Construction method of echinococcus granulosus immunogenic protein EG95 constitutive expression vector

A technology of Echinococcus granulosus and immunogenicity, applied in the field of genetic engineering, to achieve the effect of reducing the production cycle

Pending Publication Date: 2021-01-22
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When expressing, glycerol is used as the carbon source, which completely overcomes the safety hazards of the existing Pichia pastoris expression system that requires methanol induction

Method used

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  • Construction method of echinococcus granulosus immunogenic protein EG95 constitutive expression vector
  • Construction method of echinococcus granulosus immunogenic protein EG95 constitutive expression vector
  • Construction method of echinococcus granulosus immunogenic protein EG95 constitutive expression vector

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Embodiment 1

[0030] 1. Construction method of expression vector pGAPZαA-Eg95

[0031] 1.1 Modification of the main immunogenic protein EG95 gene of Echinococcus granulosus

[0032]On the basis of the complete EG95 protein, the signal peptide sequence of 16 amino acids was removed from the N-terminus, and 21 amino acids were truncated from the C-terminus, and the codon usage bias and codon degeneracy of the yeast expression system were compared. The Eg95 gene is codon optimized, and the modified Eg95 gene sequence (5'-3') is as follows:

[0033] CAGGAATACAAGGGTATGGGTGTTGAGACTCGTACGACTGAGACTCCTTTGAGGAAACACTTTAACCTGACCCCAGTTGGATCGCAAGGTATCAGACTTTCATGGGAAGTGCAACATCTGTCTGATTTGAAAGGTACCGACATCTCCCTGAAAGCTGTCAATCCTTCCGATCCCTTGGTTTACAAGAGACAAACAGCAAAGTTTAGCGACGGTCAGCTAACAATTGGAGAGTTAAAACCATCTACTTTGTATAAGATGACCGTTGAAGCTGTGAAGGCTAAAAAGACAATTCTTGGCTTCACTGTGGATATTGAGACTCCAAGAGCCGGAAAAAAGGAAAGTACTGTAATGACA

[0034] The amino acid sequence of the EG95 protein is as follows:

[0035] QEYKGMGVETRTTETPLRK...

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Abstract

The invention provides an echinococcus granulosus immunogenic protein EG95 constitutive expression vector and a pichia pastoris genetically engineered bacterium containing the expression vector. The applicant successfully realizes secretory expression of EG95 protein in a pichia pastoris constitutive expression system, the concentration of the protein expressed by a triangular flask shaking tablereaches 0.5g / L, and the purity of the target protein reaches 90% or above. Westernblotting tests prove that the expressed recombinant protein has good reactogenicity and specificity (no reaction withother recombinant protein antibodies) with existing sheep echinococcosis EG95 subunit vaccine immune positive serum. The engineered bacterium capable of efficiently and stably secreting and expressingthe EG95 recombinant protein is screened out from constructed p-GAPZ alpha A-EG95 recombinant plasmid transformed pichia pastoris SMD1168, no inducer is needed, the production period is remarkably shortened, the production cost is remarkably reduced, and the production complexity is remarkably reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a constitutive expression vector of Echinococcus granulosus immunogenic protein EG95. Background technique [0002] Echinococcosis, commonly known as echinococcosis, is a serious zoonotic disease caused by the larvae of the Echinococcus tapeworm, Echinococcus parasitizing in the liver, lungs and other tissues of animals (including humans). Suffering from parasitic diseases is one of the global important public health problems. my country is one of the countries with the highest incidence of echinococcosis. Echinococcosis is classified as a globally notified infectious disease by the World Organization for Animal Health (OIE), and is listed as one of 17 neglected tropical diseases (Neglected tropical diseases, NTDs) by the World Health Organization (WHO). Echinococcosis not only affects poor people in developing countries and reg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/81C12N15/66C12N1/19C12R1/84
CPCC07K14/43554C12N15/815C12N2800/22
Inventor 贾万忠闫鸿斌李立郭爱民李建秋李文静李文卉李乐张林生张念章付宝权
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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