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Construction method and application of DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J

A subgroup, microbial strain technology, applied in the field of veterinary nucleic acid vaccines, can solve the problems of low level and slow antibody production of vaccines

Inactive Publication Date: 2014-03-05
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Nucleic acid vaccine is one of the new vaccines developed after the 1990s. It has the advantages of convenient preparation, long-lasting immunity, less side effects and the ability to induce nuclear humoral immunity mediated by T cells and B cells. However, the vaccine has slow production of antibodies. , the shortcomings of low levels, need effective adjuvants to overcome
At present, the development of ALV-J DNA vaccine has not been reported

Method used

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  • Construction method and application of DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J
  • Construction method and application of DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J
  • Construction method and application of DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of J subgroup avian leukemia DNA vaccine pcDNA3.1-Fc-env

[0032] (1) Cloning and identification of chicken IgG-Fc gene

[0033] According to the chicken IgG heavy chain sequence (X07174.1) published by NCBI, the primers were designed, the EcoR I restriction site and ATG start codon were added to the upstream primer, the terminator was removed from the downstream primer, and the chicken was obtained by RT-PCR method. IgG-Fc gene.

[0034] 1. Extraction of total RNA from chicken spleen

[0035] 1) Weigh an appropriate amount of spleen tissue into a 1.5ml centrifuge tube, add 500ul of Buffer LY and 10ul of β-mercaptoethanol, and shake vigorously on a shaker for 2 minutes. Then let stand for 2 minutes to sink the tissue fragments to the bottom of the tube.

[0036] 2) Pour the supernatant into a DNA gap column, 13000rpm for two minutes, discard the DNA column (filter the DNA in the solution), and keep the liquid in the collection tube below.

[0...

Embodiment 2

[0113] Example 2 Evaluation of the immune effect of DNA vaccine pcDNA3.1-Fc-env.

[0114] 1. Mass extraction of recombinant plasmids

[0115] (1) Take the recombinant ALVFc E. coli constructed in Example 1, inoculate 150 ml of LB medium, add the overnight cultured recombinant E. coli bacterial solution into a centrifuge tube, centrifuge at 10,000 rpm for 2 to 3 minutes to collect bacteria, and discard all the supernatant as much as possible.

[0116] (2) Add 12ml DNA purification buffer Buffer P1 (included in the commercial DNA purification kit) to the centrifuge tube with the bacterial cell pellets according to the instructions of the commercial DNA purification kit, and mix thoroughly with a pipette or a vortex shaker , suspended bacterial pellets.

[0117] (3) Add 12ml of Buffer P2 (included in a commercial DNA purification kit) to the centrifuge tube, gently invert and mix for 6-8 times to fully lyse the cells, and leave at room temperature for 3-5 minutes. At this point...

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Abstract

The invention relates to a construction method and application of a DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J. A recombinant eukaryotic expression plasmid pcDNA3.1-Fc-env of an Fc fragment gene for expressing chicken immunoglobulin G and an envelope protein (env protein) gene of the avian leukosis virus subgroup J is constructed; transient transfection and indirect immunofluorescence assay prove that the pcDNA3.1-Fc-env can be accurately expressed in a 293T cell; a great number of plasmids are extracted, purified and quantified to 1mg / ml, then, the recombinant plasmids are used for immunizing mice, each mouse is immunized three times, 100mu g of recombinant plasmids are used in each immunization, and one immunization is carried out every two weeks; the level of an ALV-J env protein-specific antibody in serum is detected to show that the pcDNA3.1-Fc-env has the effect of preventing the avian leukosis virus subgroup J.

Description

1. Technical field [0001] The invention relates to the construction and application of a J subgroup avian leukemia DNA vaccine, and belongs to the field of veterinary nucleic acid vaccines. 2. Background technology [0002] Avian Leukosis virus subgroup J (ALV-J) is a new subgroup of avian leukemia virus first isolated from British white-feathered broilers in 1988, which mainly causes myeloid hyperplasia and tumors in white-feathered broilers. Like subgroups A and B, ALV-J is an exogenous avian leukemia virus, which can be transmitted both vertically and horizontally in chickens, and the horizontal transmission speed of subgroup J is much higher than that of subgroups A and B. The disease has spread rapidly to many countries, posing a serious problem for the chicken industry. [0003] In my country, ALV-J was brought into our country's white-feather broiler flock with the introduction of white-feather broiler breeders. In 1999, ALV-J was isolated and detected for the first...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K48/00A61P35/02C12R1/19
Inventor 常维山崔治中郭浩
Owner SHANDONG AGRICULTURAL UNIVERSITY
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