Chicken Newcastle Disease Vaccine Virus Strain rclone30‑chil15 and Its Application

A technology of chicken Newcastle disease virus and strains, which is applied in the direction of antiviral agents, virus/bacteriophage, virus antigen components, etc., can solve the problem that chickens cannot get immune protection, shorten the immune blank period, improve immune effect, and improve immune protection rate effect

Active Publication Date: 2018-03-02
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such vaccines often need to produce antibodies 7 days after the first vaccination until the antibody titer reaches the protective level two weeks after immunization. It takes more than 2 weeks from immunization to protection. If Newcastle disease breaks out at the time of vaccination During this blank period of immunity, chickens will not be able to receive effective immune protection

Method used

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  • Chicken Newcastle Disease Vaccine Virus Strain rclone30‑chil15 and Its Application
  • Chicken Newcastle Disease Vaccine Virus Strain rclone30‑chil15 and Its Application
  • Chicken Newcastle Disease Vaccine Virus Strain rclone30‑chil15 and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, preparation of rClone30-chIL15 virus liquid

[0039] 1. Construction of recombinant plasmids

[0040] 1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

[0041]In the sequence 1 of the synthetic sequence list, the 1st to 6th nucleotides from the 5′ end are the recognition sequence of the restriction endonuclease SacII, the 9th to 14th nucleotides are the Kozac sequence, and the 15th to 578th nucleotides It is the coding gene of chIL15 polypeptide, and the 579th to 584th nucleotides are the recognition sequence of the restriction endonuclease MluI;

[0042] 2. Double-digest the double-stranded DNA molecule obtained in step 1 with restriction enzymes SacII and MluI, and recover the digested product;

[0043] 3. Digest the pBrClone30 plasmid with restriction endonucleases SacII and MluI, and recover the vector backbone fragment, which is about 16000bp in size;

[0044] 4. Ligate the digested product of step 2 with ...

Embodiment 2

[0055] Example 2, Detection of the expression level of chIL15 in the virus fluid

[0056] 1. Inoculate DF-1 cells in the logarithmic growth phase on a six-well plate, infect the rClone30-chIL15 virus solution prepared in Example 1 at a dose of 0.1 MOI (rClone30-chIL15 virus solution is diluted with complete DMEM medium), 37°C Incubate statically for 1 hour, wash with complete DMEM medium three times, add complete DMEM medium containing 1 μg / mL trypsin and place in 5% CO2, 37°C for 48 hours, then freeze and thaw repeatedly 3 times, and collect supernatant by centrifugation liquid;

[0057] 2. Replace the rClone30-chIL15 virus solution with the rClone30 virus solution prepared in Example 1, and the others are the same as step 1;

[0058] 3. Use the chIL15 kit (Antibodies online, ABIN414131) and operate according to the instructions to detect the concentration of chIL15 in the supernatant obtained in step 1 and the supernatant obtained in step 2;

[0059] see results Figure 4...

Embodiment 3

[0060] Example 3, Dynamic growth curves of rClone30-chIL15 virus and rClone30 virus in host cells

[0061] 1. Inoculate DF-1 cells in the logarithmic growth phase on a six-well plate, and inoculate the rClone30-chIL15 virus solution prepared in Example 1 (rClone30-chIL15 virus solution diluted with complete DMEM medium) at a dose of 0.1 MOI in Cell monolayer, using complete DMEM medium containing 1 μg / mL trypsin, placed in 5% CO2, 37°C for static culture for 96 hours, and the supernatant was taken every 24 hours and measured for TCID 50 ;

[0062] 2. The rClone30 virus solution prepared in Example 1 was used instead of the rClone30-chIL15 virus solution, and the rest were the same as step 1.

[0063] see results Figure 5 . The reproduction titer of rClone30-chIL15 virus was consistent with that of rClone30 virus. The results showed that the proliferative ability of empty vector rClone30 was not changed after inserting exogenous chIL15 gene.

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Abstract

The invention discloses a novel chicken Newcastle disease virus strain and application thereof. The preparation method of the chicken Newcastle disease virus strain provided by the invention comprises the following steps: co-transfecting mammalian cell BHK-21 with recombinant plasmid pBrClone30-chIL15 and helper plasmids pBL-N, pBL-P and pBL-L and culturing to obtain the obtained The chicken Newcastle disease virus strain; the recombinant plasmid pBrClone30-chIL15 is a plasmid having a DNA molecule shown in Sequence 1 of the sequence listing. Sequence 3 of the sequence listing is the genomic DNA of the rClone30-chIL15 virus from the 2703-18546th nucleotide at the 5' end. The invention introduces molecular adjuvant chIL15 into the genome of the existing Newcastle disease live vaccine, which can effectively improve the immune effect and improve the immune protection rate. The invention has great significance for the prevention and treatment of Newcastle disease.

Description

technical field [0001] The invention relates to a novel chicken Newcastle disease virus strain rClone30-chIL15 and application thereof. Background technique [0002] Newcastle Disease (ND) is a highly contagious and acute septic infectious disease of poultry caused by Newcastle Disease Virus (NDV), which is extremely harmful to the world's poultry industry. As a single-strand negative-strand RNA virus, NDV virus has a wide range of hosts. Among them, chickens are the most susceptible, and the morbidity and mortality of young chickens are significantly higher than those of older chickens. They can occur throughout the year, but mainly in spring more. In the past three years alone, 1,252 cases of Newcastle disease have occurred, involving 19 provinces, municipalities, and autonomous regions. Nearly 400,000 poultry birds were infected, 200,000 birds died, and more than 150,000 birds were killed. [0003] At present, my country has been adopting vaccination-based measures supp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01A61K39/17A61P31/14C12R1/93
Inventor 何金娇李德山张天援王卉刘云野
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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