Purification method of recombinant herpes simplex virus

A technology of herpes simplex virus and purification method, which is applied in the field of purification of recombinant herpes simplex virus, can solve the problems of high price, high cost, and difficult promotion, and achieve the effect of no virus damage, low cost, and good purification effect

Inactive Publication Date: 2015-08-19
HUBEI CHUANGRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purification effect of precipitation and dialysis is not good, and the purification effect of affinity chromatography and cesiu

Method used

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  • Purification method of recombinant herpes simplex virus
  • Purification method of recombinant herpes simplex virus
  • Purification method of recombinant herpes simplex virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] I. Virus Proliferation

[0063] Use a 10 mL sterile pipette to draw 6 mL of PBS from Vero cells with a cell density of 80%-90% to wash the cells three times; o C adsorption for 1 hour; add virus growth solution and maintenance solution to the cell bottle, and culture it in a 37 ℃ constant temperature incubator until 75-100% of the cells have CPE lesions. Harvest. Before harvesting, the cells can be placed in a -70 ℃ refrigerator and frozen Melt 2 to 3 times, 4 o Centrifuge at 6000g for 20 minutes, remove the precipitate, and collect 50 mL of virus supernatant.

[0064] II. Virus Concentration

[0065] Add 5% PEG8000 to the supernatant, 4 o C stirring for 6 hours or overnight, 4 o Centrifuge at 13500g for 2 hours; dissolve the precipitate with TNMC buffer, and add n-dodecyl-β-D-maltoside to the solution to a concentration of 20mM to promote the dissolution of the precipitate, and finally make it centrifuged to purify the virus sample The final volume is 1% of that b...

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Abstract

The invention relates to a purification method of recombinant herpes simplex virus. The method comprises: adding 5% of PEG 8000 to the recombinant herpes simplex virus supernatant obtained through amplification in Vero cells, and carrying out centrifugation; dissolving the precipitate with a TNMC buffer solution, and adding n-dodecyl-beta-D-maltoside to the solution to promote the precipitate dissolving; and adding the concentrated mtHSV solution to the sucrose density gradient top layer, and carrying out centrifugation identification analysis. The method of the present invention combines the precipitation method and the sucrose density gradient centrifugation method, has characteristics of low cost, no damage on virus, good purification effect, and the like, and is the effective virus purification method capable of be promoted.

Description

technical field [0001] The present invention relates to the technical field of virus purification, more specifically to a purification method of recombinant herpes simplex virus, Background technique [0002] Recombinant herpes simplex virus (mtHSV) transformed from herpes simplex virus type 1 (HSV-1) is an oncolytic virus, which can specifically kill tumor cells. Animal experiments with intratumoral injection of mtHSV It has shown good safety and anti-tumor effect on glioma, cervical cancer, melanoma and rhabdomyosarcoma in clinical phase I trials. At present, the HSV-1 mutant deletion virus G207 has passed the phase I clinical trial. After being used in patients with malignant glioma, it was found that the survival time of the patients was prolonged, which initially proved its safety and effectiveness. However, for a long time, there is still a lack of efficient, simple and low-cost purification methods. At present, the conventional methods for purifying viruses mainly i...

Claims

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Application Information

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IPC IPC(8): C12N7/02C12R1/93
Inventor 刘为勇王涛
Owner HUBEI CHUANGRUI BIOTECH CO LTD
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