Duck Tembusu virus E protein-LTB fusion protein and application thereof

A technology of duck Tembusu virus and fusion protein, which is applied in antiviral agents, virus antigen components, hybrid peptides, etc.

Inactive Publication Date: 2015-02-04
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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At the same time, as a new virus, there is no

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  • Duck Tembusu virus E protein-LTB fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 The acquisition of duck Tembusu virus novel fusion protein LTB-Es

[0017] 1. Using biological software to analyze the epitope of the envelope protein E of duck Tembusu representative strain ZJ-6 (GenBank accession number: JF459991.1), and select the Domain III position with strong antigenicity of E protein (303-410aa) Splicing with the LTB sequence, and designing Lingker (GGGSGGGS) in the middle for connection, a new type of fusion protein LTB-Es of Duck Tambusu virus E protein Domain III and LTB was obtained. The amino acid sequence of the encoded protein is SEQ ID NO: 1, The rare codons of Escherichia coli were optimized using the online biological software DNAWorks to obtain the nucleotide sequence SEQ ID NO:2 of the fusion protein LTB-Es.

[0018] The nucleotide sequence of the newly obtained fusion protein LTB-Es was subjected to whole gene synthesis, and BamH I and Hind III restriction sites were added to both ends respectively.

Embodiment 2

[0019] The construction of embodiment 2 genetic engineering protein expression vector and the acquisition of engineering bacteria

[0020] 1. The fusion protein LTB-Es gene synthesized by the above-mentioned whole gene was digested with BamH I and Hind III, and then ligated into the corresponding restriction site of pET30a vector to construct pET30a / Es expression vector.

[0021] 2. Use CaCl 2 The pET30a / Es expression vector was transformed into Escherichia coli BL21(DE3), spread on an agar plate containing 50 μg / ml kanamycin, and cultured overnight at 37°C. 10 single colonies were selected to extract plasmids, and the positive colonies verified by BamH I and Hind III double enzyme digestion were further sequenced and identified. The positive clones verified by sequencing were fermented and cultured in LB medium to 0.6-0.8 hours and induced by adding 0.3mM IPTG for 4-5 hours, and the bacteria were collected by centrifugation for SDS-PAGE electrophoresis, and uninduced bacte...

Embodiment 3

[0022] Example 3 Fermentation, Purification and Procedures for Preparation of Duck Tembusu Virus Genetic Engineering Subunit Vaccine

[0023] 1 strain of bacteria

[0024] 1.1 The strain used for manufacturing is duck Tembusu virus genetically engineered subunit vaccine producing strain LE; the virulent strain used for detection is Tembusu SD strain from goose.

[0025] 1.2 Standards for strains used in production

[0026] 1.2.1 Morphological and biochemical properties

[0027] Cultivate overnight on the LB agar plate containing kanamycin, round, smooth colonies with neat edges, protrusions, milky white luster appear on the culture plate, and after Gram staining, they are Gram-negative short bacilli under the microscope; The biochemical test results were glucose fermentation +, indole test +, methyl red test +, VP -, citric acid utilization test -.

[0028] 1.2.2 Culture characteristics It can grow in the medium containing kanamycin.

[0029] 1.2.3 Identification test

...

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Abstract

The invention aims to provide a duck Tembusu virus gene engineering subunit vaccine which is prepared by the following steps: screening to obtain duck Tembusu virus E protein with Domain III structure field of dominant antigen epitope, constituting a novel fusion protein LTB-Es from Lingker and enterotoxin LTB, and preparing the duck Tembusu virus gene engineering subunit vaccine by using the fusion protein as the antigen. The amino acid sequence of the coding protein of the duck Tembusu virus novel fusion protein LTB-Es is SEQ ID NO:1, and one of the nucleotide sequences is SEQ ID NO:2. The prokaryotic expression vector is utilized to construct the Escherichia coli BL21(DE3) host bacterium capable of expressing the duck Tembusu virus novel fusion protein LTB-Es. The gene engineering subunit vaccine prepared from the purified recombinant expressed protein can enable the immunized duck group to obtain immunoprotection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion protein of duck Tembusu virus E protein and LTB and application thereof. Background technique [0002] my country is a big duck raising country in the world. The annual production of duck meat accounts for about 70% of the world's total. It increased from 1.866 million tons in 2000 to 2.581 million tons in 2008, with an average annual growth rate of about 3.8%, much higher than the world's. Average. Duck Tembusu virus is a new virus that was first discovered in China in 2010. It mainly causes duck feed intake and egg production to drop sharply, ovarian bleeding, neurological symptoms, etc. Therefore, the disease is also called duck hemorrhagic ovary at the early stage. Inflammation, egg drop syndrome, etc. Through virus isolation and gene analysis, the duck Tembusu virus is currently located in the Flaviviridae, Flavivirus genus, and Entayavirus group of mosquit...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/70C07K19/00A61K39/12A61P31/14
Inventor 侯竹美
Owner QINGDAO AGRI UNIV
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