Novel Newcastle disease vaccine virus strain rClone30-chIL15 and applications thereof

A chicken Newcastle disease virus and Newcastle disease technology, which is applied to the new chicken Newcastle disease vaccine virus strain rClone30-chIL15 and its application fields, can solve the problem that chickens cannot be protected by immunity, shorten the blank period of immunity, improve the immune effect, and improve The effect of immune protection rate

Active Publication Date: 2015-03-11
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 we

Method used

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  • Novel Newcastle disease vaccine virus strain rClone30-chIL15 and applications thereof
  • Novel Newcastle disease vaccine virus strain rClone30-chIL15 and applications thereof
  • Novel Newcastle disease vaccine virus strain rClone30-chIL15 and applications thereof

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 Newcastle disease virus strain and applications thereof. The preparation method of the provided Newcastle disease virus strain comprises the following steps: co-transfecting recombinant plasmid pBrClone30-chIL15 and auxiliary plasmid pBL-N, pBL-P, and pBL-L to a mammal cell BHK-21, and then culturing the mammal cell BHK-21 so as to obtain the Newcastle disease virus strain, wherein the recombinant plasmid pBrClone30-chIL15 is from a DNA molecule represented by the sequence 1 in the sequence table. The nucleotide on the positions from 2703 to 18546 of the 5' end of the sequence 3 in the sequence table is the genome DNA of rClone30-chIL15 virus. A molecular adjuvant chIL15 is introduced into the conventional Newcastle disease live vaccine genome so as to effectively improve the immunity effect and increase the immunity protection rate. The provided Newcastle disease virus strain has an important meaning for the prevention and treatment on 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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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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