Method for constructing recombinant virus for expressing ALV-K envelope protein

A technology of ALV-K and envelope protein, applied in the direction of retroRNA virus, virus, viral peptide, etc., can solve the problem of low expression level of viral protein, no commercial specific ALV-K antigen and antibody detection kit, The detection effect is not very good and other problems, to achieve the effect of speeding up the purification process

Active Publication Date: 2019-02-01
YANGZHOU UNIV
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  • Claims
  • Application Information

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Problems solved by technology

Due to the weak replication ability of ALV-K virus and the low level of viral protein expression, the current general detect...

Method used

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  • Method for constructing recombinant virus for expressing ALV-K envelope protein
  • Method for constructing recombinant virus for expressing ALV-K envelope protein
  • Method for constructing recombinant virus for expressing ALV-K envelope protein

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Embodiment

[0021] (1) Preparation of ALV-K proviral DNA:

[0022] Take 400uL of the supernatant after ALV-K infection of the cells in a 1.5mL finger tube, add 400uL of cell lysate (50mmol / L Tris-HCl pH=8.0, 20mmol / L EDTA pH=8.0, 2% SDS and proteinase K), fully After mixing, place it in a 56°C water bath for 4 hours; add 400uL Tris to balance the phenol, mix well and centrifuge at 10000rpm for 10min, take the supernatant into another 1.5mL finger tube; add 400uL phenol: chloroform: isoamyl alcohol, and mix well Then centrifuge at 10,000rpm for 5min, take the supernatant into another 1.5mL finger tube; add 800uL of absolute ethanol, invert and mix well, incubate at -20°C for 30 minutes, centrifuge at 12,000rpm for 15 minutes, discard the supernatant; after natural drying at room temperature Add 30uL sterilized ultrapure water and 2uL RNase to the precipitate, fully dissolve to obtain ALV-K proviral DNA, and store at -20°C for later use.

[0023] (2) PCR amplification of ALV-K env gene fra...

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Abstract

The invention discloses a method for constructing a recombinant virus for expressing an ALV-K envelope protein, specifically comprising the following steps: designing a PCR amplification ALV-J infectious clone linear expression vector primer and a PCR amplification ALV-K-env gene primer sequence; carrying out PCR amplification on an ALV-K envelope protein gene fragment and the ALV-J infectious clone linear vector, constructing a recombinant infectious cloning plasmid, and transfecting the recombinant plasmid into DF-1 cells to rescue the recombinant virus for expression of the ALV-K envelope protein. By ALV-J infectious cloning, the recombinant virus for efficient replication and expression of the ALV-K envelope protein is constructed. The acquisition of the recombinant virus not only laysa foundation for studying functional receptors of the ALV-K envelope protein and its pathogenicity, but also provides a target virus for the clinical detection of neutralizing antibodies of the ALV-K.

Description

technical field [0001] The invention relates to a method for constructing a recombinant virus expressing ALV-K envelope protein. This invention not only lays the foundation for the study of the ALV-K envelope protein functional receptor and its pathogenicity, but also provides a target virus for clinical detection of ALV-K neutralizing antibodies. Therefore, the present invention has certain application value. Background technique [0002] Avian leukemia virus (ALV) is a retrovirus that can cause a variety of tumors and immunosuppressive diseases in poultry. According to the characteristics of its envelope protein, the virus can be divided into 11 subgroups, A-K. Among them, A, B, and J subgroups are more common, and J subgroup is currently the most common in China. ALV-J infection mainly causes malignant proliferation of hematopoietic cells in chickens, causing myeloid cell tumors, hemangiomas and severe immunosuppression, causing huge economic losses to poultry industry...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/48C12N15/63
CPCC07K14/005C12N7/00C12N15/63C12N2740/11021C12N2740/11022
Inventor 叶建强吕璐李拓凡谢菁邵红霞秦爱建
Owner YANGZHOU UNIV
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