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Recombinant A subgroup avian leukosis virus being able to express ALV-J envelope protein, and construction method and use of virus

An avian leukemia virus and envelope protein technology, applied in the biological field, can solve the problems of low virus titer and poor replication ability of ALV-J strain, and achieve the effects of high virus titer, strong replication ability and high detection sensitivity

Active Publication Date: 2016-12-21
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve the problems of poor replication ability and low virus titer of the traditional ALV-J strain, the present invention uses the ALV-A infectious clone preserved in our laboratory as the backbone, and replaces the envelope protein gene with the envelope protein gene of ALV-J , in order to further improve virus detection sensitivity, introduce sea cucumber luciferase (luciferase) reporter gene after its envelope protein gene, construct the recombinant ALV-A virus that the envelope protein with luciferase reporter gene is ALV-J envelope protein

Method used

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  • Recombinant A subgroup avian leukosis virus being able to express ALV-J envelope protein, and construction method and use of virus
  • Recombinant A subgroup avian leukosis virus being able to express ALV-J envelope protein, and construction method and use of virus
  • Recombinant A subgroup avian leukosis virus being able to express ALV-J envelope protein, and construction method and use of virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The construction of the recombinant ALV-A virus infectious clone ALV-A(J)-luciferase that embodiment 1 expresses ALV-J envelope protein and luciferase

[0039] 1. Construction of ALV-A(J)-luciferase recombinant plasmid:

[0040] The pRAV-1 vector containing the infectious clone of the ALV-A strain (nucleotide sequence shown in SEQ ID NO.2) was double digested with KpnI / StuI, and a vector backbone fragment with a size of about 11 000 bp was recovered from the gel. Using pHPRS103 (nucleotide sequence as shown in SEQ ID NO.1) as a template, according to the primer PF1 / PR1 in Table 1, PCR amplifies the target fragment with a size of about 1700bp (including part of the pol gene and the entire env gene fragment) . Pass the PCR product through ClonExpress TM The II One StepCloning Kit was homologously recombined into the vector backbone, and the constructed plasmid expressing the envelope protein of ALV-J was named pALV-A(J). Subsequently, the pALV-A(J) plasmid was digeste...

Embodiment 2

[0045] Example 2 Rescue of recombinant subgroup A avian leukosis virus expressing ALV-J envelope protein and luciferase

[0046] 1. Method

[0047] 1.1 Rescue of recombinant virus

[0048] The viral infectious clone pALV-A(J)-luciferase constructed in Example 1 was purified, and transfected into DF-1 cells forming 80% monolayer using Polyjet transfection reagent according to the manual, and the supernatant was discarded after 6h , after washing twice with serum-free DMEM, add DMEM containing 10% fetal bovine serum, place at 37°C, 5% CO 2 cultivated under conditions. 72 hours after transfection, the cells were harvested, frozen in a -80°C refrigerator, and serially passaged on DF-1 cells after freezing and thawing twice.

[0049] 1.2 Western blot identification of rescued virus

[0050] Get the rescued ALV-A(J)-luciferase virus of two passages, carry out SDS-PAGE electrophoresis, transfer to nitrocellulose membrane, block with 5% skimmed milk, with anti-ALV-J 4A3MAb (1:200 ...

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Abstract

The invention discloses a recombinant A subgroup avian leukosis virus being able to express an ALV-J envelope protein, and a construction method and a use of the virus. The virus adopts ALV-A infectious clone as a skeleton, the envelope protein of the ALV-A infectious clone is replaced by the ALV-J envelope protein, and the 3' end of the envelope protein carries luciferrase report gene, so the infectious clone of the recombinant ALV-A virus being able to express the ALV-J envelope protein is constructed. Experiments prove that the recombinant virus obtained through infectious clone rescuing has high replication ability, the tilter of the virus reaches 10<5.21> TCID50 / ml, and is 125 times that of an ALV-J original strain, and the luciferrase report gene is carried out in order to realize easy quantification. The recombinant virus can infect 293T cells for expressing chNHE1, and can be detected 8h after infection at the earliest, so the recombinant virus has high sensitivity. The a recombinant A subgroup avian leukosis virus solves the problems of low virus titer, low replication ability, and adverseness of a small amount of the virus in the early stage of virus infection to detection of traditional ALV-J viruses, and is of great significance to carry out researches related with viruses and virus receptors and anti-ALV-J antibody detection.

Description

technical field [0001] The invention relates to a recombinant virus and a construction method thereof, in particular to an ALV-A recombinant virus capable of expressing ALV-J envelope protein, and also to a construction method of the recombinant virus. The invention belongs to the field of biotechnology. Background technique [0002] Avian leukemia virus (ALV) can cause a variety of tumor diseases in poultry. ALV is divided into A-J a total of 10 subgroups. At present, the most prevalent in our country are A, B, J subgroups, among them, ALV-J is more serious harm to my country's poultry industry. Therefore, it is particularly important to study the pathogenic mechanism of ALV-J. Infection of host cells by retroviruses requires the interaction of viral surface envelope proteins with their specific cellular receptors. The differences in the viral envelope proteins determine the host range of the virus. Viral receptors mediate viral entry into host cells and are the first ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N7/01G01N33/68C12R1/93
Inventor 高玉龙王笑梅祁小乐王永强
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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