Newcastle disease virus strain and application thereof in preparation of Newcastle disease vaccine

A technology for chicken Newcastle disease virus and strain, which is applied to the chicken Newcastle disease virus strain and its application field in preparing chicken Newcastle disease vaccine, can solve the problem that chicken flocks cannot obtain immune protection and the like, and achieves shortening the immune blank period, improving the immune protection rate, The effect of improving immunity

Active Publication Date: 2014-11-26
JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If Newcastle disease breaks out in the blank period of vaccination, chickens will not be able to get effective immune protection

Method used

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  • Newcastle disease virus strain and application thereof in preparation of Newcastle disease vaccine
  • Newcastle disease virus strain and application thereof in preparation of Newcastle disease vaccine
  • Newcastle disease virus strain and application thereof in preparation of Newcastle disease vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the preparation of rClone30-GM-CSF virus liquid

[0043] 1. Construction of recombinant plasmids

[0044] 1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

[0045] In sequence 1 of the sequence listing, the 1st to 6th nucleotides from the 5' end are the recognition sequence of the restriction endonuclease HpaI, the 10th to 15th nucleotides are Kozac sequences, and the 16th to 450th nucleotides It is the coding gene of chGM-CSF polypeptide, and the 451st to 456th nucleotides are the recognition sequence of restriction endonuclease Mlu I.

[0046] 2. Digest the double-stranded DNA molecule obtained in step 1 with restriction endonucleases HpaI and MluI, and recover the digested product.

[0047] 3. Digest the pBrClone30 plasmid with restriction endonucleases HpaI and MluI, and recover the vector backbone of about 16000 bp.

[0048] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obt...

Embodiment 2

[0059] Embodiment 2, detection of the expression level of chGM-CSF in virus liquid

[0060] 1. Inoculate the DF-1 cells in the logarithmic growth phase on a six-well plate, and infect the rClone30-GM-CSF virus solution prepared in Example 1 at a dose of 0.1 MOI (the rClone30-GM-CSF virus solution is diluted with complete DMEM medium ), incubated at 37°C for 1 h, then washed three times with complete DMEM medium, then added complete DMEM medium containing 1 μg / mL trypsin and placed in 5% CO 2 , Cultured at 37°C for 48 hours, then repeated freezing and thawing 3 times, and the supernatant was taken.

[0061] 2. Use the rClone30 virus solution prepared in Example 1 to replace the rClone30-GM-CSF virus solution, and the other steps are the same as step 1.

[0062] 3. Using the GM-CSF kit (CSB-E17363C, Cusabio) and operating according to the instructions, detect the concentration of chGM-CSF in the supernatant obtained in step 1 and the supernatant obtained in step 2, respectively...

Embodiment 3

[0064] Example 3, Dynamic growth curves of rClone30-GM-CSF virus and rClone30 virus in host cells

[0065] 1. Inoculate the DF-1 cells in the logarithmic growth phase on a six-well plate, and dilute the rClone30-GM-CSF virus solution prepared in Example 1 (the rClone30-GM-CSF virus solution is diluted with complete DMEM medium) at a dose of 0.1 MOI. diluted) inoculated on cell monolayer, using complete DMEM medium containing 1 μg / mL trypsin, placed in 5% CO 2 , Culture at 37°C for 96 hours, take supernatant every 24 hours and measure TCID 50 .

[0066] 2. Use the rClone30 virus solution prepared in Example 1 to replace the rClone30-GM-CSF virus solution, and the other steps are the same as step 1.

[0067] see results Figure 5 . The rClone30-GM-CSF virus maintained the same reproductive titer as the rClone30 virus. The results showed that the insertion of the gene encoding chGM-CSF did not affect the proliferation ability of rClone30 virus.

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Abstract

The invention discloses a Newcastle disease virus strain and an application thereof in preparation of a Newcastle disease vaccine. A preparation method of the Newcastle disease virus strain comprises the following step: culturing recombinant plasmids pBrClone30-GM-CSF, pBL-N plasmids, pBL-P plasmids and pBL-L plasmid cotransfection mammalian cells together so as to obtain the Newcastle disease virus strain, wherein the recombinant plasmids pBrClone30-GM-CSF are of the plasmids of DNA molecules shown in No.2703 to No.18408 nucleotides from the 5' tail end of a sequence 3 with a sequence table. According to the Newcastle disease virus strain, molecular adjuvants GM-CSF are led into the genomes of the existing Newcastle disease vaccine, so that the immune effect is effectively improved and the immune blank period is greatly shortened. Meanwhile, the Newcastle disease virus strain can be used for resisting a maternal antibody so as to improve the immune protective rate. Thus, the Newcastle disease virus strain has an important value for preventing and treating Newcastle diseases.

Description

technical field [0001] The invention relates to a chicken Newcastle disease virus strain and its application in preparing chicken Newcastle disease vaccine. Background technique [0002] Newcastle disease (ND), also known as Asian chicken plague, is a highly contagious and acute septic infectious disease of poultry caused by Newcastle disease virus (NDV), which seriously endangers the poultry industry in the world. One of the major severe infectious diseases. NDV is an RNA virus that can infect almost all poultry. Sick chickens are the main source of infection of the disease. 24 hours before clinical symptoms appear after chicken infection, the virus is excreted in the secretions and feces of the mouth and nose. The virus exists in all tissues and organs, body fluids, secretions and excreta of sick chickens. Poisoned chickens in the intermission period of the epidemic are also the source of infection of the disease. Birds are also important communicators. The virus can i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/63A61K39/17A61P31/14C12R1/93
Inventor 李德山王卉张天援何金娇
Owner JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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