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A mutant strain of heat-resistant Newcastle disease virus and its preparation method and application

A technology for Newcastle disease virus and mutant strains, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve the problem of not being able to further improve the thermal stability of heat-resistant strains, and achieve improved thermal stability and thermal stability Good, cost reduction effect

Active Publication Date: 2021-04-20
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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Problems solved by technology

The thermal stability of non-heat-resistant viruses can be improved to a certain extent by HN gene exchange, but how to improve the thermal stability of existing heat-resistant strains? Since there is no more heat-resistant virus strain HN gene can be exchanged, therefore, the HN gene exchange method cannot further improve the heat stability of the existing heat-resistant strains

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  • A mutant strain of heat-resistant Newcastle disease virus and its preparation method and application
  • A mutant strain of heat-resistant Newcastle disease virus and its preparation method and application
  • A mutant strain of heat-resistant Newcastle disease virus and its preparation method and application

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preparation example Construction

[0019] The present invention also provides a method for preparing the heat-resistant Newcastle disease virus mutant strain rTS-HN-PU4, comprising the following steps: a. constructing a transcription plasmid of the Newcastle disease virus TS09-C strain; b. converting the TS09-C strain Amino acids 3, 197, 203, and 495 of the HN gene in the transcription plasmid were mutated to serine, isoleucine, aspartic acid, and valine, respectively; c. Co-transfected the mutated transcription plasmid with three helper plasmids Host cells to obtain mutant strains of Newcastle disease virus.

[0020] The invention also provides the application of the heat-resistant Newcastle disease virus mutant strain rTS-HN-PU4 in preparing a heat-resistant live vaccine for Newcastle disease.

Embodiment 1

[0023] Construction of Transcription Plasmid and Virus Rescue of Mutant Newcastle Disease Virus

[0024] According to the mutation protocol, four mutated HN gene sequences (HN-P4, HN-PU4, HN-NU4 and HN-UP4) were obtained by gene synthesis. The mutation scheme of HN-P4 is A at position 62 to R, N at position 98 to K, E at position 293 to K, and D at position 494 to G. The mutation scheme of HN-PU4 was R at position 3 to S, R at position 197 to I, H at position 203 to N, and K at position 495 to V. The mutation scheme of HN-UP4 is A at position 62 to R, Q at position 280 to R, Q at position 353 to R, and G at position 570 to R. The mutation scheme of HN-NU4 is D at position 147 to G, E at position 293 to G, D at position 309 to N, and D at position 494 to G. It replaced the HN gene in the TS09-C strain transcription plasmid respectively to obtain four transformed transcription plasmids (pTS-HN-P4, pTS-HN-PU4, pTS-HN-NU4 and pTS-HN-UP4), as figure 1 shown. The transformed tra...

Embodiment 2

[0036] Thermal Stability Test of Newcastle Disease Virus Mutants

[0037] Allantoic fluid infected with 4 mutant strains of Newcastle disease virus, 100 μL / tube, was heat-treated in a 56°C water bath, and three replicates were set up at 0, 2, 5, 10, 15, 30, 60, 120, and 180 min respectively. Take out the virus allantoic fluid at the time, put it on ice quickly, detect the hemagglutination (HA) titer of the virus, and count the change of the titer, and obtain Table 1. As shown in Table 1, compared with the parental TS09-C strain, the HA thermostability of rTS-HN-P4, rTS-HN-NU4 and rTS-HN-UP4 strains all decreased significantly, and only rTS-HN-PU4 The thermal stability of HA is significantly improved. After the strain was heat-treated at 56 degrees for 180 minutes, the HA titer only decreased by 3log 2 , while the female parent TS09-C strain has dropped to 0. The control LaSota strain has dropped to 1 log after heat treatment for 5 minutes 2 . Therefore, among the four NDV...

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Abstract

The invention discloses a heat-resistant Newcastle disease virus mutant strain and a preparation method and application thereof. The mutant strain is deposited in the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: V202041. The preparation method uses the Newcastle disease virus TS09-C strain as the parent strain, and introduces 4 amino acid mutations into its HN protein: the 3rd arginine is mutated to serine, and the 197th arginine is mutated to isoleucine. acid, histidine at position 203 was mutated to asparagine and lysine at position 495 was mutated to valine. The heat resistance test results show that the heat resistance of the virus mutant strain is significantly higher than that of the parent strain, and is much higher than that of the commonly used vaccine strains, which proves that the present invention further improves the thermal stability of the existing heat-resistant strains.

Description

technical field [0001] The invention belongs to the field of vaccine genetic engineering, in particular to a heat-resistant Newcastle disease virus mutant strain rTS-HN-NU4. More specifically, the present invention relates to the use of reverse genetic manipulation technology to carry out heat-resistant transformation on Newcastle disease virus in the form of point mutation, to obtain a new mutant strain of heat-resistant transformation, and the application of the strain in vaccine preparation . Background technique [0002] Newcastle disease is a highly contagious disease caused by Newcastle disease virus (NDV), which poses a huge threat to the poultry industry all over the world. NDV belongs to the Paramyxoviridae family and a member of the genus Avian Mumps virus. It is a single-stranded, negative-strand, non-segmented RNA virus containing an envelope. The NDV genome encodes six major structural proteins, namely nucleocapsid protein (NP), phosphorylated protein (P), mat...

Claims

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Application Information

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IPC IPC(8): C12N7/00C07K14/125A61K39/17A61P31/14
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2760/18121C12N2760/18122A61K2039/5254A61K2039/552
Inventor 温国元商雨李丽邵华斌罗青平王红琳罗玲张蓉蓉汪宏才张腾飞张文婷卢琴
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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