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Hydatidovis soluble antigen preparation method and product thereof

A hydatid and soluble technology, applied in the field of genetically engineered vaccines for hydatid, can solve problems such as limited popularization and application, reduced antigenicity, and increased immune effect

Active Publication Date: 2012-01-11
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When preparing vaccines, it still needs to be dissolved with high-concentration urea, which not only reduces its antigenicity due to repeated denaturation and refolding, but also greatly increases the production cost of antigens
In order to improve its immunogenicity, Quil-A also needs to be used as an adjuvant. Therefore, each dose of vaccine needs up to 50 μg of antigen, resulting in high production costs, which greatly limits its application (Lightowlers, Parasitology, 2006, S27 -S42), in addition, although GST is a good immune adjuvant, it also has a good effect of increasing immunity (Scott JC, Parasitol.Int., 2000, 49 (4): 289-300), but derived from The GST of Schistosoma japonicum is far less effective against echinococcosis than the GST derived from echinococcosis itself

Method used

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  • Hydatidovis soluble antigen preparation method and product thereof
  • Hydatidovis soluble antigen preparation method and product thereof
  • Hydatidovis soluble antigen preparation method and product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Soluble expression and immunization experiment of hydatid hydatid Sj-GST-eg95 antigen gene (SEQ ID NO: 3) in silkworm bioreactor

[0054] 1 Experimental method

[0055] 1.1. About the configuration of solution and medium

[0056] Solution I: 50mmol / L glucose, 25mmol / L Tris-HCl (pH8.0), 10mmol / L EDTA.

[0057] Solution II: 0.2mol / L NaOH, 1% SDS (prepared and used immediately).

[0058] Solution III: 100mL system, 5mol / L potassium acetate 80mL, glacial acetic acid 12mL, ddH 2 O8mL.

[0059] TAE (50×): 242g Tris base, 57.1mL glacial acetic acid, 100mL 0.5mol / LEDTA (pH8.0), dilute to 1000mL with sterile water.

[0060] TER solution: Pancreatic RNAse (RNAse A) was dissolved in 10mM Tris-HCl, 15mMNaCl, made into a 10mg / mL storage solution and stored at -20°C, then diluted with 1×TE buf to a 20μg / mL working solution and stored at 4°C .

[0061] PPt Buffer: isopropanol 22mL; 5mol / mL KAc 1mL; ddw 2mL.

[0062] PEG solution: Weigh a certain amount of NaCl to prep...

Embodiment 2

[0252] Example 2 Soluble Expression and Immunization Experiment of Echinococcosis Eg-GST-eg95 Antigen Gene (SEQ ID NO: 5) in Bombyx mori Bioreactor

[0253] GSTs (EC2.5.1.18) is a group of isoenzymes encoded by multiple genes and having multiple functions widely present in various organisms, with a molecular weight of 23-29KDa. GSTs protein as a vaccine can not only induce strong immune protection, but also effectively reduce the number of adults and the number of eggs laid. In this experiment, the eg-GST gene and EG95 gene of hydatid ovis were fused to obtain the Eg-GST-eg95 fusion antigen Gene, and obtain soluble high-expression product in silkworm bioreactor, animal experiment shows that it has good protective effect.

[0254] 2.1. Cloning of eg-GST gene, vector construction and antibody preparation

[0255] The preparation of hydatid hydatid RNA, the preparation of reverse transcription and cDNA template is the same as embodiment 1, in order to amplify the eg-GST gene, the ...

Embodiment 3

[0277] Example 3 Soluble expression and immunization experiment of echinococcosis EG95 antigen gene (SEQ ID NO: 1) in silkworm bioreactor

[0278] Over the years, domestic and foreign scholars have done a lot of work in the development of vaccines in order to effectively control the disease, and have made great progress. The early work was mainly on the study of parasite immunogens. Although it was confirmed that hydatid echinococcoids were grinded as a whole and excreted and secreted antigens can be used as vaccination to obtain good immune prevention effect, but the life history of the pathogen is complex, the source is limited, and it is difficult to batch Production is thus limited in actual work. In recent years, with the rapid development of immunology, biochemistry and molecular biology, Lightowlers et al. found that the main protective antigen in this sheep echinococcosis and its excretion and secretion antigen as a vaccine is EG95 protein (Lightowlers MW, et al., Int....

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Abstract

The invention discloses a hydatidovis soluble antigen preparation method and a product thereof. The preparation method comprises: expressing gene represented by SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 in an insect bioreactor or a pichia pastoris bioreactor; and collecting and purifying the antigen to obtain the hydatidovis soluble antigen. In the method disclosed by the invention, a domestic silkworm bioreactor or the pichia pastoris bioreactor is used to prepare the hydatidovis antigen, the product is soluble, immune experiments prove the antigen has high antigen performance, the unit expression is 10 to 100 times higher than that of Escherichia coli, and animal experiments prove the antigen has high insect reducing efficiency. When the method is used, the production cost of the hydatidovis antigen can be reduced greatly, and the purification process of the conventional production of antigen by Escherichia coli is simplified; and the method has the advantages of safety, high efficiency, low cost and the like.

Description

technical field [0001] The invention relates to a preparation method of an antigen, in particular to a preparation method of a hydatid worm soluble antigen and a product prepared by the preparation method, and belongs to the field of genetic engineering vaccines of the hydatid hydatid. Background technique [0002] Echinococcosis of sheep is commonly known as hydatidosis (Hydatidosis,), also known as echinococcosis or cystic echinococcosis (cystic echinococcosis; CE). Echinococcus granulosus (commonly known as sheep hydatid) (Echinococcus granulosus) or multilocularis (E.multilocularis) larvae infecting sheep is a zoonotic disease. It is a zoonotic parasitic disease that seriously endangers health and is caused by the continuation stage larvae of Echinococcus granulosus (Eg) parasitic in human liver, lung and other organs. One of the five major parasitic diseases. Most of the patients are children and young adults, and they grow slowly in the human body. Symptoms often app...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/866C12N15/81C07K14/435A61K39/00A61K38/17A61P33/10
Inventor 张志芳李轶女易咏竹贾如韩宾邹媛媛沈桂芳舒惠国
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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