Recombinant cell line for stable expression of porcine epidemic diarrhea virus S1 protein, vaccine and application

A porcine epidemic diarrhea, recombinant cell line technology, applied in the fields of biomedical genetic engineering and immunology, to achieve the effects of rapid proliferation, easy culture and good protein expression level

Inactive Publication Date: 2018-01-23
GUANGZHOU BONIZZI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the immune prevention and control of PEDV in China mainly uses inactivated vaccines and attenuated vaccines, and there are many domestic researches on these two vaccines; although inactivated vaccines and attenuated vaccines play a certain role in the prevention and control of PEDV, but Because these two vaccines have certain deficiencies, there is an urgent need for a new type of vaccine with high biological safety and good immune effect on the market.

Method used

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  • Recombinant cell line for stable expression of porcine epidemic diarrhea virus S1 protein, vaccine and application
  • Recombinant cell line for stable expression of porcine epidemic diarrhea virus S1 protein, vaccine and application
  • Recombinant cell line for stable expression of porcine epidemic diarrhea virus S1 protein, vaccine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction and detection of a recombinant cell line stably expressing porcine epidemic diarrhea virus S1 protein

[0069] 1. Design and preparation of gene sequence encoding porcine epidemic diarrhea virus S1 protein

[0070] 1.1 Design of gene sequence encoding porcine epidemic diarrhea virus S1 protein

[0071] According to the S1 protein gene of porcine epidemic diarrhea virus epidemic strain CH / GDZQ / 2014 strain (GenBank: KM242131.1), the following sequence optimization and expression design were carried out:

[0072] (1) Sequence optimization

[0073] According to the S1 protein gene nucleotide sequence of porcine epidemic diarrhea virus epidemic strain CH / GDZQ / 2014 strain (GenBank: KM242131.1), add Kozak sequence and Nhe I restriction site before the start codon of S1 protein gene Point and protective base, add a terminator, Nhe I restriction site and protective base at the coding end of the S1 protein gene, add IgK signal peptide at the 5' end, and a...

Embodiment 2

[0127] Example 2 Purification and Analysis of Recombinant Porcine Epidemic Diarrhea Virus S1 Protein

[0128] 1. Purification of porcine epidemic diarrhea virus S1 protein

[0129] Filter the cell culture supernatant of porcine epidemic diarrhea virus S1 protein recombinant cell line 239T-S1 through a 0.45 μm filter membrane to obtain a pretreated sample;

[0130] His Trap TM HP column with BioLogic LP After the protein purification instrument is connected correctly, equilibrate the column with 3 times the column bed volume of loading buffer (20mM pH7.4 phosphate buffer, 0.5M NaCl), and 1ml / min to equilibrate the column. After loading the sample, wash it with loading buffer (flow rate 1ml / min, 5 times the column bed volume), and then use 20mM pH7.4 phosphate buffer (containing 200mM imidazole) to elute the recombinant protein, and at the same time use BioLogic LP Monitor and start collecting when the baseline rises, that is, when the elution peak appears, collect the elu...

Embodiment 3

[0134] Example 3 Porcine epidemic diarrhea virus subunit vaccine

[0135] The porcine epidemic diarrhea virus subunit vaccine of this example is composed of the recombinant cell line 293T-S1 constructed and screened in Example 1 and an adjuvant. The specific experimental steps are as follows:

[0136] When the recombinant cell line 293T-S1 constructed and screened in Example 1 was normally passaged and grown to reach a confluence of more than 90%, the serum-free medium was changed to continue culturing for 4 days; the culture supernatant was collected and centrifuged at 2000 rpm for 10 min, and the supernatant was absorbed;

[0137] The above culture supernatant and mineral oil adjuvant were emulsified at a weight ratio of 1:1, the emulsification temperature was 30°C, the emulsification speed was 350 rpm, and the emulsification time was 5 minutes, and the porcine epidemic diarrhea virus sub unit vaccine.

[0138] The above-mentioned serum-free medium is a common reagent used ...

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Abstract

The invention discloses a recombinant cell line for stable expression of porcine epidemic diarrhea virus S1 protein, a vaccine and an application. The recombinant cell line is constructed by transfecting HEK-293T cells by virtue of recombinant plasmid which is constructed by carrying a target gene on a lentiviral vector, and then transfecting the HEK-293T cells by virtue of generated high-titer virus particles; and the recombinant cell line, which can achieve stable expression, can still keep an excellent protein expression level after several passages. The recombinant cell line for stable expression of the porcine epidemic diarrhea virus S1 protein provided by the invention has the characteristics of being easy for culture, rapid in proliferation, unlimited in expansion, stable in property and high in protein expression amount; and when the vaccine, which is prepared from the expression protein and adjuvants, is used for immunizing pigs, the generation of a high-titer porcine epidemicdiarrhea virus neutralizing antibody can be induced from animal bodies, and the piglets (the pigs) can resist strong attack of porcine epidemic diarrhea viruses.

Description

technical field [0001] The invention relates to the fields of biomedical genetic engineering and immunology, in particular to the immune research of porcine epidemic diarrhea virus, and in particular to a recombinant cell line stably expressing porcine epidemic diarrhea virus S1 protein, a vaccine and its application. Background technique [0002] Porcine epidemic diarrhea (PED) is an infectious enteric disease caused by porcine epidemic diarrhea virus (PEDV), clinically characterized by acute enteritis, vomiting, watery diarrhea, dehydration and It is characterized by high mortality in suckling piglets. The disease first occurred in Belgium and the United Kingdom in 1978. In 1984, Xuanhua and others confirmed the existence of the disease in my country through fluorescent antibody test and serum neutralization test, and it is now widely prevalent in my country. [0003] PEDV can destroy the jejunum and ileum epithelial cells and intestinal villi of infected pigs, cause ente...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/86A61K39/215A61P31/14
Inventor 李安迪高永新颜仁和王升尧刘军李红卫
Owner GUANGZHOU BONIZZI BIOTECH CO LTD
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