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Purification and concentration method and system for recombinant avian influenza virus

An avian influenza virus, ultrafiltration concentration technology, which is applied to the purification and concentration method of recombinant avian influenza virus and the system field thereof, can solve the problems of difficult removal of impurities, low vaccine purity, large side effects, etc. Simple process and the effect of reducing damage

Active Publication Date: 2018-05-11
吉林冠界生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The first purpose of the present invention is to provide a method for purifying and concentrating recombinant avian influenza virus to solve technical bottlenecks such as difficulty in removing impurities, resulting in low vaccine purity, poor safety, and large side effects

Method used

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  • Purification and concentration method and system for recombinant avian influenza virus

Examples

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Embodiment 1

[0041] A method for purifying and concentrating recombinant avian influenza virus, comprising the following steps:

[0042] 1) The recombinant avian influenza virus (cell source) H7N9 H7-Re1 strain to be treated was left to settle at low temperature, and then the supernatant was extracted to obtain the pretreated recombinant avian influenza virus. It was determined that the HA titer was 2 9 ;EID 50 for 10 8.0 / 0.1ml; TCID 50 for 10 8.1 / 1ml.

[0043] 2) Use a continuous flow centrifuge 1 to process the virus to remove large particles in the virus

[0044] ①Use continuous flow centrifuge 1, set the rotating speed as 15000 rev / min.

[0045] ②Collect the supernatant after centrifugation, discard the heavy liquid, and put the supernatant into figure 1 Medium tank A.

[0046] ③Send samples to test HA and EID 50 、TCID 50 and endotoxins.

[0047] HA, EID after centrifugation in continuous flow centrifuge 1 50 、TCID 50 and endotoxin indicators have not changed, no loss.

...

Embodiment 2

[0061] A method for purifying and concentrating recombinant avian influenza virus, comprising the following steps:

[0062] 1) The recombinant avian influenza virus (cell source) H7N9H7-Re1 strain to be treated was left to settle at low temperature, and then the supernatant was extracted to obtain the pretreated recombinant avian influenza virus. It was determined that the HA titer was 2 9.1 ;EID 50 for 10 8.1 / 0.1ml; TCID 50 for 10 8.1 / 1ml.

[0063] 2) Use a continuous flow centrifuge 1 to process the virus to remove large particles in the virus

[0064] ①Use continuous flow centrifuge 1, set the rotating speed at 10000 rpm.

[0065] ②Collect the supernatant after centrifugation, discard the heavy liquid, and put the supernatant into figure 1 Medium tank A.

[0066] ③Send samples to test HA and EID 50 、TCID 50 and endotoxins.

[0067] HA, EID after centrifugation in continuous flow centrifuge 1 50 、TCID 50 and endotoxin indicators have not changed, no loss.

[0...

Embodiment 3

[0081] A method for purifying and concentrating recombinant avian influenza virus, comprising the following steps:

[0082] 1) The recombinant avian influenza virus (cell source) H7N9H7-Re1 strain to be treated was left to settle at low temperature, and then the supernatant was extracted to obtain the pretreated recombinant avian influenza virus. It was determined that the HA titer was 2 9 ;EID 50 for 10 8.0 / 0.1ml; TCID 50 for 10 8.1 / 1ml.

[0083] 2) Use a continuous flow centrifuge 1 to process the virus to remove large particles in the virus

[0084] ①Use a continuous flow centrifuge 1, set the speed at 12000 rpm.

[0085] ②Collect the supernatant after centrifugation, discard the heavy liquid, and put the supernatant into figure 1 Medium tank A.

[0086] ③Send samples to test HA and EID 50 、TCID 50 and endotoxins.

[0087] HA, EID after centrifugation in continuous flow centrifuge 1 50 、TCID 50 and endotoxin indicators have not changed, no loss.

[0088] 3) T...

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Abstract

The invention relates to the field of virus purification and concentration, in particular to a purification and concentration method and system for recombinant avian influenza virus. The purificationand concentration method for the recombinant avian influenza virus comprises the steps as follows: pretreated recombinant avian influenza virus is centrifuged, large-particle substances and cell debris are removed, and a clear liquid is obtained; the clear liquid is treated with a hollow fiber microfiltration system, and a permeate liquid is obtained; the permeate liquid is treated with a membraneultrafiltration concentration system, a circulation liquid is collected, concentration is performed, and the recombinant avian influenza virus is obtained. According to the purification and concentration method, cell debris and large-particle substances are removed through high-speed centrifugation, small-particle protein is removed through purification and clarification by the hollow fiber microfiltration system, then, concentration is performed with the membrane ultrafiltration concentration system, and the purified and concentrated recombinant avian influenza virus is obtained. The wholeprocess is simple and easy to implement, the content of impurities and impure protein in the virus can be effectively reduced, and the removal rate of impure protein in the virus is 90% or above; thetiter of the concentrated virus is remarkably improved.

Description

technical field [0001] The invention relates to the field of virus purification and concentration, in particular to a purification and concentration method and system for recombinant avian influenza virus. Background technique [0002] Existing recombinant avian influenza vaccines mainly use chicken embryo technology for antigen production. In the production process of avian influenza embryotoxin vaccine, due to the large amount of miscellaneous protein, endotoxin, urate, etc. in the allantoic fluid, the quality of the antigen is unstable. During the use of the vaccine, the chicken body has stress response, local ulceration, death and other immune side effects. . In particular, miscellaneous proteins and endotoxins not only interfere with the production of vaccine immune antibodies, but also greatly reduce the immune effect of the unit dose of antigen. At the same time, clinically, immune failure and animal death caused by vaccine quality are not uncommon. In addition, too...

Claims

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Application Information

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IPC IPC(8): C12N7/02C12M1/12
CPCC12M47/12C12N7/00C12N2760/16151
Inventor 陈宏赵海源曾显营陈化兰李金祥田国彬冯玉强李莉朱长动王玉红蒋晓梅张天舒王博孙佰强崔凯赵博刘金伟曾晓敏王仁君
Owner 吉林冠界生物技术有限公司