Recombination cell line for stable expression of classical swine fever virus E2 and application thereof

A recombinant cell line and swine fever virus technology, which is applied in the fields of biomedical genetic engineering and immunology, can solve the problems of increased difficulty and cell lysis in production processes such as folding and modification, which are inferior to mammalian cell expression systems and downstream purification. Conducive to later purification, easy mass production, rapid proliferation effect

Inactive Publication Date: 2016-02-17
GUANGZHOU BONIZZI BIOTECH CO LTD
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the vaccine is expressed through the insect baculovirus expression system. Compared with the prokaryotic expression system, the expression system can undergo post-translational processing and modification to a certain extent after expressing the protein, but it still has a certain structure with the natural antigenic protein of the virus. The difference, protein folding and modification after expression is not as good as mammalian cell expression system, and when the system produces and prepares antigens, cells will lyse and die after virus infection, which increases the difficulty of downstream purification and other production processes

Method used

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  • Recombination cell line for stable expression of classical swine fever virus E2 and application thereof
  • Recombination cell line for stable expression of classical swine fever virus E2 and application thereof
  • Recombination cell line for stable expression of classical swine fever virus E2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Construction and detection of recombinant cell lines stably expressing classical swine fever virus E2 protein

[0060] 1. Design and preparation of gene sequence encoding classical swine fever virus E2 protein

[0061] 1.1 Design of gene sequence encoding classical swine fever virus E2 protein

[0062] The amino acid sequence of classical swine fever virus E2 protein (CSFVE2) refers to the protein sequence of CSFV Shimen strain (GenBank: AAC68902.2). After the nucleic acid sequence is deduced from the reference protein sequence, the C-terminal transmembrane sequence of the protein sequence is removed and his tag is added Nucleotide sequence, and when designing the gene, add a Kozak sequence, NheI restriction site and protective base before the start codon, and add a terminator and NheI restriction site at the end of the E2 protein gene coding and protective bases; the nucleic acid sequence of the obtained E2 protein is shown in SEQ ID NO.1, and SEQ ID NO.2 i...

Embodiment 2

[0116] Example 2. Experiment of the immune efficacy of swine fever subunit vaccine on pigs

[0117] The classical swine fever subunit vaccine of this example is composed of the recombinant cell line HEK-293T-E2 constructed and screened in Example 1 and an adjuvant, and the specific experimental steps are as follows.

[0118] 1. Vaccine preparation

[0119] When the recombinant cell line HEK-293T-E2 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 7 days; liquid;

[0120] The above culture supernatant was mixed with mineral oil adjuvant at a weight ratio of 1:1 and fully emulsified to prepare the swine fever subunit vaccine of this embodiment.

[0121] The above-mentioned serum-free medium is a common reagent used by those skilled in the art, and commercially available products can be used.

[0122] 2. Experiment grouping and immunization operation

[012...

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Abstract

The invention discloses a recombination cell line for stable expression of classical swine fever virus E2 and the application thereof. The recombination cell line is established after a lentivirus vector carries target genes to establish recombinant plasmids, then HEK-293T cell transfection is conducted, and generated high-titer virions are reinfected with HEK-293T cells. Stable expression is achieved, and a high protein expression level is kept after several passages. When a vaccine prepared with the recombination cell line is used for pig immunization, an animal organism can be induced to generate a high-potency classical swine fever virus neutralizing antibody, so that virulent attack of the classical swine fever virus is resisted.

Description

technical field [0001] The invention relates to a recombinant mammalian expression cell line, in particular to a recombinant cell line stably expressing classical swine fever virus E2 protein. The invention also discloses the method for preparing the recombinant cell line and the application of the recombinant cell line in preparing the swine fever subunit vaccine. It belongs to the field of biomedical genetic engineering and immunology. Background technique [0002] Classical swine fever (CSF) is an acute and highly fatal disease of pigs caused by classical swine fever virus (CSFV). animal diseases. The economic loss caused by swine fever to my country is nearly 10 billion every year, and the harm to my country's pig industry is extremely serious. [0003] The main measure for the effective prevention and control of the disease at present is to immunize. The attenuated swine fever vaccine (strain C) successfully researched by Chinese scientists has made outstanding cont...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K39/187A61P31/14C12R1/91
Inventor 李安迪高永新李红卫颜仁和王升尧
Owner GUANGZHOU BONIZZI BIOTECH CO LTD
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