Unlock instant, AI-driven research and patent intelligence for your innovation.

Production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein

A porcine epidemic diarrhea, secretory expression technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve problems such as inability to meet actual production needs, increase the production cost of S1 protein, and inability to increase lentivirus production, and achieve savings The effect of labor and material cost, high cost performance and high production capacity

Active Publication Date: 2022-04-29
SOUTHERN MEDICAL UNIVERSITY +1
View PDF12 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on this, Chinese patent CN109890959A discloses a serum-free suspension system for lentivirus production, in which culture supplements are added including: amino acids and / or dipeptides and sugar sources, sugar alcohols and / or carbon sources, amino acids and Glucose, etc., also added a lentivirus production enhancer, including sodium propionate, sodium butyrate and caffeine, but because sodium butyrate is easy to cause cell damage, it is necessary to choose a low concentration during application, and it cannot be directly added It is used in culture medium, therefore, the system provided by this patent cannot increase the yield of lentivirus, and in the process of using sodium butyrate, it is easy to cause cell damage and lead to cell death
[0006] At the same time, Chinese patent CN104248762B discloses a porcine epidemic diarrhea vaccine composition and its preparation method and application. The vaccine composition includes porcine epidemic diarrhea virus subunit antigen, porcine epidemic diarrhea whole virus inactivated antigen and carrier, etc. The S1 protein in the vaccine composition can be recombined and expressed in a large number of components of the vaccine composition by genetic engineering, which not only takes a short time, but also facilitates large-scale production. However, the S1 protein obtained by this method is not directly produced by the virus Therefore, its structure and function may be damaged, and in large-scale production, genetic engineering is required, and each step is required to be fine, which greatly increases the production cost of S1 protein
[0007] To sum up, it can be seen that when using viruses to produce S1 protein in the prior art, there are generally disadvantages such as low production capacity, easy to cause cell damage, high production cost, and inability to meet actual production needs.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein
  • Production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein
  • Production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 A production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein

[0035] The production method for the efficient secretion and expression of porcine epidemic diarrhea virus S1 protein comprises the following steps:

[0036] S1. After washing the cell bioreactor with PBS, add 2L of DMEM complete medium containing 8% fetal bovine serum, 1% human serum albumin, and 4.4g / L glucose, and pretreat overnight at 37°C And do a sterility test to obtain a sterile cell bioreactor;

[0037] S2. Use the cell factory to produce the total amount of HEK-293T cells required, and then dilute the cells with 2L of the DMEM complete medium, and slowly inoculate them into the perfusion bag in the sterile cell bioreactor obtained in step S1, and inoculate The amount is 1.5×10 9 One, static adsorption for 1 hour, and then carry out low-speed and high-speed circulation respectively, wherein the low-speed circulation is 130mL / min circulation for ...

Embodiment 2

[0041] Example 2 A Production Method for Efficient Secretion and Expression of Porcine Epidemic Diarrhea Virus S1 Protein

[0042] The production method for the efficient secretion and expression of porcine epidemic diarrhea virus S1 protein comprises the following steps:

[0043] S1. After washing the cell bioreactor with phosphate buffered solution PBS, add 2L of DMEM complete medium containing 12% fetal bovine serum, 2% human serum albumin, and 4.6g / L glucose, and pretreat overnight at 37°C And do a sterility test to obtain a sterile cell bioreactor;

[0044] S2. Use the cell factory to produce the total amount of HEK-293T cells required, and then dilute the cells with 2L of the DMEM complete medium, and slowly inoculate them into the perfusion bag in the sterile cell bioreactor obtained in step S1, and inoculate The amount is 2.5×10 9 One, static adsorption for 1 hour, and then carry out low-speed and high-speed circulation respectively, wherein the low-speed circulation...

Embodiment 3

[0048] Example 3 A Production Method for Efficient Secretion and Expression of Porcine Epidemic Diarrhea Virus S1 Protein

[0049] The production method for the efficient secretion and expression of porcine epidemic diarrhea virus S1 protein comprises the following steps:

[0050] S1. After washing the cell bioreactor with PBS, add 2L of DMEM complete medium containing 10% fetal bovine serum, 1.5% human serum albumin, and 4.5g / L glucose, and pretreat overnight at 37°C And do a sterility test to obtain a sterile cell bioreactor;

[0051] S2. Use the cell factory to produce the total amount of HEK-293T cells required, and then dilute the cells with 2L of the DMEM complete medium, and slowly inoculate them into the perfusion bag in the sterile cell bioreactor obtained in step S1, and inoculate The amount is 2.0×10 9 One, static adsorption for 1 hour, and then carry out low-speed and high-speed circulation respectively, among which the low-speed circulation is 150mL / min circulat...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of biology, and particularly relates to a production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein. The method comprises the following steps: amplifying and expressing the porcine epidemic diarrhea virus S1 protein by using a poly-fiber paper carrier and a cell bioreactor, culturing by using a culture medium containing 10% of fetal calf serum, and producing and expressing the porcine epidemic diarrhea virus S1 protein by using a serum-free culture medium containing 7mM of caffeine, 2mM of KH2PO4, 25-50mg / L of dextran sulfate, 5mM of sodium butyrate, 0.5 mM of hydroxyethyl starch and 1mM of L-arginine. Part of the culture medium is collected every 5-7 days, the serum-free culture medium is added again, glucose is supplemented according to the sugar consumption of cells every day, and the concentration of the glucose is kept at 4 g / L. By adopting the method provided by the invention, the expression level of cells is effectively improved, the manufacturing cost of vaccines is reduced, and a foundation is laid for realizing large-scale production of vaccines.

Description

[0001] This application enjoys the priority of the patent application number "2021106269219", the application date is "June 4, 2021", and the invention title is "A Production Method for Efficient Secretion and Expression of Porcine Epidemic Diarrhea Virus S1 Protein". technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein. Background technique [0003] Porcine epidemic diarrhea (PED) is an acute, highly contagious disease characterized by vomiting, watery diarrhea, dehydration, and high mortality in suckling piglets caused by porcine epidemic diarrhea virus (PEDV). Sexual intestinal diseases. In the 1970s, the disease was mainly prevalent in major pig producing countries in Europe; in 1982, it was first discovered in Japan in Asia and spread rapidly to neighboring countries. In 1984, Xuanhua and others confirmed the exis...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N5/10
CPCC07K14/005C12N5/0686C12N2770/20022C12N2770/20051C12N2510/02
Inventor 毛莹莹颜仁和李红卫万鹏飞仇珍珍梁铁坤李堪贺
Owner SOUTHERN MEDICAL UNIVERSITY