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Cell strain for culture of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof

A respiratory system and virus culture technology, applied in animal cells, viruses/phages, vertebrate cells, etc., can solve the problems that the virus content cannot meet the requirements of vaccines and diagnostic antigens, and the virus titer is low, and achieve stable cell morphology and growth performance. Good and safe effect

Active Publication Date: 2014-08-06
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that although the existing Marc145 cells are suitable for the proliferation of PRRSV, the virus titer is relatively low, and the virus content cannot meet the preparation of vaccines and diagnostic antigens

Method used

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  • Cell strain for culture of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof
  • Cell strain for culture of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof
  • Cell strain for culture of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning and screening of Marc145 cell line

[0040] 1. Cloning culture of Marc145 cells by limiting dilution method

[0041] Marc145 cells (Harbin Veterinary Research Institute) preserved in the laboratory without contamination by mycoplasma, bacteria, fungi, and exogenous viruses were digested with trypsin to make a single-cell suspension. After counting cells, they were serially diluted, and then inoculated in 96 wells Cell culture plate, 37°C 5% CO 2 Cultivate in an incubator for 10-14 days, select the cells in the cell wells with single cell growth and transfer them to 24-well plates for culture, and expand the cells to 12-well plates, 6-well plates, and T25 cell culture flasks in turn to obtain subclonal cell lines ;

[0042] 2. Screening of Marc145 cell line sensitive to porcine reproductive and respiratory syndrome virus

[0043] Select several clonal cells with better cell status and inoculate them in 96-well cell culture plates, inoculate NVDC-JXA...

Embodiment 2

[0044] Example 2: Research on the biological properties of the monkey kidney cell line Marc145-1 strain

[0045] 1. Cell Morphology

[0046] The Marc145 mother cells and the monkey kidney cell line Marc145-1 strain grown into a single layer were respectively prepared into a cell suspension. 4 piece / cm 2 Inoculate in T25 cell culture flasks, add DMEM growth solution containing 8%~10% fetal bovine serum, and store at 37°C in 5% CO 2 Under the condition of culture for 48h, observe the cell morphology of the two. The cells are well adherent, plump in shape, clear in boundary and smooth in surface. The monkey kidney cell line Marc145-1 was smaller than the Marc145 mother cell, and the density was increased. The boundary between cells was more obvious and neat.

[0047] 2. Cell Kinetic Detection

[0048] The monolayer of Marc145 mother cells and the monkey kidney cell line Marc145-1 were prepared into a cell suspension. After counting the cells, the 4 piece / cm 2 Inoculate in ...

Embodiment 3

[0092] Example 3: Detection of Proliferation Characteristics of Porcine Reproductive and Respiratory Syndrome Virus NVDC-JXA1 Strain

[0093] Take Marc145 mother cells and monkey kidney cell line Marc145-1 each about 1×10 6 Cells were inoculated in T25 cell culture flasks, and the same amount of NVDC-JXA1 virus (0.001MOI) was inoculated into each flask after the cells had grown to a monolayer, and an equal amount of maintenance solution was added to 5ml, and the cells were incubated at 37°C in 5% CO 2 Cultivate in an incubator, take 3 bottles of cells at 24h, 48h, 72h, and 96h after inoculation, and obtain virus liquid after repeated freezing and thawing, and use TCID 50 Virus content was determined at four time points.

[0094] Make NVDC-JXA1 virus solution 10 -1 to 10 -8 After doubling dilution, the Marc145 mother cells and the monkey kidney cell line Marc145-1 were respectively inoculated in a 96-well plate to form a monolayer. Each dilution had 6 wells, and a negative ...

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Abstract

The invention relates to a cell strain for culture of PRRSV and application thereof. The cell strain is highly sensitive to PRRSV, is stable and can continuously and stably proliferate high-titer PRRSV. The cell strain is free of pollution by mycoplasma, bacteria, fungi and exogenous viruses, does not has mutagenicity, teratogenicity and carcinogenicity and has high security. When used in enrichment culture of PRRSV, the PRRSV is more beneficial for reproduction and proliferation of PRRSV compared with Marc145 blast cells. Thus, application of the cell strain for enrichment culture of high-titer PRRSV is favorable for obtainment of more comprehensive information about research and epidemic prevention of PRRSV and for more direct and effective research. The monkey kidney cell line Marc145-1 strain highly sensitive to PRRSV and a high-titer PRRSV proliferation method have wide development and application prospects in research and epidemic prevention of PRRSV.

Description

technical field [0001] The invention belongs to the technical field of animal husbandry biopharmaceuticals, and relates to a cell strain used for cultivating porcine reproductive and respiratory syndrome virus and the application of the cell strain in cultivating porcine reproductive and respiratory syndrome virus. Background technique [0002] Porcine reproductive and respiratory syndrome is a highly contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV), also known as porcine blue ear disease. The disease is characterized by reproductive impairment (abortion, stillbirth, mummification) in pregnant sows and respiratory symptoms in pigs of all ages, especially in piglets. Since the disease was discovered in the United States in 1987, it has rapidly spread to various pig-raising countries in the world, and it is more likely to be popular in areas with dense pig herds and frequent movements, causing serious economic losses. In recent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/91C12R1/93
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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