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

A technology for porcine epidemic diarrhea and secretion expression, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve problems such as losses and unsatisfactory effects in the pig industry, save labor and material costs, cost-effective, and improve stability. sexual effect

Inactive Publication Date: 2021-09-03
GUANGZHOU BONIZZI BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the vaccines at home and abroad are mainly attenuated or inactivated vaccines. These vaccines play a certain role in the control of PED in my country, but the effect is not ideal. The occurrence of PED every year causes huge losses to my country's pig industry, and urgently needs research and development. A new type of PEDV vaccine with high biological safety and good immune effect can effectively control it

Method used

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  • 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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 A production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein

[0030] The production method for the efficient secretion and expression of porcine epidemic diarrhea virus S1 protein comprises the following steps:

[0031] S1. Treatment of the cell bioreactor: Check whether the consumables are damaged, record the batch, inflate the torrent bag and the perfusion bag for leak detection overnight (note that the inflation flow rate is not too large, the filter membrane will be crushed and the bag will be discarded); DO electrode Power on the electrode for more than 6 hours; the next day, after the calibration electrode is sterilized, fill the tank and connect the pipeline. After the system is connected, check the air tightness, inject 2.5L of PBS buffer solution, and soak the paper carrier in the reactor overnight. On the third day, pour out PBS; add 2L DMEM complete medium to the above reactor, pretreat the polyfiber disc ...

Embodiment 2

[0041] Example 2 Comparison experiment of bioreactor expression and cell factory expression

[0042] Petri dish expression method:

[0043] 1) According to the culture conditions of the bioreactor in Example 1, inoculate in the 10-layer cell factory after cultivating enough cells in the cell culture dish, and use 300g / L glucose and 7.5%NaHCO 3 Adjust the reaction conditions in the petri dish to be consistent with the bioreactor;

[0044] 2) After the cells are full, replace the serum-free medium according to the ratio of the number of cells, and use 300g / L glucose and 7.5% NaHCO 3 Adjust the reaction conditions in the petri dish to be consistent with the bioreactor; collect 1 / 2-2 / 3 volume of serum-free medium expression supernatant every 4 days and supplement the corresponding volume of serum-free medium, which is the same as that of the bioreactor every 4 days. Batch comparison of protein expression.

[0045] As a result, only 2 batches of expression samples can be collect...

Embodiment 3

[0046] The impact of embodiment 3 different pH values ​​on protein yield

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein. According to the invention, amplifying and expressing porcine epidemic diarrhea virus S1 protein through a poly-fiber paper carrier by using a cell bioreactor, culturing for 2-3 days by using a DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal calf serum in a cell culture stage, discarding all the culture medium, washing with PBS (Phosphate Buffer Solution) for 1-2 times to remove the serum, then completely replacing the culture medium with a serum-free culture medium and starting to produce and express the porcine epidemic diarrhea virus S1 protein; collecting part of the culture medium every 3-5 days, adding serum-free culture medium again, and supplementing glucose according to the sugar consumption of cells every day to keep 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

technical field [0001] 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 [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) is characterized by vomiting of pigs, watery diarrhea, dehydration and high mortality of suckling piglets. 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 existence of the disease in our country through fluorescent antibody test and serum neutralization test, and it is widely prevalent in our country at present. The epidemic of PEDV affects pig herds of all ages in many provinces of China, with ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N5/10
CPCC07K14/005C12N5/0686C12N2770/20022C12N2770/20051C12N2510/02
Inventor 颜仁和毛莹莹李红卫万鹏飞仇珍珍梁铁坤梁文翰陈泽典苏海龙李虎林
Owner GUANGZHOU BONIZZI BIOTECH CO LTD
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