Bovine Newbutton virus recombinant VP1 gene, recombinant protein and application thereof

A virus and gene technology, applied in the field of genetic engineering, can solve the problems of restricting the development of NeV antibodies, lack of serological detection methods, and not suitable for large-scale operations, and achieve the effect of being suitable for popularization and use, shortening expression time, and time-saving cultivation methods.

Pending Publication Date: 2021-05-28
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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Problems solved by technology

[0005] At present, pathogenic detection methods such as RT-PCR are widely used in the screening and detection of NeV. The method involves long time-consuming nucleic acid extraction, complicated operations and is not suitable for large-scale operations; there is a lack of serological detection for large-scale screening of antibodies containing Neurubivirus method, which limits the development of research on NeV and related antibodies

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  • Bovine Newbutton virus recombinant VP1 gene, recombinant protein and application thereof
  • Bovine Newbutton virus recombinant VP1 gene, recombinant protein and application thereof
  • Bovine Newbutton virus recombinant VP1 gene, recombinant protein and application thereof

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Embodiment 1

[0054] The present embodiment provides a kind of method for preparing bovine Newbury virus recombinant VP1 gene protein, comprising the following steps:

[0055] 1. Amplification of the VP1 gene: Take an appropriate amount of nucleic acid-positive feces samples of bovine Newbull BO / LN-13 / 18 / CH strain virus (GenBank accession number MH718898) preserved by the Laboratory of Veterinary Medicine, Southwest University for Nationalities, and extract them by conventional methods The total RNA was reverse-transcribed to synthesize cDNA, amplified by RT-PCR, and the gene fragments were spliced ​​using biological software to obtain a complete ORF sequence of the VP1 gene with a size of 1647bp. The nucleic acid sequence is shown in SEQ ID NO: 1. It encodes 549 amino acids, and its amino acid sequence is shown by SEQ ID NO:2.

[0056] 2 Optimize the obtained VP1 gene sequence, without changing any amino acid, make its codon preference close to that of Escherichia coli, the optimized VP1 c...

Embodiment 2

[0062] The present embodiment provides an indirect ELISA detection method of a non-diagnostic bovine Newburgh virus antibody, comprising the following steps:

[0063] 1ELISA operation process

[0064] Dilute the recombinant VP1 protein of the present invention with 50mmol / L pH 7.6 phosphate buffer and coat it on a microtiter plate, 100 μL / well, overnight at 4°C. The coating solution was discarded the next day, and washed three times with PBST, 200 μL / well. Add 2% bovine serum albumin (BSA) as a blocking solution, 100 μL / well, 7° C. for 1 h. The coating solution was discarded, and after washing 3 times, the negative and positive sera were diluted in proportion with 1% bovine serum albumin (BSA), respectively, and added to the microtiter plate, 100 μL / well, 37°C for 1h. The liquid in the plate was discarded, and after washing three times, HRP-labeled rabbit anti-bovine IgG diluted with 1% bovine serum albumin (BSA) was added in a certain proportion, 100 μL / well, 37°C for 1h. ...

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Abstract

The invention provides a bovine Neurovirus VP1 gene, a recombinant protein and application of the bovine Neurovirus VP1 gene and the recombinant protein. The VP1 gene has a nucleotide sequence as shown in SEQ ID NO: 1. The amino acid sequence of the recombinant VP1 gene protein is as shown in SEQ ID NO: 2. A prokaryotic expression system constructed by using the amino acid sequences can obtain a large amount of excellent recombinant proteins in a short time, the expression time of the recombinant proteins is shortened, and the production efficiency of the recombinant proteins is improved. An indirect ELISA detection method of the bovine Neurovirus antibody is successfully established by using the expressed recombinant protein to coat an antigen, the optimal condition of the method is determined, and a rapid and simple serological method is provided for immune cattle antibody detection and epidemiological investigation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant VP1 gene of bovine Newbury virus, a recombinant protein and an application thereof. Background technique [0002] Nebovirus (NeV) is the only member of the genus Nebovirus (Nebovirus) in the Caliciviridae family (Caliciviridae). NeV-containing diarrheal feces infected newborn sterile calves after aseptic treatment, causing intestinal damage (especially in the duodenum and jejunum) and severe diarrhea, proving that it is a diarrhea-causing virus. At present, the virus has been detected in calf diarrhea fecal samples in 12 countries including Brazil, Italy, and the United States, with the detection rate ranging from 3.0% to 25.2%. Our laboratory has confirmed that NeV existing in domestic dairy cows and yaks is a new virus that causes cattle diarrhea in China; this laboratory has further investigated the epidemics of dairy cows and yaks in Xin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C07K14/08C12N15/70G01N33/569G01N33/543
CPCC07K14/005C12N15/70G01N33/56983G01N33/543C12N2770/16022G01N2333/08
Inventor 汤承岳华王远微王珍贤
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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