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Construction of stable expression cell line of capsid protein of peste des petits ruminant virus

A technology of Peste des petits ruminants and capsid protein, applied in the direction of antisense single-stranded RNA virus, virus, viral peptide, etc., can solve the problems of virus sensitivity, limit the large-scale field use of vaccines, and distinguish naturally infected animals, etc.

Inactive Publication Date: 2019-09-10
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Peste des petits ruminants mainly relies on vaccine immunization prevention. Attenuated vaccines can induce the body to produce strong neutralizing antibodies, but PPR virus is very sensitive to heat, which has become the biggest obstacle to the use of live virus vaccines in tropical epidemic areas
In addition, live attenuated vaccines also have the risk of strong virulence, which limits the large-scale field use of vaccines
At the same time, attenuated vaccines cannot serologically distinguish naturally infected and vaccinated animals, so there is an urgent need to develop a safe and effective vaccine candidate with good thermal stability.

Method used

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  • Construction of stable expression cell line of capsid protein of peste des petits ruminant virus
  • Construction of stable expression cell line of capsid protein of peste des petits ruminant virus
  • Construction of stable expression cell line of capsid protein of peste des petits ruminant virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The construction of embodiment 1 eukaryotic expression vector

[0031] connecting the N protein gene fragment (shown in SEQ ID NO: 1) to the expression vector to construct a recombinant expression vector;

[0032] The capsid protein gene fragment is obtained by optimizing the capsid protein gene, inserting KpnI at the N-terminus, inserting HA at the C-terminus in turn, and obtaining the EcoR I restriction site. Perform PCR to obtain a sufficient amount of the target fragment; extract the plasmid to obtain the vector pcDNA3.1; double enzyme digestion reaction to expose the corresponding cohesive ends of the vector and the target fragment; connect the target fragment to the vector through the ligation reaction of T4 DNA ligase; transform Formation of clones; extraction of recombinant plasmids by plasmid mini-extraction; double-enzyme digestion verification; sequencing. The specific experimental methods and steps are as follows:

[0033] (1) Preparation of Escherichia co...

Embodiment 2

[0054] Example 2 Cell Transfection

[0055] After testing that the liposome method was not feasible, the experiment switched to the electroporation method for cell transfection. The main steps are as follows: plant 2×10 cells in T175 cell culture flasks before electroporation. 6 MDBK cells, add 20mL complete medium (DMEM+10%FBS), place in 5% CO 2 Incubate for 24 h at 37°C in an incubator. Take out the cultured cells and put them on the ultra-clean workbench, discard the old medium, wash 2 times with 5mL PBS, add 4mL 37°C preheated trypsin and let it stand for digestion for 2min, and then beat the adherent cells out of the culture by external force The bottle is in a suspended state, and finally blown and broken up by a pipette gun to make it a single distribution. The cell suspension was transferred to a 50mL centrifuge tube, placed on ice for 1min and then centrifuged at 1200rpm for 1min. Remove the supernatant after taking out the centrifuge tube, add 10 mL of pre-cooled ...

Embodiment 3

[0056] The establishment of embodiment 3 stable cell lines

[0057] Determine the optimal screening concentration of G418: select the concentration of 250 μg / mL, 500 μg / mL, and 800 μg / mL for experiments, and use cells without G418 as a control to screen for the lowest concentration that can kill all MDBK cells within 10 to 14 days , finally choose 600μg / mL as the experimental screening concentration.

[0058] Cells are replaced with screening medium 4 hours after transfection. During the screening process, the cells should be treated separately according to the growth status, density and color of the medium. Generally, the medium is replaced every 3 days, and you can see it in about 5 days. When the cells begin to die, a large number of cells begin to die in about 7 days, but adherent cells in good growth state can still be seen at the bottom of the culture flask.

[0059] After 15 days, the cells at the bottom of the cell culture dish can be seen distributed in clusters, tha...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a method based on application of genetic engineering. The method is used for preparing a stable expression cell lineof capsid protein of peste des petits ruminant virus. A capsid protein gene fragment is obtained in a laboratory by optimizing a capsid protein gene, inserting KpnI into an N end and sequentially inserting HA and EcoRI enzyme cutting sites into a C end. The obtained stable cell line is stable in protein yield and high in efficiency, and an auxiliary research tool is provided for constructing novel live virus vaccines and primary infection type vaccines of peste des petits ruminant virus.

Description

[0001] Technical field: [0002] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a cell line stably expressing capsid protein of Peste des petits ruminants virus by using a genetic engineering method. [0003] Background technique: [0004] Peste des petits ruminants is an acute, virulent and highly contagious viral disease caused by Peste des petits ruminants virus, which mainly infects small ruminants such as sheep and goats. It can lead to 100% morbidity and high mortality in sheep and goats. Serious impact on food security, especially in developing countries dependent on small ruminants. A global plan to eradicate Peste des petits ruminants by 2030. The capsid protein (N protein) is the most expressed among all the proteins of Peste des petits ruminants virus, and plays an important role in diagnosis. At present, Peste des petits ruminants mainly relies on vaccine immunization prevention. Attenuated vaccines can ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N15/85
CPCC07K14/005C12N15/85C12N2760/18422
Inventor 樊振川孟德梅贾雪霞杨可心
Owner TIANJIN UNIV OF SCI & TECH
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