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Hemangioma pathotype subgroup J avian leukosis virus gene and construction of infectious clone thereof

A technique for infectious cloning of avian leukosis virus, applied in genetic engineering, plant gene improvement, virus/bacteriophage, etc., can solve the problems of ALV-J cloned virus without hemangioma lesion, to ensure integrity and purity, The effect of high conversion rate, rich content and research tools

Inactive Publication Date: 2010-10-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, only the British ALV-J prototype strain HPRS-103 of the myeloma lesion type and the domestic NX0101 strain have established infectious molecular clones, and there is no report of the hemangioma lesion type ALV-J cloned virus.

Method used

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  • Hemangioma pathotype subgroup J avian leukosis virus gene and construction of infectious clone thereof
  • Hemangioma pathotype subgroup J avian leukosis virus gene and construction of infectious clone thereof
  • Hemangioma pathotype subgroup J avian leukosis virus gene and construction of infectious clone thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Acquisition of hemangioma lesion type J subgroup avian leukosis virus gene

[0031] The present inventors isolated hemangioma leukosis type J subgroup avian leukosis virus SCAU-HN06 strain from Hailan brown layer hens with hemangioma leukosis type, and the virus strain is now preserved by Veterinary College of South China Agricultural University.

[0032] The present inventors carried out molecular biology research on hemangioma pathological type J subgroup avian leukosis virus SCAU-HN06 strain, at first with reference to the relevant sequence disclosed by GenBank, after comparative analysis, utilize Primer Premier 5.0 professional primer design software to design merger primers, to The gene was amplified in three sections A, B, and C. The specific primers were shown in Table 1, and the amplification electrophoresis results were as follows: figure 1 shown.

[0033] The above-mentioned three fragments A, B and C were subjected to gel cutting recovery, T carrie...

Embodiment 2

[0037] Embodiment 2 Specific primer design

[0038] In this example, specific primers were designed according to the hemangiomatous type J subgroup avian leukosis virus gene obtained in Example 1. The nucleotide sequences of the primers are shown in SEQ ID NO: 2-7, and were sent to the biological company for synthesis.

Embodiment 3

[0039] Example 3 Construction of Hemangioma Lesion Type ALV-J Infectious Clones

[0040] 1. Experimental materials

[0041] Chicken embryo fibroblasts and ALV-J subgroup-specific monoclonal antibody JE9 were obtained from the Veterinary College of Yangzhou University; FITC-goat anti-mouse IgG was produced by ROCLAND; T4DNA ligase was produced by Promega; Prime STAR TM HS DNA Polymerase, pMD18-Tsimple vector, etc. are all products of TaKaRa Company; pBlueskriptII(-) is a product of Generay Company; restriction enzymes Sal I, Not I, Kpn I, EcoR I are products of Fermentas Company; ALV-Ag ELISA reagent The box is a product of IDEXX company.

[0042] 2. Construction method of infectious clone

[0043]Using the hemangiomatosis type J subgroup avian leukosis virus gene (its nucleotide sequence shown in SEQ ID NO: 1) obtained in Example 1 as a template, utilize the six specific primers synthesized in Example 2 to divide the gene sequence into three The segments were amplified and...

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Abstract

The invention discloses a hemangioma pathotype subgroup J avian leukosis virus gene and the construction of infectious clone thereof. A full-length genome sequence of a subgroup J avian leukosis virus of the invention is shown as SEQ ID NO:1. Then, the hemangioma pathotype subgroup J avian leukosis virus gene shown as SEQ ID NO:1 is subjected to amplification by adopting a specific primer; and pBlueskript SK (I) is used as a vector and chick embryo fibroblast is used as a host cell to construct the infectious clone of the hemangioma pathotype subgroup J avian leukosis virus gene so as to lay the foundation for subsequent research on relation among pathogenic mechanism, a gene structure and functions, virus reproduction, genetic variation rules and the like of the hemangioma pathotype subgroup J avian leukosis virus ALV-J.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the research of ALV-J virus, in particular to the construction of a hemangiomatous leukosis type J subgroup avian leukosis virus gene and its infectious clone. Background technique [0002] A notable feature of avian leukosis virus (ALV) as a retrovirus is that the RNA polymerase of the virus lacks a proofreading mechanism. Regardless of whether immune pressure exists or not, it will cause genetic variation during the passage process. Therefore, reverse genetic technology is used to construct a single origin Infectious clones are very important for studying viruses. [0003] Subgroup J avian leukosis virus (ALV-J) is a new exogenous avian leukosis virus that emerged in the early 1990s. Different ALV-J can cause a variety of different clinical phenotypes, such as myeloma lesion type, hemangioma lesion type and so on. But so far, only the British ALV-J prototype strain HPRS-103 of th...

Claims

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Application Information

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IPC IPC(8): C12N15/48C12N15/63C12N7/00
Inventor 廖明张贺楠赖汉漳曹伟胜罗开健徐成刚辛朝安
Owner SOUTH CHINA AGRI UNIV
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