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Modification of echinococcus granulosus EG95 protein, and expression thereof in yeast

A technology of Echinococcus granulosus and EG95, which is applied in fermentation, microbe-based methods, microbiological measurement/testing, etc., can solve the problems of affecting the immune protection effect of vaccines, difficulty in maintaining the natural structure of proteins, lack of protein processing and modification processes, etc.

Active Publication Date: 2012-10-17
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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Problems solved by technology

However, the existing recombinant EG95 recombinant protein vaccine antigen is produced by Escherichia coli, and its expression products mostly exist in the form of insoluble inclusion bodies, which makes it difficult to maintain the natural structure of the protein and lacks post-translational processing and modification of the protein, thus affecting the immunity of the vaccine protective effect

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  • Modification of echinococcus granulosus EG95 protein, and expression thereof in yeast
  • Modification of echinococcus granulosus EG95 protein, and expression thereof in yeast
  • Modification of echinococcus granulosus EG95 protein, and expression thereof in yeast

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Embodiment Construction

[0018] 1 Construction method of expression vector pPIC9K-tEG95

[0019] 1.1 Perform sequence synthesis according to the codon-optimized and truncated tEG95 gene sequence provided by SEQ №1, and connect it to the pMD18T-Simple vector (TRKARA, Dalian, Liaoning) to construct the pMD18-tEG95 cloning vector, and express pMD18-tEG95 and The vector pPIC9K was digested with EcoR I and Not I respectively, the target product was recovered from the gel, ligated with T4DNA ligase, and the expression vector pPIC9K-tEG95 was obtained.

[0020] 1.2 Linearize pPIC9K-tEG95 with Sal I digestion and prepare to mix with Pichia pastoris competent cells for electrotransformation.

[0021] 2 Construction methods of recombinant yeast transformants

[0022] 2.1 Pick a single colony of Pichia pastoris GS115 and inoculate it into YPD medium. For the YPD medium used, refer to the above content, and cultivate it to OD at 28°C 600 After reaching 1.3-1.5, collect the bacteria by centrifugation, wash the b...

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Abstract

The invention discloses a modification method of echinococcus granulosus EG95 protein, methods for preparing recombinant expression plasmids and recombinant engineering yeast strains by using the modified echinococcus granulosus EG95 protein, and a recombinant engineering yeast strain induction expression method. The modification method of the echinococcus granulosus major immunogenic protein EG95 comprises the steps that: on the basis of a complete EG95 protein, 23 amino acids are removed from an N- terminal, and 22 amino acids are truncated from a C- terminal. A modified tEG95 gene sequence is shown as SEQ ID No.1, and the protein sequence is shown as SEQ ID No.2. An yeast monoclonal strain EG95-1 is established and screened by using the method provided by the invention, and is collected in China General Microbiological Culture Collection Center on February 16th, 2012, with a collection number of CGMCC No.5764.

Description

technical field [0001] The invention relates to a method for modifying the Echinococcus granulosus EG95 protein, and using the modified Echinococcus granulosus EG95 protein to prepare a recombinant expression plasmid, a recombinant yeast engineering bacterium, and a method for inducing expression of the recombinant yeast engineering bacterium. Background technique [0002] Echinococcosis, also known as hydatid disease, is a kind of harm to humans and animals caused by the parasitic larvae of Echinococcus tapeworm in the liver, lung and other organs in mammals (including humans). Extremely serious zoonotic parasitic disease. The disease is distributed worldwide, and my country is one of the countries with the highest incidence of echinococcosis. Cystic echinococcosis (CE) caused by Echinococcus granulosus and alveolar echinococcosis (AE) caused by Echinococcus multilocularis are the most widely distributed in China. Disease (CE) predominance. CE is extremely harmful to hum...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/81C12N15/66C12N1/19C12P21/02C12Q1/68C12N15/11C12R1/84
Inventor 贾万忠娄忠子李宏民闫鸿斌倪兴维
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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