Recombinant J subgroup avian leucosis virus infective cloned plasmids and preparation method and application thereof

A technology for infectious cloning of avian leukosis virus, applied in the field of infectious cloning of avian leukosis virus, can solve problems such as increasing cell migration and affecting downstream signaling pathways, and achieve the effect of enhancing versatility and repeatability

Inactive Publication Date: 2010-12-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

In addition, the occurrence of tumors may also be related to the interaction between ALV-J and its receptor NHE1. NHE1 is regulated by various intracellular or extracellular signals, which can promote cell division and proliferation, reduce cell adhesion, and increase cell migration. Necessary for tumorigenesis and invasion, therefore, ALV-J infection may activate NHE1, affecting its downstream signaling pathways, resulting in the transformation of infected cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The construction of embodiment 1 infectious clone plasmid

[0032] 1. Obtaining the target gene fragments of ALV-J and ALV-E proviruses

[0033] 1.1 Amplification primers of ALV-J and ALV-E provirus target gene fragments

[0034] Referring to the complete gene sequences of ALV-J and ALV-E, 5 pairs of primers were respectively designed to amplify the target gene fragments of ALV-J and ALV-E proviruses, and at the same time, necessary restriction sites were introduced at the 5' end of some primers, 5 The primer sequences are shown in SEQ ID NO: 2-7, 11-14.

[0035] 1.2 PCR amplification of ALV-J target gene fragment

[0036] Using the DNA of CEF cells infected with ALV-J as a template, use PrimeSTARTM HS DNA polymerase (TaKaRa) and the following 3 pairs of primers to amplify the target fragment of ALV-J proviral DNA:

[0037] The PCR reaction conditions of the first pair of primers were as follows: 98°C for 3min; 30 cycles of 98°C for 15s, 55°C for 10s, and 72°C for 3m...

Embodiment 2

[0053] Example 2 Cell Transfection and Virus Harvesting

[0054] 1. Preparation of plasmids and cells for transfection

[0055] Refer to the instructions of the OMEGA Endotoxin-Removing Plasmid Extraction Kit, the details are as follows: collect 3-5mL bacterial liquid in a 1.5mL centrifuge tube, and centrifuge at 8000r / min for 3min. Discard the supernatant, add 250 μL Solution I solution (containing RNase A), vortex to resuspend the bacteria; then add 250 μL Solution II solution, gently invert and mix 6 times; if the solution is relatively clear at this time, immediately Add 350 μL Solution III solution, if it is still relatively turbid, add 350 μL Solution III solution after 2~5 minutes of action, invert and mix 6 times. Centrifuge at 12000r / min for 15min. Transfer the supernatant to a spin column, centrifuge at 10,000r / min for 30s, and discard the filtrate in the collection tube. Add 500 μL of HB Buffer to the column, centrifuge at 10,000 r / min for 30 s, and discard the f...

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Abstract

The invention discloses recombinant J subgroup avian leucosis virus infective cloned plasmids and a preparation method and application thereof. The preparation method comprises the following steps of: replacing long terminal repetitive sequences of two ends of a provirus gene of a J subgroup avian leucosis virus with long terminal repetitive sequences of two ends of a provirus gene of an E subgroup avian leucosis virus; and inserting the long terminal repetitive sequences of the two ends of the provirus gene of the E subgroup avian leucosis virus into vectors, so that the J subgroup avian leucosis virus infective cloned plasmids are constructed, and the obtained sequence is shown as SEQ ID NO:1. The replication capacity and the pathogenicity of recombinant virus strains obtained by transfecting the J subgroup avian leucosis virus infective cloned plasmids are obviously weaker than those of natural J subgroup avian leucosis viruses, but the recombinant virus strains can express structural proteins of the J subgroup avian leucosis viruses, have the antigenicity of the J subgroup avian leucosis viruses and can be identified by a specificity monoclonal antibody JE9 of the J subgroup avian leucosis viruses. The invention provides reference for a material and a method for preparing low-toxicity live vaccines of the J subgroup avian leucosis viruses.

Description

technical field [0001] The invention relates to the field of infectious cloning of avian leukemia virus, in particular to a recombinant J subgroup avian leukemia virus infectious cloning plasmid and its preparation method and application. Background technique [0002] Avian leukemia virus (ALV) belongs to the Orthoretroviridae subfamily, Retroviruses (Fauquet et al, 2005). Avian leukemia (AL) is a general term for a variety of neoplastic diseases in poultry caused by ALV, which has various clinical manifestations, including lymphocytic leukemia, erythroblastic leukemia, myeloblastic leukemia, myeloid leukemia, Cellular leukemia, etc. (Ewert, 1988; Coffin, 1992; Payne et al, 1997; Payne, 1998; Fadly et al, 2003). [0003] Before the 1980s, the variation of ALV subgroups was very small, mainly the 5 subgroups identified in the 1960s (Levy, 1992). Afterwards, subgroup J avian leukemia epidemics occurred successively in broilers in the United States, Israel, France and other...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/48C12N15/66C12N7/01C12N7/04A61K39/21A61P31/14A61P35/02
Inventor 廖明赖汉漳曹伟胜张贺楠辛朝安
Owner SOUTH CHINA AGRI UNIV
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