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Duck plague virus transfer vector pUC-deltagC-EGFP, and gC-deleted recombinant strain DPV-deltagC-EGFP

A transfer vector, duck plague virus technology, applied in the field of genetic engineering

Active Publication Date: 2012-10-17
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on duck plague virus is still in its infancy, and the functions of various glycoproteins still need to be further studied

Method used

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  • Duck plague virus transfer vector pUC-deltagC-EGFP, and gC-deleted recombinant strain DPV-deltagC-EGFP
  • Duck plague virus transfer vector pUC-deltagC-EGFP, and gC-deleted recombinant strain DPV-deltagC-EGFP
  • Duck plague virus transfer vector pUC-deltagC-EGFP, and gC-deleted recombinant strain DPV-deltagC-EGFP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1. Construction of the EGFP expression cassette containing CMV promoter and SV40polyA

[0064] 1.1 Extraction of plasmid pEGFP-Cl

[0065] Streak inoculate the Escherichia coli carrying the plasmid pEGFP-Cl in the LB solid medium containing kanamycin, culture at 37 °C for 20 h, pick a single colony and inoculate it in the LB liquid medium containing kanamycin at 37 °C Cultivate overnight, and then follow the instructions of the AxyPrep Plasmid DNA Mini Kit:

[0066] Take 1-4mL of the bacterial solution cultured overnight in LB medium, centrifuge at 12000×g for 1min, and discard the supernatant;

[0067] Add 250 μL Buffer S1 to suspend the bacterial pellet, the suspension should be uniform, and no small bacterial lumps should be left;

[0068] Add 250μL Buffer S2, gently and fully turn it up and down 4-6 times to mix evenly to fully lyse the bacteria until a clear solution is formed;

[0069] Add 350μL Buffer S3, gently and thoroughly mix up and down 6-8 ti...

Embodiment 2

[0131] Embodiment 2. Containing the construction of EGFP gene transfer plasmid vector

[0132] 2.1 Construction of the left and right plasmids containing UL44

[0133] According to the UL43-UL45 fragment sequence in the whole genome sequence of DPV CHv strain (GenBank accession number: JQ647509), two pairs of primers were designed ( figure 2 ),they are, respectively:

[0134] gCLF: 5′-TTGCCCAAGCTTGGGACAAGAACGGAGGTCAAGAC-3′

[0135] gCLR: 5′-CTAGCTCTAGAGCGATATAGAATCGTTAAGTATT-3′

[0136] gCRF: 5′-ATACGGGATCCCGTGGCCGTTTGTTTCTATTAT-3′

[0137] gCRR: 5′-ATCGGAATTCCATACACGGATTAGCCAGA-3′

[0138] They respectively amplify the left and right fragments of the UL44 fragment of the duck plague virus genome.

[0139] After extracting duck plague virus CHv strain genome by conventional methods, PCR amplified the left and right fragments of UL44 respectively, T-cloned into pMD19-T vector, and then transformed into Escherichia coli DH5α competent cells, picked positive clones for pro...

Embodiment 3

[0144] Example 3. Construction and purification of recombinant duck plague virus DPV-ΔgC-EGFP

[0145] 3.1 Construction of recombinant duck plague virus DPV-ΔgC-EGFP containing EGFP gene

[0146] Prepare primary duck embryo fibroblasts by conventional methods, culture them in a six-well cell culture plate until a monolayer is formed, inoculate DPV CHv strain, and inoculate at 37°C for 2 hours; after about 1 hour, add 240 μL serum-free OPTI-MEM and Calculate the ratio of 10 μL Lipofectamine 2000 to prepare solution 1, then add 240 μL serum-free OPTI-MEM and 10 μL containing 4 μg pUC-ΔgC-EGFP transfer plasmid DNA to each well to calculate and prepare solution 2, then mix solution 1 and solution 2, and let them react at room temperature for 20 minutes During this period, gently wash the cells in the six-well plate twice with serum-free OPTI-MEM, add 1.5mL serum-free OPTI-MEM to each well, then add 0.5mL of the incubated mixture dropwise, and leave for a well as a control; 37°C 5...

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Abstract

The invention discloses a duck plague virus transfer vector pUC-deltagC-EGFP and a duck plague virus gC-deleted recombinant strain DPV-deltagC-EGFP. An escherichia coli DH5alpha containing the transfer vector pUC-deltagC-EGFP is collected on November 30th, 2011, in China Center for Type Culture Collection at Wuhan University of China, and has a collection number of CCTCC NO:M2011432. After the transfer vector is subjected to homologous recombination with the duck plague virus, and after plaque purification, the EGFP-labeled recombinant virus DPV-deltagC-EGFP is obtained. As a result of experiments, same as an attenuated vaccine, the recombinant virus DPV-deltagC-EGFP with three dosages can provide complete protection for ducklings attacked by virulent virus. Therefore, the recombinant strain has certain potential to be developed into a novel vaccine which can effectively prevent duck plague.

Description

technical field [0001] The invention belongs to the field of genetic engineering in the biotechnology pharmaceutical industry, in particular to a duck plague virus transfer vector and its application. Background technique [0002] Using genetic engineering technology, the foreign DNA is transferred to the corresponding vector, and through recombination with the virus gene, the function of the virus gene can be lost, or the foreign protein can be expressed. In this way, on the one hand, the function of the missing gene can be understood more clearly, and on the other hand, the virus can be developed as a live-vector vaccine by using the virus to express foreign DNA proteins. The transfection efficiency of viral live vector vaccine is high, and the expressed protective foreign protein is close to the post-translational mature protein; this vaccine can simultaneously activate the body's cellular and humoral immunity, and can prevent multiple diseases through one immunization, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N1/21C12N15/66C12N7/00C12R1/19C12R1/93
Inventor 程安春孙昆峰汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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