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A kind of chicken Newcastle disease virus strain and its application in the preparation of chicken Newcastle disease vaccine

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: 2016-07-13
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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  • A kind of chicken Newcastle disease virus strain and its application in the preparation of chicken Newcastle disease vaccine
  • A kind of chicken Newcastle disease virus strain and its application in the preparation of chicken Newcastle disease vaccine
  • A kind of chicken Newcastle disease virus strain and its application in the preparation of chicken 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 the Kozac sequence, and the 16th to 450th nucleotides It is the coding gene of the chGM-CSF polypeptide, and the 451st to 456th nucleotides are the recognition sequence of the restriction endonuclease MluI.

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

[0047] 3. The pBrClone30 plasmid was digested with restriction enzymes HpaI and MluI, and the vector backbone of about 16000bp was recovered.

[0048] 4. Connect the enzyme-digested product of step 2 to the vect...

Embodiment 2

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

[0060] 1. The DF-1 cells in the logarithmic growth phase were inoculated in a six-well plate, and the rClone30-GM-CSF virus solution prepared in Example 1 was infected with a dose of 0.1MOI (the rClone30-GM-CSF virus solution was diluted with complete DMEM medium). ), incubate at 37°C for 1 h, then wash three times with complete DMEM medium, then add complete DMEM medium containing 1 μg / mL trypsin and place in 5% CO 2 , 37 ℃ environment for 48 hours of static culture, and then repeated freezing and thawing 3 times, take the supernatant.

[0061] 2. The rClone30 virus solution prepared in Example 1 was used instead of the rClone30-GM-CSF virus solution, and the other steps were the same as in step 1.

[0062] 3. Use a GM-CSF kit (CSB-E17363C, Cusabio) and operate according to the instructions to detect the concentration of chGM-CSF in the supernatant obtained in step 1 and the supernatant obtained i...

Embodiment 3

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

[0065] 1. The DF-1 cells in the logarithmic growth phase were inoculated into a six-well plate, and the rClone30-GM-CSF virus solution (rClone30-GM-CSF virus solution was diluted with complete DMEM medium) prepared in Example 1 at a dose of 0.1 MOI. dilution) were seeded on the cell monolayer in complete DMEM medium containing 1 μg / mL trypsin, placed in 5% CO 2 , 37 ℃ environment for 96h static culture, take the supernatant every 24h and measure TCID 50 .

[0066] 2. The rClone30 virus solution prepared in Example 1 was used instead of the rClone30-GM-CSF virus solution, and the other steps were the same as in step 1.

[0067] see the results Figure 5 . rClone30-GM-CSF virus maintained the same reproductive titer as 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 chicken Newcastle disease virus strain and its application in preparing chicken Newcastle disease vaccine. The preparation method of chicken Newcastle disease virus strain provided by the invention comprises the steps of: co-transfecting mammalian cells with recombinant plasmids pBrClone30-GM-CSF, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid and culturing to obtain the obtained The chicken Newcastle disease virus strain; the recombinant plasmid pBrClone30-GM-CSF is a plasmid having the DNA molecule shown in the 2703-18408th nucleotide at the 5' end of the sequence 3 of the sequence listing. The invention introduces the molecular adjuvant GM-CSF into the genome of the existing Newcastle disease live vaccine, which can effectively improve the immune effect, greatly shorten the immune blank period, resist maternal antibodies, and improve the immune protection rate. The invention has great value for the prevention and treatment of Newcastle disease.

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 septicemic infectious disease caused by Newcastle disease virus (NDV) in poultry. one of the diseases. NDV is an RNA virus that can infect almost all birds. Sick chickens are the main source of infection of the disease. 24 hours before the clinical symptoms of chickens appear, the virus is excreted in their mouth, nasal secretions and feces. The virus is present in all tissues and organs, body fluids, secretions and excreta of sick chickens. Poisoned chickens in the intermittent period of the epidemic are also the source of infection of this disease. Birds are also important communicators. The virus can enter the body through the digestive tract, respiratory tract, conjunctiva, injured skin and c...

Claims

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Application Information

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Patent Type & Authority Patents(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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