGTTGGAA-3′ (EcoR I)

[0044] Downstream primer: 5′-CCC AAGCTT CTCTCTACACGCACACATTTAT-3′ (Hind III)

[0045] Primers were purchased from Shanghai Bioengineering Company.

[0046] 1.2 Vectors and strains

[0047] The vector pFBDM-MBP inserts the MBP tag between the BamH I and EcoR I restriction sites of the pFBDM vector. For the physical map of pFBDM, see figure 1 .

[0048] The transposable plasmid pFBDM-MBP contains two multi-cloning sites, and the original multi-cloning site can be inserted again between the two reverse promoters, so this plasmid has the ability to express four foreign genes at the same time. genetic potent...

Embodiment 2

[0189] Example 2 Expression of IBV-HN99 membrane protein gene in insect-baculovirus system

[0190] 1. Construction of recombinant expression vector Bacmid-egfp-MBP / M

[0191] The method of Example 1 was repeated to construct the recombinant expression vector Bacmid-egfp-MBP / M.

[0192] 2. Transfection

[0193] Sf9 cells were cultured in TNM-FH medium containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% fetal bovine serum, and were usually passaged once every 3 days, with three or four passages each time. Transfection experiments were used when 95% of Sf9 cells were in logarithmic growth phase. Sf9 cells were transfected with the recombinant vector Bacmid-egfp-MBP / M by lipofection method, normal Sf9 cells were used as the blank control, and Sf9 cells were infected with wild-type baculovirus AcNPV as the negative control. The specific operation steps are as follows:

[0194] (1) Take 10 μL of the recombinant plasmid Bacmid-egfp-MBP / M and add it to an Eppendorf tu...

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Abstract

The invention relates to a method for expressing an IBV-HN99 membrane protein gene in an insect-rhabdovirus system, which belongs to the field of biological engineering. The invention also discloses a recombinant expression carrier of the membrane protein gene, and the recombinant expression carrier simultaneously contains a maltose label and a green fluorescent protein gene named ACB acmid-egfp-MBP-M. The method for expressing the IBV-HN99 membrane protein gene in the insect-rhabdovirus system is as follows: an expression carrier Bacmid-egfp-MBP-M is used for transfecting an Sf9 cell by a lipidosome transfect method through two generations of blind passages. The construction of the method for expressing the IBV-HN99 membrane protein gene in the insect-rhabdovirus system has important significance for developing a gene engineered vaccine of an IBV M gene or an ELISA diagnostic reagent kit of the M protein.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a membrane protein gene recombinant expression vector and a method for expressing the IBV-HN99 membrane protein gene in an insect-baculovirus system. Background technique [0002] Chicken infectious bronchitis (Infectious Bronchitis, IB) is an acute, highly contagious infectious disease of chickens caused by Infectious Bronchitis Virus (Infectious Bronchitis Virus, IBV), mainly invading the respiratory, digestive and genitourinary systems of chickens, The number of eggs laid by laying hens is reduced, and the quality of eggs is reduced; chicks die in large numbers due to respiratory or kidney infections. The membrane protein gene of IBV encodes the M protein. The N-terminus of the M protein is located outside the virion and is glycosylated. Its main component transmembrane 3 times. The M protein contains 224-225 amino acid residues with a molecular weight of 23ku. Across the capsul...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/50C12N15/10C07K14/165
Inventor 贺秀媛张淑霞李玉峰杜恩岐崔沛张君涛张志平崔保安杨明凡陈红英邓立新贺丛董海聚仝宗喜张惠茹白延杰安森亚朱翠娟原维李小波
Owner HENAN AGRICULTURAL UNIVERSITY
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