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Eukaryotic coexpression vector of gB gene of avian infectious laryngotracheitis virus and chicken interleukin-18 gene

A technology of laryngotracheitis virus and co-expression vector, which is applied in the field of animal medicine and bioengineering, can solve the problems of large reaction, strong vaccine virulence, and strong virulence reversion, and achieves the advantages of improving the commissioning efficiency, enhancing the immune effect and shortening the incubation period. Effect

Inactive Publication Date: 2011-10-19
ZHENGZHOU HOUYI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the vaccines used to prevent chicken infectious laryngotracheitis virus both at home and abroad are attenuated vaccines, but all attenuated vaccines have the following disadvantages: 1) because the virulence of the virus is positively correlated with immunogenicity, the current vaccines The virulence is generally strong, and the reaction after vaccination is strong; 2) Like the strong ILTV virus, its attenuated vaccine strain can also cause latent infection, and latently infected chickens will eventually be infected; 3) The vaccine virus can be transmitted from vaccinated chickens to non-virulent chickens. Inoculated chickens, the virulence will become stronger during the transmission process between chickens, chickens and chickens, thus becoming a new source of infection; 4) So far there is no effective method to identify strong and weak strains

Method used

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  • Eukaryotic coexpression vector of gB gene of avian infectious laryngotracheitis virus and chicken interleukin-18 gene
  • Eukaryotic coexpression vector of gB gene of avian infectious laryngotracheitis virus and chicken interleukin-18 gene
  • Eukaryotic coexpression vector of gB gene of avian infectious laryngotracheitis virus and chicken interleukin-18 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Construction of recombinant expression plasmid pIRES / IL-18

[0019] 1) Primer design: Design a pair of primers based on pGEM-ChIL-18. The upstream and downstream primers add SalI and NotI restriction sites respectively. The amplification length of the pair of primers is 594bp. The primers were synthesized by Shanghai Bioengineering Co., Ltd. The sequence is as follows:

[0020] Upstream primer P1: 5'-GAC GTC GAC ATGAGCTGTGAAGAGATC-3',

[0021] Downstream primer P2: 5'-TAT GCGGCCGC TTATAGGTTGTGCCTTT-3';

[0022] 2) PCR amplification of IL-18 gene: Use the pGEM-ChIL-18 plasmid as a template and P1 and P2 as primers for PCR amplification reaction. The amplification system is: Premix Ex Taq DNA polymerase 25 μL, template plasmid 1 μL, upper, 1 μL of each downstream primer, supplemented with 50 μL of ultrapure water, mixed evenly, and then amplified on the PCR machine, and no DNA control was set up. The amplification program was: 94°C pre-denaturation for 5 m...

Embodiment 2

[0029] Example 2 Construction of recombinant expression plasmid pIRES / gB

[0030]1) Primer design: Refer to the ILTVgB gene nucleotide sequence (M64927) published by GenBank, and use Primer 5.0 gene analysis software to design a pair of primers. The primer amplification length is 2.6kb, and the primers were synthesized by Shanghai Bioengineering Co., Ltd. The specific sequences of the primers are as follows:

[0031] Upstream primer P3: 5'-GGTA CTCGAG ATGGCTAGCTTGAAA-3',

[0032] Downstream primer P4: 5'-TGGT ACGCGT TTATTCGTCTTCGCT-3';

[0033] 2) Amplify the target fragment of gB whole gene by PCR: use pGEM-T-gB as template and P3 and P4 as primers for PCR amplification reaction. The amplification system is: Premix Ex Taq DNA polymerase 25 μL, template plasmid 1 μL, upper, 1 μL each of the downstream primers, 50 μL of sterilized deionized water was added, mixed evenly, and then amplified on a PCR instrument. The amplification program was: 94°C pre-denaturation for 5 min...

Embodiment 3

[0040] Embodiment 3 Construction of recombinant plasmid pIRE / gB / IL-18

[0041] 1) pIRES / IL-18 double enzyme digestion: extract the correct recombinant expression plasmid pIRES / IL-18 verified by sequencing, enzyme digestion system: pIRES / IL-18 30 μL, MluI 2.5 μL, XhoⅠ 2.5 μL, sterilized water to make up 50 μL , digested in a water bath at 37°C for 3 hours, and the digested product was subjected to gel agarose electrophoresis and slicing to recover pIRES / IL-18 fragments. For specific steps, refer to the instructions of the V-gene DNA gel recovery kit to purify and recover specific DNA bands Observe the recovery situation through gel agarose electrophoresis;

[0042] 2) Ligation of the gB target fragment with the expression plasmid pIRES / IL-18: take 1 μL of the double-enzyme-digested plasmid pIRES / IL-18, and 7 μL of the gB target fragment recovered by Mlu I and XhoI double-enzyme digestion, and react in 200 μL In the tube, add 10×ligation buffer 1 μL, T 4 DNA ligase 1 μL, mix ...

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Abstract

The invention discloses a eukaryotic coexpression vector of a gB gene of avian infectious laryngotracheitis virus and a chicken interleukin-18 gene. The eukaryotic coexpression vector is constructed by respectively or simultaneously inserting the gB gene of avian infectious laryngotracheitis virus (ILTV) and the chicken interleukin 18 (IL-18) gene into a eukaryotic coexpression vector (p1RES), wherein a fragment of the gB gene of the avian infectious laryngotracheitis virus is inserted between the restriction sites XhoI and M1uI of an MCSA (Multiple Cloning Site A) at the downstream of a promoter and a fragment of the chicken interleukin-18 gene is inserted between the restriction sites Sa1I and NotI of an MCSB (Multiple Cloning Site B) to obtain the coexpression plasmid pIRES / gB / IL18. According to the eukaryotic compression vector disclosed by the invention, after a nucleic acid vaccine immune organism is obtained, the expressed gB glycoprotein can stimulate the organism to have an immune protection function on the avian infectious laryngotracheitis virus; the expressed chicken interleukin 18 can give play to the obvious immune adjustment; and the immune effect is enhanced through the synergistic effect of the avian infectious laryngotracheitis virus and the chicken interleukin 18.

Description

technical field [0001] The invention belongs to the technical field of animal medicine bioengineering, in particular to a eukaryotic co-expression carrier of chicken infectious laryngotracheitis virus gB gene and chicken interleukin-18 gene. Background technique [0002] Chicken infectious laryngotracheitis is an acute upper respiratory contact infectious disease of chickens caused by chicken infectious laryngotracheitis virus (ILTV). Seriously affect the egg production of laying hens. The disease has the characteristics of acute outbreak and long-term latent infection, and is distributed all over the world. It is one of the important epidemic diseases that endanger the poultry industry. [0003] At present, the vaccines used to prevent chicken infectious laryngotracheitis virus both at home and abroad are attenuated vaccines, but all attenuated vaccines have the following disadvantages: 1) because the virulence of the virus is positively correlated with immunogenicity, the...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/38C12N15/24
Inventor 吴红云李厚伟李建正
Owner ZHENGZHOU HOUYI PHARMA
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