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Genetic modification method for changing thermal stability of envelope virus, recombinant virus and application of recombinant virus

A technology of genetic modification and thermal stability, applied in the field of molecular biology technology and microbiology, can solve the problems such as the thermal stability relationship of unknown viruses, and achieve the effects of weakening viability, reducing cost and reducing virulence

Active Publication Date: 2021-06-22
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research report on the relationship between protein charge properties and virus thermal stability

Method used

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  • Genetic modification method for changing thermal stability of envelope virus, recombinant virus and application of recombinant virus
  • Genetic modification method for changing thermal stability of envelope virus, recombinant virus and application of recombinant virus
  • Genetic modification method for changing thermal stability of envelope virus, recombinant virus and application of recombinant virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This embodiment provides a genetic modification method for improving the thermal stability of Newcastle disease virus, comprising the following steps:

[0039] S101:

[0040] Compare the HN protein amino acid sequences of 3 heat-resistant strains of Newcastle disease virus, 4 non-heat-resistant strains and the virus strain TS09-C to be transformed; the 3 heat-resistant strains are V4, I-2, Ulster strain, and the The 4 non-heat resistant strains are LaSota, Mukteswwar, HB1103, HN1107 strains;

[0041] Such as figure 1 As shown, the 3 heat-resistant strains and the 4 non-heat-resistant strains have a total of 85 sites with amino acid mutations compared with the same sites of the TS09-C strain. However, because the mutations at 42 of the 85 sites in the heat-resistant strain and the non-heat-resistant strain will not change the charge value of the adsorbed protein, they are excluded, and only the remaining N is 43 mutations that will affect the charge of the HN protein. ...

Embodiment 2

[0067] Such as Figure 4 As shown, this example is roughly the same as Example 1, except that the other four sites of the 10 amino acid mutation sites in step S103 are selected to obtain two different mutation schemes, and the mutations constructed and rescued using the same method Viruses rTS-HN-UN4 and rTS-HN-PU4. At the same time, the other four sites of the 19 amino acid mutation sites in step S103 were selected to obtain two different mutation schemes, using the same method to construct and rescue mutant viruses rTS-HN-UP4 and rTS-HN-NU4. The mutation method of rTS-HN-UN4 is that all four sites are mutated from uncharged amino acids to negatively charged amino acids, and the mutation method of rTS-HN-PU4 is that all four sites are mutated from positively charged amino acids to uncharged amino acids. In addition, the mutation method of rTS-HN-UP4 is that all four sites are mutated from uncharged amino acids to positively charged amino acids, while the mutation method of r...

Embodiment 3

[0069] Heat-resistant Transformation of Newcastle Disease Virus LaSota Strain

[0070] Newcastle disease virus LaSota strain, as a classic strain for the preparation of low-virulence live vaccines, has been widely promoted and applied worldwide, making important contributions to the prevention and control of Newcastle disease. However, due to the poor thermal stability of this strain, it is a non-heat-resistant strain. Therefore, live vaccines prepared from this strain need to be stored and transported in a cold chain system, which increases the cost of immunization and the risk of vaccine failure. Apply the method of the present invention to carry out heat resistance transformation and effect test on LaSota strain.

[0071] According to the same method of embodiment 1, compare the HN protein amino acids of 4 strains of Newcastle disease virus heat-resistant strains, 3 strains of non-heat-resistant strains and the virus LaSota strain to be transformed; the 4 strains of heat-re...

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Abstract

The invention provides a genetic modification method for changing thermal stability of an envelope virus, a recombinant virus and an application of the recombinant virus. The method comprises the following steps: comparing multiple sequences of enveloped viruses to obtain amino acid mutation which obviously influences an adsorptive protein charge value of a virus strain to be modified; analyzing the change value of the amino acid mutation to the adsorbed protein charge, and screening an amino acid mutation combination capable of remarkably increasing or reducing the adsorbed protein charge; applying the genetic manipulation technology of the virus, performing mutation modification on the envelope virus at the transcription plasmid level, and saving and obtaining the recombinant envelope virus subjected to heat-resistant modification. According to the invention, the heat-resistant characteristic of the envelope virus is obviously changed in an artificial mutation mode for the first time. The method can be widely applied to heat-resistant transformation of envelope virus vaccine strains, and has a wide application prospect in the aspect of research and development of heat-resistant, safe and efficient envelope virus vaccines.

Description

technical field [0001] The invention relates to the fields of molecular biology technology and microbiology, in particular to a genetic modification method for changing the thermal stability of enveloped viruses and its application. More specifically, the present invention relates to a genetic modification method for changing the thermostability of the virus through the mutation of the charge-related amino acid combination of the adsorption protein gene, the recombinant virus modified for heat resistance, and the application of the modified recombinant virus in the preparation of heat-resistant vaccines . Background technique [0002] Envelope refers to a lipid bilayer membrane composed of proteins, polysaccharides and lipids coated in the virus shell, also known as envelope. Enveloped viruses are collectively referred to as enveloped viruses. The envelope is mainly derived from the host cell membrane (phospholipid layer and membrane protein), and also contains some glycop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C07K14/125C07K14/11
CPCC12N7/00C07K14/005C12N2760/18122C12N2760/16122C12N2760/16222C12N2760/16311C12N2760/18134C12N2760/16134C12N2760/16234C12N2760/16334
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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