Preparation of Newcastle disease virus strain rClone30-fliC and application of the Newcastle disease virus strain rClone30-fliC in prevention and treatment of Newcastle disease

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 blank period of immunity, improve the rate of immune protection, and improve immunity effect of effect

Active Publication Date: 2015-02-04
JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If Newcastle disease breaks out in the blank period of vaccin...

Method used

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  • Preparation of Newcastle disease virus strain rClone30-fliC and application of the Newcastle disease virus strain rClone30-fliC in prevention and treatment of Newcastle disease
  • Preparation of Newcastle disease virus strain rClone30-fliC and application of the Newcastle disease virus strain rClone30-fliC in prevention and treatment of Newcastle disease
  • Preparation of Newcastle disease virus strain rClone30-fliC and application of the Newcastle disease virus strain rClone30-fliC in prevention and treatment of Newcastle disease

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, the preparation of rClone30-fliC virus liquid

[0044] 1. Construction of recombinant plasmids

[0045] 1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

[0046] 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-15th nucleotides are Kozac sequences, and the 16th to 1503rd nucleotides are In the coding gene of fliC protein, the 1504th to 1509th nucleotide is the recognition sequence of restriction endonuclease Mlu I.

[0047] 2. Double-digest the double-stranded DNA fragment obtained in step 1 with restriction endonucleases HpaI and MluI, and recover the target fragment in the digested product.

[0048] 3. Digest the pBrClone30 plasmid with restriction endonucleases HpaI and MluI, and recover a vector fragment of about 16000 bp.

[0049] 4. Ligate the digested product fragment in step 2 with the vector fra...

Embodiment 2

[0061] Example 2, Detection of the expression level of fliC in the virus fluid

[0062] 1. Inoculate DF-1 cells in the logarithmic growth phase on a six-well plate, infect the cells with the rClone30-fliC virus solution prepared in Example 1 at a MOI of 0.1 (rClone30-fliC virus solution is diluted with complete medium), and statically at 37°C. Incubate for 1 h, then wash three times with complete medium, add complete medium containing 1 μg / mL trypsin and place in 5% CO 2 , and cultured at 37°C for 48 hours, then repeatedly freeze-thawed at -80°C for 3 times, and took the supernatant.

[0063] 2. Use the rClone30 virus solution prepared in Example 1 to replace the rClone30-fliC virus solution, and the others are the same as step 1.

[0064] 3. Use fliC antibody (629702, BioLegend) for ELISA detection, and detect the concentration of fliC protein in the supernatant obtained in step 1 and step 2, respectively.

[0065] see results Figure 4 . The results showed that rClone30-...

Embodiment 3

[0067] Example 3, Dynamic growth curves of rClone30-fliC virus and rClone30 virus in host cells

[0068] 1. Inoculate DF-1 cells in the logarithmic growth phase on a six-well plate, inoculate the rClone30-fliC virus solution prepared in Example 1 (rClone30-fliC virus solution is diluted with complete medium) at a dose of 0.1 MOI in a monolayer cells in complete medium containing 1 μg / mL trypsin in 5% CO 2 , Culture at 37°C for 96 hours, take the supernatant every 24 hours and measure the TCID 50 .

[0069] 2. Use the rClone30 virus solution prepared in Example 1 to replace the rClone30-fliC virus solution, and the others are the same as step 1.

[0070] see results Figure 5 . The rClone30-fliC virus maintained the same reproductive titer as the rClone30 virus. The results showed that the insertion of the gene encoding fliC protein did not affect the proliferation ability of rClone30 virus.

[0071]

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Abstract

The invention discloses a Newcastle disease virus strain rClone30-fliC and an application thereof in prevention and treatment of Newcastle disease. The Newcastle disease virus strain rClone30-fliC and a preparation method thereof are disclosed. The preparation method comprises a step of co-transfecting a constructed recombinant plasmid pBrClone30-fliC and corresponding auxiliary plasmids pBL-N, pBL-P and pBL-L to a BHK-21 cell which can stably express a T7 polymerase with rescue cultivation to obtain the Newcastle disease virus strain. In the invention, by means of introducing a molecular adjuvant fliC to a conventional Newcastle disease live vaccine genome, immunoreactions of an inoculated chicken is enhanced and an HI antibody is quickly generated beyond expectation so that an early-stage immune effect is achieved and an immune blanking period is reduced. Meanwhile, the Newcastle disease virus strain rClone30-fliC is free of interference of a maternal antibody and an immune protective effect is increased. The invention has an important value on prevention and treatment of the Newcastle disease.

Description

technical field [0001] The invention relates to the preparation of chicken Newcastle disease virus strain rClone30-fliC and its application in the prevention and treatment of chicken Newcastle disease. Background technique [0002] Newcastle Disease (ND) is an acute, febrile, septic and highly contagious infectious disease caused by Newcastle Disease Virus (NDV), which has high morbidity and mortality , is one of the major severe infectious diseases that seriously endanger the poultry industry in the world. OIE lists it as a class A disease, and my country lists it as a class I animal disease. NDV is a non-segmented single-stranded negative-sense RNA virus, which mainly harms the respiratory system, nervous system and digestive system of chickens and turkeys, and can infect almost all poultry. The disease can occur throughout the year, but more in spring and autumn. Once the disease occurs in the chickens in the chicken farm, it can affect the whole flock within 4 to 5 da...

Claims

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Application Information

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