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Rotavirus strain and application thereof as vaccine

A technology of sequences and amino acids, applied in the field of rotavirus strains and their use as vaccines, can solve the problems that the protection effect of vaccines needs to be improved, pollution, and cannot be used for immunodeficiency, etc.

Pending Publication Date: 2021-10-08
山东威高利彤生物制品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protective efficiency of all currently used attenuated live rotavirus vaccines is about 75%, and the protective effect of the vaccine needs to be improved; secondly, the detoxification of live attenuated rotavirus vaccines after preparation, use and immunization makes the virus in the environment Long-term survival, there are potential safety hazards, including the risk of virulence reversion, intussusception, etc.; the risk of exogenous factor contamination in the preparation process of the attenuated live vaccine and the disadvantages that it cannot be used for immunodeficiency or immunosuppressive treatment groups, so It is of great significance and application prospect to develop inactivated rotavirus vaccine

Method used

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  • Rotavirus strain and application thereof as vaccine
  • Rotavirus strain and application thereof as vaccine
  • Rotavirus strain and application thereof as vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Isolation and characterization of embodiment 1LSR-8 strain

[0121] Get a virus sample

[0122] Collect diarrheal excrement samples from children with pediatric diarrhea in the hospital, resuspend in 0.01M PBS solution with pH 7.2, centrifuge at 8000rpm and 4°C for 20 minutes to get the supernatant, and test positive for rotavirus by colloidal gold kit, use 0.22 Sterilize and filter with a μm filter, aliquot into 1.5ml sterile centrifuge tubes, and store at -80°C for later use.

[0123] Plaque purification of virus morphology of LSR-8 strain

[0124] Add trypsin to the rotavirus harvest solution and activate it in a constant temperature water bath at 37°C for 1 hour, then dilute to 10 times with maintenance solution 10 times. -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 .

[0125] MA104 cells were inoculated into a 6-well plate, and when the cells grew into a dense monolayer, the growth medium was discarded, and the cell surface was washed 3 times with ...

Embodiment 2

[0138] Adaptability and passage of embodiment 2LSR-8 strain

[0139] Adaptation of LSR-8 Strain Virus on VERO Cells

[0140] Add trypsin to the rotavirus harvest solution and activate it in a constant temperature water bath at 37°C for 1 hour.

[0141] Take the VERO cells that have grown into a dense monolayer in the T25 flask, discard the growth medium, and wash the cell surface with the maintenance solution. Aspirate the remaining maintenance solution, add 500ul of activated rotavirus solution, absorb at 37°C for 60-120 minutes, add 10ml of maintenance solution containing trypsin with a final concentration of 4μlg / ml, and incubate at 37°C, 5% CO 2 Cultured in medium to observe the cytopathic effect CPE.

[0142] When cells above 75% CPE appeared, virus was harvested. Freeze and thaw the harvested virus culture solution twice at -20°C and 25°C alternately, centrifuge at 8000 rpm and 4°C for 20 minutes, take the supernatant, and inoculate VERO cells for passage in the same ...

Embodiment 3

[0143] The identification method of embodiment 3LSR-8 strain

[0144] LSR-8 strain virus titer determination:

[0145] Add trypsin to the rotavirus harvest solution and activate it in a constant temperature water bath at 37°C for 1 hour, then dilute it to 10 times with the maintenance solution. -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 .

[0146] MA104 cells were inoculated into a 6-well plate, and when the cells grew into a dense monolayer, the growth medium was discarded, and the cell surface was washed 3 times with maintenance medium. Aspirate the remaining maintenance solution, add 500 μl of activated rotavirus solution, absorb at 37°C for 60-120 minutes, discard the virus solution, add 3ml of maintenance solution containing 0.3% agar, each dilution gradient is 2 wells, wait After the agar has solidified, transfer it to the incubator. After 72 hours, a maintenance solution containing 0.006% neutral red and 0.3% agar was covered on the original agar laye...

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Abstract

The invention relates to the field of viruses, in particular to separation, culture and identification of a group A rotavirus G9P[8] strain and application of the group A rotavirus G9P[8] strain as a vaccine. Specifically, the invention relates to a group A rotavirus seed LSR-8 strain, and relates to a group A rotavirus seed LSR-8 strain; the total amino acid sequences of the group A rotavirus seed LSR-8 strain are shown as SEQ ID No.1 to SEQ ID No.11; and the total nucleic acid sequences of the group A rotavirus seed LSR-8 strain are shown as SEQ ID No.12 to SEQ ID No.22. The invention further relates to application of the LSR-8 strain in production of inactivated human rotavirus vaccines, human rotavirus attenuated live vaccines and combined vaccines; and the LSR-8 strain can be used for preventing, relieving or treating rotavirus infection and diseases caused by rotavirus infection, such as rotavirus gastroenteritis, diarrhea and the like.

Description

technical field [0001] The invention relates to the field of viruses, in particular to rotavirus strains and their use as vaccines. Background technique [0002] Group A rotavirus can cause more than 90% of human rotavirus gastroenteritis. In 2016, about 128,500 children under the age of 5 died of rotavirus infection worldwide, and children under the age of 5 worldwide were infected with rotavirus About 260 million people. Group A rotaviruses are highly contagious zoonotic pathogens that pass the fecal-oral route and can also be transmitted through the respiratory tract in the form of aerosols. As far as the infection rate and incidence of rotavirus are concerned, there is no significant difference between developed countries and developing countries. Differences in materials and living environments are not important factors affecting the inhibition of rotavirus infection. There is no specific treatment for rotavirus infection. Vaccine is the first choice to prevent and co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/14C12N15/46C12N7/00G01N33/569G01N33/58A61K39/15A61K39/39A61K38/16A61P1/00A61P1/12A61P31/14C12R1/93
CPCC07K14/005C12N7/00G01N33/56983G01N33/581A61K39/12A61K39/39A61P31/14A61P1/00A61P1/12C12N2720/12322C12N2720/12321C12N2720/12334G01N2333/14G01N2469/10A61K2039/5252A61K38/00Y02A50/30
Inventor 倪世利邢苗苗李慧娟于菲邹水燕曲衍洪孙萌
Owner 山东威高利彤生物制品有限公司
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