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A method and application of cultivating hek-293t cell line to efficiently secrete and express classical swine fever e2 protein

A HEK-293T, secretory expression technology, applied in the field of high-efficiency secretory expression of swine fever E2 protein, can solve the problems of uncontrollable cell culture conditions, unstable culture dish production conditions, and inability to carry out process optimization, achieving high cost performance and repeatability. High, low-cost effects

Active Publication Date: 2020-08-14
GUANGZHOU BONIZZI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing bioreactor expression method has the following disadvantages for cell expression of virus E2 protein: the expression level is low, and the vaccine cost is high; Carry out process optimization; there is contact inhibition of cell growth in the culture dish in cells, the cell density is low, and the growth cycle is short; the production conditions of the culture dish are unstable, and the difference between batches is large

Method used

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  • A method and application of cultivating hek-293t cell line to efficiently secrete and express classical swine fever e2 protein
  • A method and application of cultivating hek-293t cell line to efficiently secrete and express classical swine fever e2 protein
  • A method and application of cultivating hek-293t cell line to efficiently secrete and express classical swine fever e2 protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 The cultivation and production method of the HEK-293T cell line that efficiently secretes and expresses the classical swine fever E2 protein

[0033] 1. Expansion of seed cells

[0034] Take out the preserved HEK-293T cell strain expressing classical swine fever E2 protein from the liquid nitrogen storage tank, place it in a constant temperature water bath at 37°C to thaw rapidly, and centrifuge at 800rpm for 5min to remove the cell cryopreservation solution. The DMEM complete medium was resuspended and placed in a 150mM cell culture dish and cultured in a carbon dioxide incubator. Every 2-3 days, when the cell density reaches more than 90%, expand it into a 150mM cell culture dish at a ratio of 1:5 until the number of cells reaches 10 9 Transfer to the bioreactor when above.

[0035] 2. The cultivation process of cell bioreactor

[0036] 1) Check whether the consumables are damaged, and record the batch. Inflate the torrent bag and the perfusion bag fo...

Embodiment 2

[0047] Embodiment 2 Bioreactor expression and petri dish expression comparative experiment

[0048] Petri dish expression method:

[0049] 1) According to the culture conditions of the bioreactor in Example 1, different cell numbers were inserted in proportion in the cell culture dish, and 300g / L glucose and 7.5%NaHCO were used to 3 Adjust the reaction conditions in the petri dish to be the same as those in the bioreactor.

[0050] 2) After the cells are overgrown, replace with 20mL serum-free medium, and use 300g / L glucose and 7.5% NaHCO 3 Adjust the reaction conditions in the petri dish to be the same as those in the bioreactor. Replace 15mL of serum-free medium every 4 days, and compare the protein expression level with each batch in the bioreactor.

[0051] As a result, only 3 batches of expression samples can be collected with cell culture dish expression, and the expression amount can only reach about 0.2g / L (such as figure 2 shown).

Embodiment 3

[0052] The impact of embodiment 3 different pH values ​​on protein yield

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Abstract

The invention discloses a method for culturing HEK-293T cell strains for efficient secretion expression of an E2 protein of a swine fever. The method comprises the following steps: adding serum-containing medium into a cell bioreactor, inoculating HEK-293T cells and detecting the concentration of glucose after the medium is circulated at low speed and then at high speed; detecting the consumptionof sugar of the cells at an interval of 22-26 hours and adding glucose to 3.8-4.2 g / L when residual glucose is lower than 2 g / L; removing the medium when the consumption of sugar exceeds 1.5 g / L eachday and adding a serum-free medium; supplementing glucose according to the consumption of sugar of the cells; collecting a part of volume of the serum-free medium at an interval of 4-5d and supplementing a new serum-free medium. According to the method, by optimizing all parameters, fifteen batches of expression proteins can be collected at most in one experiment, so that the method is high in capacity. Additionally, the method is stable in culture conditions and high in repeatability. According to the method, the cultured cells are high in in density and high in expression quantity. The method is low in production cost of a unit vaccine, high in performance cost ratio and wide in application prospect.

Description

technical field [0001] The invention relates to a method and application for cultivating HEK-293T cell lines to efficiently secrete and express hog fever E2 protein. Background technique [0002] Classical swine fever (CSF), commonly known as "rotten intestinal fever", is an acute, febrile, contagious infectious disease caused by classical swine fever virus (CSFV), which is highly contagious and fatal. It is one of the main infectious diseases that seriously threaten the pig industry, and the Ministry of Agriculture of my country stipulates it as a first-class animal infectious disease. Swine fever is still prevalent in our country. According to statistics, more than 30% of the pigs that died due to disease in our country were caused by swine fever, and there is currently no specific drug against swine fever. Therefore, standardized vaccination of swine fever vaccine is an effective way to reduce An effective approach to morbidity and mortality. [0003] CSFV belongs to the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073C12P21/02C12R1/91
CPCC07K14/005C12N5/0603C12N2500/02C12N2500/60C12N2501/999C12N2770/24351
Inventor 颜仁和王升尧李红卫高永新李安迪万鹏飞
Owner GUANGZHOU BONIZZI BIOTECH CO LTD
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