Newcastle disease virus heat-resistant transformation method and application

A Newcastle disease virus and heat-resistant technology, which is applied in the field of virus reverse genetic manipulation, can solve the problems of a method that does not involve heat-resistant transformation of Newcastle disease virus, a method that does not involve heat-resistant transformation of non-heat-resistant Newcastle disease virus, and the like. Convenience, reduce transportation, ensure the effect of use

Active Publication Date: 2016-03-23
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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Problems solved by technology

For example: the document "Newcastle Disease LaSota Vaccine Strain Reverse Genetic Operating System and Its Application" with patent number 200510097997.8 discloses a non-heat-resistant live vaccine carrier based on Newcastle disease virus LaSota vaccine strain, but does not involve resistance to Newcastle disease virus The method of thermal transformation; the document "Recombinant Newcastle Disease LaSota Attenuated Vaccine Strain Expressing Avian Influenza Virus H5 Subtype HA Protein" with the patent number of 200610075781.6 discloses the dual genetic eng

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  • Newcastle disease virus heat-resistant transformation method and application
  • Newcastle disease virus heat-resistant transformation method and application
  • Newcastle disease virus heat-resistant transformation method and application

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[0028] Example 1

[0029] Construction of Transcription Plasmid of Non-heat-resistant LaSota Strain of Newcastle Disease Virus

[0030] The transcription plasmid of the non-heat-resistant LaSota strain of Newcastle disease virus contains the complete genome sequence of the LaSota strain, the T7RNA polymerase promoter sequence, the T7RNA polymerase terminator sequence, the hepatitis D virus ribozyme sequence and the low copy plasmid sequence, etc. The function is to transcribe, express and accurately cut out the whole genome RNA sequence of LaSota strain. The construction strategy is as follows: firstly, the whole genome sequence is amplified by fragments, and divided into 4 fragments A-D. Among them, the T7 promoter was added to the upstream of the A segment, and the hepatitis D virus ribozyme sequence and the T7 terminator were added to the downstream of the D segment. After 4 fragments were amplified by ordinary PCR, fusion PCR and primer self-extension methods, the 4 fragments...

Example Embodiment

[0039] Example 2

[0040] Construction of HN genetically modified LaSota strain transcription plasmid and virus rescue

[0041] Replace the corresponding HN gene in the transcription plasmid of the LaSota strain with the HN gene of the heat-resistant TS09-C strain of Newcastle disease virus to obtain the HN genetically modified LaSota transcription plasmid, such as figure 1 Shown. The modified transcription plasmid and the helper plasmid are co-transfected into host cells to obtain a heat-resistant modified recombinant Newcastle disease virus.

[0042] 1. PCR amplification of HN gene of Newcastle disease virus TS09-C strain

[0043] The chicken embryo allantoic fluid of Newcastle disease virus TS09-C strain was used as the object, and the viral genomic RNA was extracted according to the kit instructions. The total RNA was dissolved in 17μl DEPC water, and the HN gene was amplified by RT-PCR. Agarose gel electrophoresis detection, the specific positive bands are purified and recovere...

Example Embodiment

[0049] Example 3

[0050] Biological characteristics test of the heat-resistant modified Newcastle disease virus rT-HN strain

[0051] After the Newcastle disease virus LaSota strain was heat-resistant, a new strain rT-HN was obtained. In order to verify whether it has similar biological characteristics to the LaSota parent strain, a biological characteristic test of the recombinant virus rT-HN was carried out.

[0052] 1. Cell growth characteristics test of Newcastle disease virus rT-HN strain

[0053] The BHK-21 cells were transferred to a 6-well plate, and the cells grew into a dense monolayer within 24 hours. The cells were inoculated with the diluted rT-HN allantoic fluid, and a LaSota strain virus control was set. The cell supernatant was aspirated at 0, 24, 48, 72, and 96 hours after inoculation, and the content of Newcastle disease virus in the supernatant was determined. Specific determination method: The supernatant was diluted by 10-fold gradient and inoculated into BHK-2...

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Abstract

The invention discloses a Newcastle disease virus heat-resistant transformation method and application of the Newcastle disease virus heat-resistant transformation method. The heat-resistant characteristic of the Newcastle disease virus is specifically and remarkably improved through an HN gene replacement method. The non-heat-resistant Newcastle disease virus is transformed at the transcription plasmid level through the RNA virus reverse genetic technology, the HN gene of the non-heat-resistant Newcastle disease virus is replaced with the HN gene of the heat-resistant Newcastle disease virus, host cells are transfected by the transformed transcription plasmid, rescue is conducted to obtain the heat-resistant transformed Newcastle disease virus, and therefore the heat-resistant characteristic of the Newcastle disease virus is greatly improved. The heat-resistant characteristic of the Newcastle disease virus is remarkably improved through the artificial recombination mode for the first time, and a non-heat-resistant Newcastle disease vaccine strain LaSota is successfully transformed into a heat-resistant Newcastle disease vaccine strain rT-HN. The method can be widely applied to heat-resistant transformation of Newcastle disease vaccine strains, and has great application prospects on the aspects of heat-resistant, safe and efficient Newcastle disease vaccine research and the like.

Description

technical field [0001] The invention belongs to the field of reverse genetic manipulation of viruses, and in particular relates to a heat-resistant transformation method and application of Newcastle disease virus. More specifically, the present invention relates to a method and heat-resistant transformation for non-heat-resistant Newcastle disease virus. The application of the recombinant virus in vaccine preparation. Background technique [0002] Newcastle Disease (ND) is a highly contagious and devastating disease caused by Newcastle Disease Virus (NDV). One, the other for bird flu. Newcastle disease virus belongs to paramyxoviridae (Paramyxoviridae) and mumps virus (Avulavirus). The genome of Newcastle disease virus is a single-stranded, negative-strand, non-staged RNA with a total length of 15,186 bases, following the six-base principle, including a 55-base 3′ leader sequence and a 5′ trailing sequence, both There are 6 structural genes between them, which are arrange...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N7/01A61K39/17A61P31/14C12R1/93
CPCA61K35/76A61K39/17A61P31/14C12N7/00C12N2760/18121C12N2760/18134C12N2760/18162
Inventor 温国元于庆忠邵华斌罗青平王红琳张蓉蓉艾地云张腾飞罗玲汪宏才
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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