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Expression vector, preparation method thereof, PEDV-S1 (Porcine Epidemic Diarrhea Virus-S1) protein, and PEDV-S1 protein-containing indirect ELISA detection kit

A technology of PEDV-S1 and expression vector, which is applied in the field of indirect ELISA detection kits, can solve the problems of time-consuming, labor-intensive, low purity and specificity of S1 protein, and achieve the effect of low cost, good antigenicity and easy purification

Active Publication Date: 2018-02-06
ZHEJIANG UNIV +1
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  • Abstract
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Problems solved by technology

[0007] The first object of the present invention is to provide an expression vector expressing porcine epidemic diarrhea virus S1 protein, the second object of the present invention is to provide the preparation method of the above-mentioned expression vector, the third object of the present invention is to provide a The porcine epidemic diarrhea virus S1 protein prepared by the above method is used to alleviate the technical problems in the prior art that the expression and purification of the S1 protein based on the prokaryotic expression system is time-consuming, labor-intensive, and the expression level, purity and specificity are low

Method used

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  • Expression vector, preparation method thereof, PEDV-S1 (Porcine Epidemic Diarrhea Virus-S1) protein, and PEDV-S1 protein-containing indirect ELISA detection kit
  • Expression vector, preparation method thereof, PEDV-S1 (Porcine Epidemic Diarrhea Virus-S1) protein, and PEDV-S1 protein-containing indirect ELISA detection kit
  • Expression vector, preparation method thereof, PEDV-S1 (Porcine Epidemic Diarrhea Virus-S1) protein, and PEDV-S1 protein-containing indirect ELISA detection kit

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preparation example Construction

[0040] The present invention also provides a method for preparing the above-mentioned expression vector, comprising:

[0041]Using the codon-optimized PEDV-S1 gene (SEQ ID NO.1) as a template, PEDV-S1-F1 (SEQ ID NO.2) and PEDV-S1-R1 (SEQ ID NO.3) as a primer combination for PEDV- The S1 gene was amplified by PCR to obtain the PEDV-S1 gene containing EcoRI and XhoI restriction sites at both ends, and the PEDV-S1 gene containing EcoRI and XhoI restriction sites was cloned into the eukaryotic expression vector pFuse-hLgG1-Fc1 The EcoRI / XhoI site was used to obtain the pFuse-PEDV-S1-Fc eukaryotic expression vector expressing porcine epidemic diarrhea virus S1 protein.

[0042] In the eukaryotic expression vector for expressing porcine epidemic diarrhea virus S1 protein provided by the present invention, the S1 gene sequence used has been codon-optimized in combination with the eukaryotic expression vector. Therefore, compared with the prior art, the expression pig provided by the ...

Embodiment 1

[0066] The construction of embodiment 1 eukaryotic expression system and the preparation of PEDV-S1 protein

[0067] 1. Construction of S1 gene expression vector

[0068] 1.1 Primer design

[0069] Referring to the PEDV (GenBank accession no.KU558701) strain in GenBank, design and synthesize a pair of specific primers, wherein PEDV-S1-F1 is shown in SEQ ID NO.2, and PEDV-S1-R1 is shown in SEQ ID NO.3 , primers were synthesized by Huada Biological Co., Ltd.

[0070] 1.2 Construction of PEDV-S1-Fc recombinant plasmid

[0071] Using the codon-optimized recombinant plasmid expressing PEDV-S as a template (constructed and stored in our laboratory), the amplified PEDV-S1 and pFuse-hLgG1-Fc1 were digested with EcoRI and XhoI respectively, and ligated to construct pFuse- PEDV-S1-Fc expression plasmid. Recombinant plasmids identified as positive (such as figure 1 shown) were sent to Huada Biological Company for sequence determination, and software was used to analyze and compare t...

Embodiment 2

[0077] Example 2 Construction of insect expression system and preparation of PEDV-S1 protein

[0078] 1. Construction of pFastbac1-PEDV-S1 recombinant plasmid

[0079] 1.1 Primer design

[0080] Through sequence alignment analysis, use the biological software DNAstar and Primer Premier to design primers, and add their respective restriction sites to the primers, wherein PEDV-S1-BamHIF is shown in SEQ ID NO.4, PEDV-S1-XhoIR-6His As shown in SEQ ID NO.5.

[0081] 1.2 Target fragment cloning

[0082] Using conventional PCR, amplify the target sequence PEDV-S1 containing preset restriction sites

[0083] (1) Add the following reagents to a 200 μL PCR reaction tube:

[0084] Reactant

Dosage (μL)

Q5High-Fidelity 2×Master Mix

25

10μM Forward Primer

2.5

10μM Reverse Primer

2.5

Template DNA

1

Nuclease-Free Water

19

[0085] (2) After mixing thoroughly, perform a brief low-speed centrifugation.

[0086] (3) Rea...

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Abstract

The invention provides an expression vector, a preparation method thereof, PEDV-S1 (Porcine Epidemic Diarrhea Virus-S1) protein, and a PEDV-S1 protein-containing indirect ELISA (Enzyme-Linked Immuno-Sorbent Assay) detection kit, and relates to the technical field of molecular biology. The expression vector for expressing the PEDV-S1 protein provided by the invention is constructed by a eukaryoticexpression vector or an insect expression vector. The PEDV-S1 which is constructed by applying the eukaryotic expression vector for expressing the PEDV-S1 provided by the invention is high in expression quantity, easy to purify and good in antigenicity. Moreover, the ELISA detection kit which is constructed by taking the S1 protein provided by the invention as a coating antigen can accurately detect an anti-porcine epidemic diarrhea virus antibody in a clinical sample. Meanwhile, the kit disclosed by the invention is high in specificity, high in sensitivity, simple, quick, simple in preparation method and low in cost, provides a new choice for the diagnosis, general survey and immune monitoring of the PEDV, and has a good clinical application prospect.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an expression vector and a preparation method thereof, PEDV-S1 protein and an indirect ELISA detection kit containing the protein. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), and is characterized by diarrhea, vomiting, dehydration and high lethality to suckling piglets. sexually transmitted intestinal diseases. As of 2017, PED has spread to most pig-raising countries in the world, causing huge economic losses to the global pig industry, and has become a common concern of the pig industry worldwide and needs to be solved urgently. [0003] At present, the clinical diagnosis methods for PEDV are mainly ELISA (enzyme-linked immunosorbent assay), which detects antigens or specific antibodies to evaluate the PEDV vaccine situation in pig farms a...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 黄耀伟雷喜梅赵鹏伟
Owner ZHEJIANG UNIV
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