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Genetic engineering subunit vaccine capable of resisting goat hydatidosis infection

A technology encoding genes and amino acids, which is applied in the field of animal immunological drugs, can solve the problems of low glycosylation level, easy formation of inclusion bodies, and poor folding type in the expression system of Pichia pastoris, and achieve strong solubility, good immune effect, and high expression level. high effect

Active Publication Date: 2020-09-18
苏州世诺生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such as CN108066755A provides a kind of method utilizing Escherichia coli expression system to obtain recombinant Eg95 protein to prepare genetic engineering subunit vaccine, but the folding type of the protein expressed by Escherichia coli expression system is poor, and easy to form inclusion bodies, so the expression level is low
As another example, CN102732436A discloses a method for recombinant expression of Eg95 protein in Pichia pastoris, which uses methanol as an inducer, which has certain hazards, and the glycosylation level of the Pichia pastoris expression system is relatively low

Method used

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  • Genetic engineering subunit vaccine capable of resisting goat hydatidosis infection
  • Genetic engineering subunit vaccine capable of resisting goat hydatidosis infection
  • Genetic engineering subunit vaccine capable of resisting goat hydatidosis infection

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preparation example Construction

[0064] In some embodiments, a method for preparing a genetically engineered subunit vaccine against echinococcosis infection specifically includes:

[0065] 1) cloning the eukaryotic expression vector comprising the optimized Eg95 protein coding gene;

[0066] 2) Transfect CHO cells, and obtain a CHO cell line that stably and efficiently expresses Eg95 protein in suspension through selection and screening;

[0067] 3) The cell strain screened out in step 2) of fermentation and cultivation is purified to obtain recombinant Eg95 protein;

[0068] 4) The recombinant Eg95 protein is thoroughly mixed, and then fully mixed with an adjuvant to obtain a recombinant expression subunit vaccine.

[0069] The method provided in the above embodiments of the present invention can harvest the target protein from the cell culture supernatant, and the yield is as high as 2-3g / L, which not only shortens the protein purification time and simplifies the vaccine production steps, but also greatly...

Embodiment 1

[0082] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-Eg95-GS

[0083] 1. Amplification and purification of Eg95 gene The codon-optimized Eg95 gene (SEQ ID NO: 1) was synthesized in Shanghai Sunny Biotechnology Co., Ltd. and cloned into the pUC-57 vector to obtain the pUC-Eg95 plasmid vector. PCR amplification was performed using pUC-Eg95 as a template and Eg95-F and Eg95-R as primers (the gene sequences of Eg95-F and Eg95-R are shown in SEQ ID NO: 3 and 4). The amplification system is shown in Table 1. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0084] Table 1 Eg95 gene amplification system

[0085]

[0086] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As shown, a band appeared near the po...

Embodiment 2

[0098] Example 2 Construction and Screening of Recombinant CHO Cells Expressing Eg95 Protein

[0099] 1. Cell Transfection

[0100] 1.1 Prepare cells Take CHO cells in the logarithmic growth phase, sample and count, and use 1×10 6 The cell density of cells / ml continues to be subcultured, maintain the seeds, centrifuge the remaining cells, centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend with about 20ml of fresh CHO-WM medium, centrifuge again, centrifuge at 1000rpm for 4 minutes, discard the supernatant After resuspending with a small amount of medium for counting, the final cell density was adjusted to 1.43×10 7 cells / ml.

[0101] 1.2 Plasmid and cell mixing Take 5 μg of the pCI-Eg95-GS plasmid vector in Example 1, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.

[0102] 1.3 Electroporation 280V 20ms electric shock for 2 pulses. Immediately after the electric shock is completed, the cells are transferred to a shaker fl...

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Abstract

The invention discloses a genetic engineering subunit vaccine capable of resisting goat hydatidosis infection. The genetic engineering subunit vaccine comprises a recombinant protein of which the encoding gene sequence is disclosed by SEQ ID NO:1. An EG (Echinococcus granulosus) 95 protein is optimized and is used for preparing the genetic engineering subunit vaccine capable of resisting the goathydatidosis infection, the antigenicity, the immunogenicity and the function are similar to the antigenicity, the immunogenicity and the function of a natural protein, an expression level of the obtained vaccine is high, the immunogenicity of the obtained vaccine is high, a good immune effect can be provided only by a small quantity, and the obtained vaccine does not have pathogenicity for goats.Meanwhile, the Eg 95 protein subjected to expression optimization by CHO cells and the like is adopted, a glycosylation level is high, solubility is high, an expression quantity (which can be 2-3 g / L) is high, and the genetic engineering subunit vaccine can be prepared in a bioreactor through large-scale serum-free suspension culture, and the production cost of the vaccine is greatly lowered.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a genetic engineering subunit vaccine against sheep echinococcosis infection. The preparation method and application thereof belong to the technical field of animal immune medicines. Background technique [0002] Hydatidosis (Hydatid Disease, Hydatid Disease), also known as Echinococcosis (Echinococcus) or Cystic Echinococcosis (CE), is caused by Echinococcus granulosus (Echinococcus granulosus, Eg) or Echinococcus multilocularis (E.multilocularis) larvae infecting sheep and parasitic in the human liver, lungs and other organs is a zoonotic parasitic disease. It is one of the five major parasitic diseases planned by the Ministry of Health of China. The disease is widespread, highly pathogenic, and difficult to prevent and control, causing greater economic losses to our country every year. Echinococcosis is distributed worldwide. my country is one of the countries with the highest...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/85C12N5/10A61K39/00A61P33/10G01N33/68
CPCA61K39/00A61K2039/552A61P33/10C07K14/43554C12N5/0682C12N15/85C12N2510/02C12N2800/107C12N2800/22G01N33/6893G01N2333/43543
Inventor 曹文龙孔迪滕小锘
Owner 苏州世诺生物技术有限公司
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