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Rotavirus VP6 gene with amino acid sequence variation and immunochromatographic test strip

A technology of immunochromatography test paper and rotavirus, which is applied in the direction of virus, virus peptide, virus/bacteriophage, etc., and can solve the problem that vaccines cannot effectively prevent rotavirus

Pending Publication Date: 2019-11-19
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the research of rotavirus recombinant antigen subunit vaccines currently used, due to the diversity of rotavirus genomes, the prepared vaccines cannot effectively prevent rotaviruses with different genetic backgrounds.

Method used

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  • Rotavirus VP6 gene with amino acid sequence variation and immunochromatographic test strip
  • Rotavirus VP6 gene with amino acid sequence variation and immunochromatographic test strip
  • Rotavirus VP6 gene with amino acid sequence variation and immunochromatographic test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Construction, expression and activity identification of rotavirus VP6 recombinant gene

[0020] Refer to the gene sequence of VP6 protein published in GenBank to construct an antigenic gene sequence, and use DNAstar software to analyze the amino acid sequence, theoretical molecular weight and isoelectric point expressed by the recombinant sequence, and add a BamHI (GGATCC) restriction site at the 5' end , 3, adding TAA termination sequence and Hind at the end (GAATTC) restriction site, the recombinant gene sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the gene sequence of VP6 is shown in SEQ ID NO.1.

[0021] Use T4 ligase to connect the VP6 gene sequence fragment with the pET-32a(+) empty vector to construct the pET-28a(+)-r VP6 recombinant plasmid, transform the recombinant plasmid into Escherichia coli BL21 (DE3), and coat it with The kanapenicillin antibody LB solid medium was cultured overnight at 37°C, and then several singl...

Embodiment 2

[0024] Example 2 Preparation of Colloidal Gold Immunochromatography Test Strips

[0025] 2.1. Protein expression: The recombinant protein VP6 was prepared, expressed and purified as described in Example 1.

[0026] 2.2. Spotting on nitrocellulose membrane: Dilute VP6 to 0.5mg / ml for marking the detection line (T line), dilute the mouse anti-His tag monoclonal antibody to 0.5mg / ml for quality control The line (C line) is dashed. Stick the NC film on the bottom plate, and then use the colloidal gold spotting system to draw the line. The amount of the line is 1µl / cm. Desiccant, valid for 15 months.

[0027] 2.3 Firing of colloidal gold

[0028] Burn colloidal gold according to the sodium citrate reduction method: soak all glassware used for firing and preserving colloidal gold in an acid tank for 24 hours, take them out, rinse them and dry them for later use. Take 100mL of deionized water and 1mL of 1% chloroauric acid, and heat to boiling. Add 1.5 mL of 1% trisodium citrate...

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Abstract

The invention provides a rotavirus VP6 gene with amino acid sequence variation and an immunochromatographic test strip. The rotavirus colloidal gold immunochromatographic test strip is provided with acarrier plate, a sample pad, a colloidal gold binding pad, a layer plate film, a detection line, a quality control line and an absorption pad. A recombinant VP6 protein is used as a detection antigen, colloidal gold particles are prepared by using the most classic trisodium citrate reduction method, development of a rotavirus immunochromatographic test strip is completed, specific binding with aporcine rotavirus positive serum can occur, and a rapid diagnostic method for the diagnosis of the rotavirus is built preliminarily.

Description

technical field [0001] The invention belongs to the technical field of antigen preparation, and in particular relates to a novel rotavirus vaccine antigen, that is, a rotavirus VP6 gene with a mutated amino acid sequence, and a rotavirus colloidal gold rapid detection test strip containing the antigen and its application. Background technique [0002] Rotavirus (Rotavirus, RV) belongs to the Reoviridae family and the genus Rotavirus. It was first discovered by Bishop et al. in 1973. It is named after its shape like a wheel spoke under an electron microscope. The virus is the main pathogen that causes viral diarrhea in humans, mammals, and birds. The high-risk group for virus infection is infants aged 6 months to 3 years. The clinical symptoms are diarrhea and vomiting that last for many days, accompanied by fever, Hematochezia, severe cases can die due to complications such as dehydration, toxic myocarditis and pneumonia. In my country, piglet diarrhea caused by porcine rota...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/558G01N33/569C07K14/005
CPCG01N33/531G01N33/56983G01N33/558C07K14/005C12N2720/12322
Inventor 朱传刚周志平冒丽刘冀江嘉欣吴思敏李嘉静纪荣毅沈元曦
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI