Yak-derived Rotavirus recombinant VP6 protein antigen and application thereof

A rotavirus and protein antigen technology, applied in the field of genetic engineering, can solve the problem of not reducing the incidence of virus transmission, and achieve the effects of being suitable for popularization, short production cycle and improving production efficiency

Inactive Publication Date: 2018-10-23
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is currently no specific drug for rotavirus, improving sanitation and providing clean water cannot reduce the incidence and prevent the spread of the virus

Method used

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  • Yak-derived Rotavirus recombinant VP6 protein antigen and application thereof
  • Yak-derived Rotavirus recombinant VP6 protein antigen and application thereof
  • Yak-derived Rotavirus recombinant VP6 protein antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of yak-derived rotavirus VP6 protein antigen

[0046] 1. Amplification of VP6 gene: Take an appropriate amount of yak-derived RVMD8 strain virus liquid, extract total RNA according to the QIAamp Viral RNA MiniKit (50) kit, and synthesize cDNA according to the instructions of the SuperScirptTMIII First-Stand Synthesis System for RT-PCR reverse transcription kit , using VP6 amplification primers to amplify two sequences of a gene fragment with a size of about 407bp (located between 931bp and 1338bp) and a gene fragment with a size of about 937bp (located between 21bp and 958bp). Use the Contig software to splice the two gene fragments together to obtain a complete ORF sequence of the VP6 gene, which is 1191bp in size. Its sequence is shown in SEQ ID NO.1, encoding 397 amino acids, and its amino acid sequence is represented by SEQ ID NO. .3 shown.

[0047] 2. Optimize the spliced ​​VP6 gene sequence to make its codon preference close to that of Esche...

Embodiment 2

[0052] The establishment of embodiment 2 indirect ELISA detection method

[0053] 1. ELISA operating procedure Dilute the purified recombinant VP6 protein with 50 mM pH 7.6 phosphate buffer and coat it on an ELISA 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, 37° 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 5 times, HRP-labeled goat anti-bovine IgG diluted 1:2500 with 1% bovine serum albumin (BSA) was added, 100 μL / well, 37°C for 1h. Discard the liquid in the plate, add TMB chromogenic solution after washing 5 times, 100 μL / well, develop color at 37°C for 15 minutes i...

Embodiment 3

[0095] Example 3 Immunization of mice with rotavirus recombinant VP6 protein can make it produce detectable serum detection antibodies

[0096] Twenty-five female BALB / c mice of SPF grade from 6 to 8 weeks were randomly divided into two groups, including 15 mice in the immune group and 10 mice in the blank control group. Before immunization, the blood of all mice was collected from the orbit, and serum IgG was detected by ELISA method. The vaccine is immunized with recombinant VP6 protein and mucosal adjuvant, and the immunization method is nasal drop. The dose of VP6 protein immunization at each immunization point is 20 μg / monkey, and the second immunization is carried out at an interval of 3 weeks after the first immunization. During immunization, after the mice were anesthetized with ether, 25 μl of the vaccine was dripped into each nostril with a micropipette gun, and the control group was immunized with an equal volume of sterile PBS. Orbital blood was collected once a w...

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Abstract

The invention discloses a yak-derived Rotavirus recombinant VP6 protein antigen and application thereof. According to the yak-derived rotavirus recombinant VP6 protein antigen and the application thereof, the nucleotide sequence of yak-derived rotavirus recombinant VP6 protein is shown in SEQ ID NO.1; the recombinant VP6 protein has good antigenicity and immunogenicity and can be efficiently expressed in a prokaryotic system; an indirect ELISA detection method is superior to a commercial detection kit, a rapid, simple, convenient and practical diagnosis method is provided for BRV antibody detection and seroepidemiology investigation in China, and the problems that an existing diagnosis method for Bovine Rotavirus is time-consuming and labor-consuming, and biological reagents and instruments are expensive can be solved; and meanwhile, a subunit vaccine has a good protection effect in mice and can be used for preventing diarrhea caused by BRV.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a yak-derived rotavirus recombinant VP6 protein antigen and its application. Background technique [0002] Bovine Rotavirus (Bovine Rotavirus, BRV), also known as Bovine Epidemic Diarrhea, belongs to Reoviridae and the genus Rotavirus, mainly causing calf diarrhea. The disease was first discovered in China in 1974, and its clinical feature is mainly jet-shaped watery diarrhea. Bovine rotavirus is mainly transmitted through the fecal-oral route, and generally 10 to 100 virions can cause infection. Therefore, rapid and accurate diagnosis is of great significance for the identification of pathogens, effective prevention and control of disease transmission. [0003] Common detection methods for bovine rotavirus mainly include virus isolation, electron microscope observation, polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay (Enzyme Linked ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/14C12N15/70C12N15/46G01N33/68G01N33/543A61K39/15A61P31/14A61P1/12
CPCA61K39/12A61K2039/543A61K2039/552A61P1/12A61P31/14C07K14/005C12N15/70C12N2720/12322C12N2720/12334G01N33/543G01N33/68G01N2333/14
Inventor 汤承岳华罗雪王远微
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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