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Fused gene, recombinant expression vector, antigen, preparation method and application

A technology of fusion antigen and fusion gene, applied in biochemical equipment and methods, fusion polypeptides, viral antigen components, etc., can solve the problems of difficult performance effect and many process steps, etc.

Active Publication Date: 2019-08-16
ZHAOQING DAHUANONG BIOLOGIC PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the problem with the above scheme is that there are many process steps for the separate production of avian influenza virus and adenovirus; if the preparation of subunit vaccines is carried out based on the principle of genetic engineering, although the process steps can be simplified, how to screen and obtain effective antigens? determinants, especially simultaneous screening of two effective epitopes is a difficult problem
[0009] A further problem is: even if two or more effective antigenic determinants are screened, how to improve their performance in subunit vaccines is a very difficult practical problem

Method used

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  • Fused gene, recombinant expression vector, antigen, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Preparation of fusion antigen based on avian influenza virus HA protein and avian adenovirus fiber2 protein:

[0058] Raw materials: Pichia pastoris GS115 and pPic9k plasmids were provided by the research group of Teacher Han Nanyu, School of Life Sciences, Yunnan Normal University; the above raw materials are also commercially available, such as: Pichia pastoris GS115 (produced by: Invitrogen TM ;Strain type: Pichia pastoris; Catalog number: C181-00; Medium: YPD medium; Growth conditions: 28-30°C, aerobic; Genotype: His4; Phenotype: Mut+; Plasmid transformation conditions: electroporation; Application: protein expression; induction method:

[0059] methanol); pPic9k plasmid (manufacturer: Invitrogen TM ;Product Name: pPIC9K Pichia Vector; Item No.: V17520)

[0060] Escherichia coli DH5α, plasmid extraction kit and restriction endonucleases ECORI and NOTI were purchased from TaKaRa-Bao Bioengineering (Dalian) Co., Ltd.; T4 DNA ligase is T4 DNA ligase, which is a comme...

Embodiment 2

[0072] The preparation of avian influenza and avian adenovirus type 4 dual genetic engineering subunit vaccine comprises the following steps:

[0073] (1) Oil phase preparation: 94 parts by weight of white oil for injection and 6 parts by weight of Siben-80 were mixed to prepare an oil phase, sterilized at 121° C. for 30 minutes, and cooled to room temperature for later use.

[0074] (2) Water phase preparation: take the fusion antigen prepared in Example 1 and dilute it into 96 parts by weight so that the content of the fusion antigen in the vaccine is 75 μg / ml, add 4 parts by weight of sterilized and cooled Tween-80, and stir until completely dissolve.

[0075] (3) Emulsification: Take 3 parts by weight of the oil phase and pour it into an emulsification tank for stirring, then slowly add 2 parts of the water phase, and emulsify for 60 minutes at an emulsification speed of 2000r / min.

[0076] (4) Subpackaging: Aseptic quantitative subpackaging, capped and sealed, and stored...

Embodiment 3

[0078] Vaccine immunity test

[0079] (1) Immune protection against bird flu (H9 subtype)

[0080] With 15 28-day-old SPF chickens, 10 of them were subcutaneously injected with 0.3 ml of avian influenza and avian adenovirus type 4 dual genetically engineered subunit vaccine (Example 2), and the other 5 were used as avian influenza challenge controls. Twenty-one days after inoculation, each chicken was intravenously injected with 0.2ml of H9 subtype avian influenza virus DA strain chicken embryotoxin (containing 2×10 7.0 EID 50). On the 5th day after the challenge, collect cotton swabs from the throat and cloaca of each chicken, mix the supernatants of the two, and inoculate 5 9-11-day-old SPF chicken embryos through the allantoic cavity, 0.2ml per embryo, and observe after incubation. At 72 hours, measure the HA titer of all chicken embryo fluids. As long as the HA titer of chicken embryo fluid in 1 chicken embryo among the 5 chicken embryos inoculated with each mixed swab...

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Abstract

The invention belongs to the technical field of veterinary biological products, and discloses a fused gene. A base sequence is as shown in a sequence table SEQ ID NO: 1. An avian influenza H9 subtypeHA gene, an adenovirus type 4 fiber2 gene and a specific connecting peptide segment gene are fused to form the fused gene. An expression protein antigen corresponding to the fused gene can induce poultry to generate high-level specific antibodies, and the poultry is protected from infection of avian influenza and avian adenoviruses. Besides, the invention further discloses a recombinant expressionvector, an antigen, a preparation method and an application.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a fusion gene, a recombinant expression vector, an antigen and a preparation method and application thereof. Background technique [0002] Avian influenza virus (Avian influenza virus AIV) is an important pathogen of the respiratory system of birds and some mammals, and belongs to the genus Influenzavirus of the family Orthomyxoviridae in taxonomy. According to the different surface protein antigenicity, avian influenza virus is divided into 17 subtypes of hemagglutinin (HA) and 10 subtypes of neuraminidase (NA), among them, the H9N2 subtype of avian influenza virus is widespread in the world The epidemic has caused a significant drop in the egg production of laying hens and a reduction in feed remuneration, which has caused huge economic losses to the poultry industry and has caused danger to human health. The pathogenicity of influenza virus to the host ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81A61K39/295A61K39/235A61K39/145A61P31/16A61P31/20
CPCC07K14/005C12N15/815A61K39/12A61P31/20A61P31/16C12N2760/16122C12N2710/10222C07K2319/00A61K2039/70A61K2039/575
Inventor 陈瑞爱李延鹏温良海蔡仕君李鹏杰
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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