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Recombinant cell line stably expressing CSFV e2 protein, preparation method, application, and CSFV subunit vaccine

A recombinant cell line and subunit vaccine technology, applied in the field of molecular biology and veterinary biological products, can solve the problems of increased difficulty in production process, poor folding and modification, cell lysis, etc., and achieve good biological safety and long duration , Good antigenic effect

Active Publication Date: 2019-02-26
北京博尔柯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still some differences with the natural antigenic protein structure of the virus, and the folding and modification of the protein after expression is not as good as that of the mammalian cell expression system
Moreover, when the system produces and prepares antigens, the cells will lyse and die after being infected by the virus, which will increase the difficulty of downstream purification and other production processes.

Method used

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  • Recombinant cell line stably expressing CSFV e2 protein, preparation method, application, and CSFV subunit vaccine
  • Recombinant cell line stably expressing CSFV e2 protein, preparation method, application, and CSFV subunit vaccine
  • Recombinant cell line stably expressing CSFV e2 protein, preparation method, application, and CSFV subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Construction and detection of a recombinant cell line stably expressing classical swine fever virus E2 protein

[0077] 1.1 Materials and methods

[0078] 1.1.1 Strains, vectors and main reagents

[0079] Transfer plasmid: pHBLV-CMVIE-IRES-ZSGreen was purchased from Hanbio.

[0080] JM109 competent cells, RNA extraction kit, PrimeStar high-fidelity polymerase, and DNA Marker are products of Takara; Reverse Transcription System, 4-12% Bis-Tris Gel, SeeBlue Plus2 pre-stained strand are products of ThermoFisher; PGEM-T is a product of Promega The company's product; the agarose gel DNA recovery kit is an Omega product; XbaI is a NEB product; DMEM / F12(1:1), Foetal BovineSerum is a GIBCO product; Anti-Mouse IgG(Fc Specific)-peroxidase antibody produced ingoat is a Sigma product; Vector VIP Peroxidase (HRP) substrate kit is a Vector Laboratories product; The Plasmid maxi Kit is a product of QIAGEN; the ELISA kit for E2 protein antibody of classical swine fever vi...

Embodiment 2

[0104] Example 2 The immune efficacy test of swine fever E2 subunit vaccine to rabbits

[0105] 2.1 Test method

[0106] 2.1.1 Vaccine preparation and immunization

[0107] Mix the E2 protein cell culture supernatant with a content of 50ug / ml and the oil adjuvant in a weight ratio of 1:1, and stir at 300RPM for 5 minutes at room temperature to prepare a vaccine. Six Japanese big-eared white rabbits were randomly divided into 2 groups, 4 of which were the immunization group, each of which was intramuscularly injected with 1ml vaccine (containing 20ug E2 protein), and the other 2 were not immunized as the control group.

[0108] 2.1.2 Antibody detection

[0109] After immunization, blood was collected on the 14th day and 21st day, and the serum was detected with a commercial ELISA kit to detect the antibody to classical swine fever.

[0110] 2.1.3 Antivirus

[0111] 21 days after immunization, the immunized group and the control group were simultaneously inoculated with 1 ml...

Embodiment 3

[0122] Example 3 The immune efficacy test of swine fever E2 subunit vaccine to pigs

[0123] 3.1.1.1 Immunity to 2-week-old piglets

[0124] (1) Grouping: 15 2-week-old piglets that were negative for CSFV antibodies were randomly divided into 3 groups, 5 pigs in each group, the first group was the weak swine fever live vaccine immunization group, and the second group was the swine fever subunit Vaccine immunization group, the third group is the blank control group.

[0125] (2) Immunization: the first group is immunized with 1 head of commercialized swine fever live vaccine according to the instruction manual; each pig of the second group is inoculated intramuscularly with 1ml of the swine fever E2 protein subunit vaccine prepared in Example 2 ; The third group was not immunized as a blank control group.

[0126] (3) Antibody detection: The swine fever live vaccine immunization group, the swine fever E2 protein subunit vaccine immunization group, and the control group were a...

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Abstract

The invention relates to a method for preparing a recombinant cell line for stably expressing a swine fever virus E2 protein. The method for preparing the recombinant cell line for stably expressing the swine fever virus E2 protein comprises the steps of providing a recombinant expression plasmid, wherein the recombinant expression plasmid contains a swine fever virus E2 protein coding sequence and a green fluorescent protein coding sequence which are effectively connected with each other; transfecting the recombinant expression plasmid and a helper plasmid into an eukaryotic cell; screening out a single cell for expressing the recombinant expression plasmid by using a fluorescence detection method and subculturing the monoclonal cell for multiple times to obtain subcultured cell clone; and detecting the expression quantity of the swine fever virus E2 protein in the cell clone and screening out the recombinant cell line which can still express the swine fever virus E2 protein after multiple subcultures, wherein the nucleotide sequence of the swine fever virus E2 protein is shown in sequence 1, and the amino acid sequence for coding the swine fever virus E2 proteinis is shown in sequence 2. The invention further relates to the recombinant cell line for stably expressing the swine fever virus E2 protein, application and a swine fever virus sub-unit vaccine.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and veterinary biological products. Specifically, the present invention relates to a recombinant cell line stably expressing the E2 protein of classical swine fever virus and a preparation method thereof, the application of the recombinant cell line in the preparation of a subunit vaccine of classical swine fever virus, and a strain of classical swine fever containing the recombinant cell line. The virus subunit vaccine and the preparation method of the swine fever virus subunit vaccine. Background technique [0002] Classical swine fever (CSF) is an acute and highly fatal disease of pigs caused by classical swine fever virus (CSFV). The mortality rate, the International Office of Epizootics (OIE) defines it as a disease that must be notified, and my country lists it as a first-class animal disease. Because pigs of different ages, sexes and breeds can be infected, swine fever causes hug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/40C12N5/10C12P21/02A61K39/12A61P31/14
CPCA61K39/12A61K2039/552C07K14/005C12N15/85C12N2770/24322C12N2770/24334
Inventor 不公告发明人
Owner 北京博尔柯生物科技有限公司
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