Hemangioma pathotype J subgroup avian leucosis mutant virus strain and construction method thereof

A technology of avian leukosis and construction method, which is applied in the field of microorganisms, can solve problems affecting E components, virus pathogenicity, etc., and achieve the effect of high conversion rate

Inactive Publication Date: 2010-09-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the study of the naturally isolated recombinant chimeric virus ALV-J / A, it was found that the E element is related to tumorigenicity, but this study is based on natural isolates, which have many sequence mutations and deletions , and these mutations or deletions may have an impact on the pathogenicity of the virus, thus affecting the research of the E element on the pathogenicity of the virus and its pathogenic mechanism

Method used

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  • Hemangioma pathotype J subgroup avian leucosis mutant virus strain and construction method thereof
  • Hemangioma pathotype J subgroup avian leucosis mutant virus strain and construction method thereof
  • Hemangioma pathotype J subgroup avian leucosis mutant virus strain and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 specific primer design

[0037] In this embodiment, specific primers are designed according to the gene sequence of the hemangiomatosis type J subgroup avian leukosis virus SCAU-HN06 strain (its nucleotide sequence is shown in SEQ ID NO: 5), and its nucleotide sequence is shown in SEQ ID NO: 1-4, the primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0038] Example 2 Hemangioma Lesion Type J Subgroup Avian Leukemia Mutant Virus Strain

[0039] 1. Experimental materials

[0040] Chicken embryo fibroblasts and ALV-J subgroup-specific monoclonal antibody JE9 were donated by Professor Qin Aijian, School of Veterinary Medicine, Yangzhou University; FITC-goat anti-mouse IgG was produced by ROCLAND; T4DNA ligase was produced by Promega; Prime STAR TM HS DNA Polymerase is the product of TaKaRa Company; restriction endonuclease CalI and Not I are the products of Fermentas Company; ALV-Ag ELISA kit is the product of IDEXX Company.

[0041] 2. Construction of hemangioma lesion type J subgroup avian leukemia mutant virus strain

[0042]First, the E (XSR) sequence between CalI-NotI of the gene of the hemangiomatosis type J subgroup avian leukemia virus SCAU-HN06 strain (its nucleotide sequence is shown in SEQ ID NO: 5) is deleted by Overlapping PCR technology , using the specific primers shown in SEQ ID NO: 1 and the specific primer...

Embodiment 3

[0048] The verification of embodiment 3 infectious virus

[0049] The transfected and infected host cell culture supernatants were detected by IDEXX ALV-Ag ELISA kit, and the ELISA detection results of the transfected and infected cell culture supernatants (shown in Table 1) were all positive.

[0050] Table 1 ELISA detection results

[0051] pSK-ALV-HN-ΔE transfection amount (μg)

transfection supernatant

Post-infection supernatant

2

1.944

1.531

0

0.032

0.044

[0052] After the infected cells were fixed with methanol, washed with PBS, specific ALV-J monoclonal antibody JE9 was added dropwise, incubated at 37°C for 30min, washed with PBS, then added with goat anti-mouse IgG fluorescently labeled antibody, incubated at 37°C for 30min, and Washed with PBS, and finally observed under a fluorescent microscope, the infected cells can be observed with obvious green fluorescence, and it is a typical cytoplasmic staining, and t...

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Abstract

The invention discloses a hemangioma pathotype J subgroup avian leucosis mutant virus strain and a construction method thereof. The virus strain is a J subgroup avian leucosis infectious cloning virus rSCAU-HN06 and preserved in the China Center for Type Culture Collection (CCTCC) on November 3th, 2009, and the preservation number is CCTCC No:V200914. In the invention, the monogenetic infectious cloning virus rSCAU-HN06 is used as a base to carry out E element deletion, thereby interferences to research results, which are caused by mutations and deletion in a naturally separated virus sequence, are avoided. The construction method is simple, convenient and easy to apply, uses a specific single enzyme cutting site on the virus and a NotI site on a carrier for carrying out sequence replacement to construct and save the infectious cloning virus, has high conversion rate and can stably carry out duplication.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to the research field of hemangioma pathological type J subgroup avian leukosis virus, in particular to an E element (XSR)-deleted hemangioma pathological type J subgroup avian leukemia mutant virus strain and a construction method thereof. Background technique [0002] The present inventor has isolated hemangioma leukosis type J subgroup avian leukemia virus SCAU-HN06 strain from hemangioma leukosis type Hailan brown layer hens, and obtained hemangioma leukosis type J subgroup avian leukosis virus SCAU-HN06 The full-length genome sequence of the strain, its nucleotide sequence is shown in SEQ ID NO:5. [0003] J subgroup avian leukosis virus SCAU-HN06 strain and ALV-J prototype strain HPRS-103 have similar characteristics in the LTR (long terminal repeat region), that is, compared with other subgroups of avian leukosis virus, there is One has a hairpin neck loop structure c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/08C12N15/85C12R1/93
Inventor 廖明张贺楠曹伟胜赖汉漳罗开健徐成刚辛朝安
Owner SOUTH CHINA AGRI UNIV
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