Method for screening virus strain

A virus and seed technology, applied in the directions of microorganism-based methods, viruses/phages, biochemical equipment and methods, etc., can solve the problem of insufficient research on rabies virus DI particles, decreased virulence and efficacy of vaccine strains, and viral strains. Toxic and potency decline and other problems, to achieve the effect of high success rate, low difficulty and simple operation

Inactive Publication Date: 2014-10-22
LANZHOU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When a virus strain contains a certain amount of DI particles, DI particles will interfere with the proliferation of normal virus particles (also known as standard virus particles, parental virus particles) in the host, resulting in a decrease in the virulence and efficacy of the virus strain
Rabies virus DI particles are still understudied, although DI particles of certain virus species, such as polio, vesicular stomatitis virus, and influenza virus, are well-studied
Moreover, the reduced virulence and potency of vaccine strains may also include other unknown causes

Method used

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  • Method for screening virus strain
  • Method for screening virus strain
  • Method for screening virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1. Obtain the virus to be screened that is beneficial to tissue culture

[0051] Take the frozen rabies virus CTN-1V strain and inoculate it in a T25 cell bottle at 0.1 MOI, discard the virus after adsorption at 37°C for 1 hour, add maintenance solution (DMEM+1% bovine serum), and culture in an incubator at 34.5°C and 5% CO2 , 8-10 days after the lesion (++-+++), it was harvested and recorded as CTN-1V-F1. CTN-1V-F1 was continuously passed on for three generations on vero cells to obtain a virus to be screened that was beneficial to tissue culture, which was denoted as CTN-1V-F4 (F4), and the virulence efficacy was detected by FAT method and M-ABT method (virulence Force 7.0LgFFU / ml, potency 1.8IU / ml), aliquot into 1.5ml centrifuge tube for later use.

Embodiment 2

[0052] Example 2. Screening the virus rabies virus CTN-1V strain to be screened by the plaque cloning method

[0053] 2.1 The first round of cloning:

[0054] The rabies virus F4 harvest liquid of harvest in embodiment 1 is diluted to 10 -4 -10 -6 , with 4ml vero (2-3X10 5 ) cells and 0.4ml diluted virus were mixed in a 6-well plate, each dilution was inoculated into 2 wells, and at the same time, 4ml vero (2-3X10 5 ) cells and 0.4ml diluent were mixed in a 6-well plate as a cell control, and cultured at 37°C for 24 hours in 2 wells.

[0055] Aspirate the supernatant and discard, add 2.5ml of 1% nutrient agarose to each well, and culture at 34.5°C for 3 days.

[0056] Add 1ml of 1% nutrient agarose containing 0.1M Hepes to each well, incubate at 34.5°C for 3 days, observe the plaques under a microscope, pick out the plaques and inoculate them into 24-well plates according to the size of the plaques, and add 1ml of cell mixture at the same time Incubate at 34.5°C.

[0057...

Embodiment 3

[0069] Example 3: Screening of viruses to be screened by limiting dilution

[0070] According to the invention of the present invention, after the ideal virus strain is screened out by the plaque cloning method described in Example 2, the purity and singleness of the virus clone can be guaranteed by further screening by the limiting dilution method. The limiting dilution method is commonly used to identify the highest dilution that produces a viral effect on cells. The viral effect may be the cytopathic effect (CPE) referred to in Example 2. In general, the wells with the highest dilution were harvested. The harvested virus is then diluted to virus-free and the process repeated. Serial 10-fold dilutions are usually prepared in appropriate medium (with or without trypsin) and 0.2 mL of each dilution is added to the wells of a plate or microtiter plate containing tissue culture cells and incubated for a period sufficient to identify The time of the CPE of the cells. Because ...

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Abstract

The invention provides a method for screening a virus strain for preparing vaccines, which comprises the following steps: (a) providing a culture cell; (b) contacting the virus strain to be screened with the culture cell for some time; (c) separating out different virus clones from the culture cell, which has contacted the virus strain to be screened in the step (b), by a plaque cloning process, and screening out a first round virus strain for preparing vaccines; (d) repeating the steps (a)-(c) by using the first round virus strain collected in the step (c), screening out a second round virus strain for preparing vaccines; (e) carrying out serial dilution on the second round virus strain collected in the step (d) to obtain a series of diluted substances with degressive virus concentrations; (f) contacting every diluted substance in the step (e) with the culture cell for some time; and (g) cloning the pathological change virus with the maximum times of diluted substance in the step (f), continuing culture, and screening out a third round virus strain for preparing vaccines.

Description

technical field [0001] The field of the present invention relates to a method for screening virus species, in particular to a method for screening dominant virus species and improving virus titer and immunogenicity in cell culture. Background technique [0002] The virus titer of the virus harvest fluid is a key quality indicator for viral vaccine products. Taking the Vero rabies vaccine for human use as an example, the "Chinese Pharmacopoeia III (2010 Edition)" stipulates that the virus titer of the rabies virus harvest liquid is ≥ 6.0LgLD50 / ml before it can be used for vaccine production, and usually it still needs to be concentrated by ultrafiltration for 10~ More than 20 times can be used to prepare finished vaccines. The ultrafiltration concentration step not only needs to invest a lot of manpower and material resources, which increases the cost, but also may cause the content of host protein residues, DNA residues and bovine serum residues in the concentrated finished...

Claims

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Application Information

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IPC IPC(8): C12N7/00G01N33/571C12R1/93
CPCY02A50/30
Inventor 李薇李建强陈军
Owner LANZHOU INST OF BIOLOGICAL PROD
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