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Method of purifying active virus

A live virus and virus technology, applied in the field of purified live virus, can solve the problems of large damage to virus particles, high cost, complex cell components, etc., and achieve significant leading and low-cost effects

Inactive Publication Date: 2006-06-21
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 1) It takes a long time for the virus to leave the culture, and centrifugation often takes several hours, or even dozens of hours, so it is not conducive to the maintenance of virus activity
[0009] 2) Incomplete resolution
This is due to the complexity of the virus-infected cells, and there must be substances with similar or even the same density as the virus, which is difficult to separate from the virus.
[0010] 3) Virus particles are more damaged during ultracentrifugation
[0011] 4) Due to the limited volume of centrifuge tubes, the sample volume is only 5%-10% of the gradient solution volume, so high-throughput preparation cannot be achieved, so industrialization cannot be realized
[0012] 5) Ultracentrifugation requires high equipment and consumes a lot, so the cost is high
Undoubtedly, these methods have greater damage to virus particles, are expensive, and most of them cannot enter industrialization
[0031] Traditional virus purification methods, such as differential centrifugation or density gradient centrifugation, on the one hand, cause greater damage to viruses, especially those relatively fragile enveloped viruses, and on the other hand, the harvested viruses always contain more or less impurities ; Another example is that affinity chromatography and some purification methods directly targeting virus particles also have greater damage to the virus

Method used

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  • Method of purifying active virus
  • Method of purifying active virus
  • Method of purifying active virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of various anti-host cell antisera

[0062] 1. Preparation of anti-bluetongue virus host cell Vero antiserum

[0063] The selected country allows the production of Vero cells to propagate bluetongue virus; at the same time, after the cultured Vero cells that have not been inoculated with the virus converge into a monolayer, the cell antigen is prepared according to the same operation steps as for harvesting the virus. The harvested sensitive cells were repeatedly frozen and thawed three times to break them; centrifuged at 5000 rpm at 10°C for 12 minutes, and the supernatant was used as Vero cell antigen to immunize animals to prepare anti-Vero cell antiserum.

[0064] Mix the Vero cell antigen with the same amount of incomplete Freund's adjuvant, and then add 9-11 mg of BCG per ml to make a milky white suspension. After complete emulsification, inject it into the quadruped pads and subcutaneously on the back of rabbits in the experimental group. 0...

Embodiment 2

[0080] Example 2 Preparation of Total Antibodies Containing Anti-Host Cell Antibodies

[0081] 1. Preparation of total antibody containing anti-bluetongue virus host cell Vero antibody

[0082] Extract and purify anti-Vero cell antibody from antiserum by saturated ammonium sulfate precipitation method, and measure its A by ultraviolet spectrophotometer 280 value, so as to obtain the concentration of IgG, and then use 0.1M NaHCO 3 / 0.5M NaCl solution to dilute it to 5mg / ml, and store it at -20°C for later use.

[0083] According to the conditions given in the product manual, incubate the initially purified antibody with the agarose beads with staphylococcal protein A at 37°C for 2 hours or overnight at 4°C, centrifuge at 1000 rpm for 10 minutes the next day, and wash gently After several times, the coupled immunoglobulin IgG was eluted and collected to realize the purification of the total antibody.

[0084] 2. Preparation of total antibody containing anti-herpes simplex vir...

Embodiment 3

[0099] The primary purification of embodiment 3 live virus

[0100] 1. Primary purification of bluetongue virus

[0101] Harvest the virus propagated on Vero cells, break the cells, centrifuge at 11,000 rpm at 4°C for 10 minutes; take the supernatant, mix it with the purified antibody at a volume ratio of 1:2, shake overnight at 4°C at 20 rpm, and make Antigens and antibodies are fully combined to form Vero cell immune complexes; the next day, the mixture is centrifuged at 12,000 rpm at 4°C for 15 minutes, and the immune complex precipitate is discarded. The supernatant is the blue tongue initially purified from Vero cell culture Virus particle suspension.

[0102] 2. Primary purification of herpes simplex virus type I and type II herpes simplex virus

[0103] Harvest the virus propagated on Vero cells, break the cells, centrifuge at 11,000 rpm at 4°C for 10 minutes; take the supernatant, mix it with the purified antibody at a volume ratio of 1:2, shake overnight at 4°C at 2...

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Abstract

The invention discloses a method for purifying live virus. The method includes preparing an antibody against host cells, and purifying it with agarose beads with staphylococcal protein A; then mixing the antibody with a cell disruption system for proliferating virus, and deprecipitating to obtain a preliminary purified live virus; The agarose beads with staphylococcal protein A are mixed with the primary purified live virus suspension, and the highly purified live virus is obtained by removing the precipitate. Compared with the traditional virus purification method, the method has the advantages of high purity of the isolated product, high activity of the isolated virus, simple operation, low cost, effective amplification, etc., and can be used in industrial production and laboratory research.

Description

technical field [0001] The invention relates to a method for purifying a live virus, in particular, the invention relates to a method for purifying a live virus from a host cell system for proliferating the virus. Background technique [0002] At present, the commonly used virus purification methods at home and abroad mainly include: density gradient centrifugation, differential centrifugation, affinity chromatography and direct immunoprecipitation techniques. The characteristics of these technologies will be described below. [0003] 1. Density gradient centrifugation technique [0004] Whether cesium chloride or sucrose is used as the medium, both have the following advantages and disadvantages. [0005] advantage: [0006] All operations involving viruses are carried out under low temperature conditions, which is conducive to the maintenance of virus activity. [0007] shortcoming: [0008] 1) It takes a long time for the virus to leave the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/02
Inventor 董长垣卢莉莉郭淑芳陈冬峨刘文培
Owner WUHAN UNIV
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