Antibody specially bound with Norovirus GII.4 genotype VP1 protein and/or VLP and preparation method and application of antibody

A genotype and virus technology, applied in antiviral immunoglobulin, chemical instruments and methods, botanical equipment and methods, etc., to achieve high specificity, good application prospects, and high affinity

Active Publication Date: 2019-01-25
NAT VACCINE & SERUM INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Since there is no more effective specific antibody against norovirus GII.4 genotype VP1 protein, we are looking f

Method used

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  • Antibody specially bound with Norovirus GII.4 genotype VP1 protein and/or VLP and preparation method and application of antibody
  • Antibody specially bound with Norovirus GII.4 genotype VP1 protein and/or VLP and preparation method and application of antibody
  • Antibody specially bound with Norovirus GII.4 genotype VP1 protein and/or VLP and preparation method and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Preparation and screening of embodiment 1 hybridoma cells

[0103] 1. Preparation of Immunogen

[0104] The immunogen used to prepare the monoclonal antibody of the Norovirus GII.4VP1 protein is the Norovirus GII.4VP1-VLP protein, which uses Hansenula cells to express the amino acid shown in SEQ ID NO.16 through in vitro recombination. Can be automatically assembled into VLP, which can be seen by transmission electron microscope observation figure 1 The shown virus-like particles are spherical in diameter between 20-40nm. Norovirus GII.4VP1-VLP protein was mixed with an equal volume of complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), and fully emulsified by ultrasound to prepare the corresponding immunogen.

[0105] 2. Animal immunization

[0106] On the 0th day, the immunogen mixed with CFA was used to subcutaneously inject mice at multiple points on the back, and immunize 3 mice at a dose of 0.2 mg / mouse, for a total of 4 times with an interv...

Embodiment 2

[0134] Example 2 Identification of 2E6-B3(302) Antibody

[0135] 1. Antibody acquisition

[0136] Adult BALB / c mice were selected, and pristane was inoculated intraperitoneally, 0.5ml per mouse. After 7-10 days, the 16th generation 2E6-B3(302) hybridoma cells were inoculated intraperitoneally, 1×10 per mouse 6 -2×10 6 indivual. After an interval of 5 days, when the abdomen was obviously enlarged and the skin felt tense when touched with hands, ascites was collected with a No. 9 needle.

[0137] 2. Antibody Purification

[0138] The ascitic fluid was centrifuged at 13000 rpm / min for 30 minutes to remove cell components and other precipitates, and the supernatant was collected. Purify with Protein A affinity chromatography, and finally obtain the monoclonal antibody 2E6-B3 (302) of the GII.4VP1 protein, with a concentration above 5 mg / ml.

[0139] 3. Antibody purity test

[0140] The purified antibody was subjected to 12% SDS-PAGE electrophoresis, and the result was that ...

Embodiment 3

[0150] Example 3 Identification of 2H7-C5(202) Antibody

[0151] 1. Antibody acquisition

[0152] Except for inoculating 2H7-C5(202) hybridoma cells, the same method as in Example 2-1 was used.

[0153] 2. Antibody Purification

[0154] Using the same method as in Example 2-1, the monoclonal antibody 2H7-C5 (202) to the GII.4VP1 protein was finally obtained with a concentration above 5 mg / ml.

[0155] 3. Detection of antibody purity

[0156] Using the same method as in Example 2-1, the results showed that the purity was above 95%.

[0157] 4. Determination of antibody light chain and heavy chain variable region gene sequences

[0158] The mRNA of 2H7-C5(202) hybridoma cells was extracted, reverse-transcribed into cDNA, high-fidelity PCR amplification was performed using universal primers for the variable region, and the PCR product fragments were inserted into T vectors for DNA sequence determination. 2H7-C5( 202) variable region gene sequence: the heavy chain is shown in S...

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Abstract

The invention discloses an antibody specially bound with Norovirus GII.4 genotype VP1 protein and/or VLP. The antibody comprises CDR1, CDR2 and CDR3 regions of a heavy chain variable region shown in SEQ ID NO. 1-3 and/or The CDR1, CDR2 and CDR3 regions of the light chain variable region shown in SEQ ID NO. 4-6. The antibody or antigen binding part of Norovirus GII.4 genotype VP1 protein and/or VLPprovided by the present invention, has no cross-reaction with GI.1 genotype, is high in specificity, has the characteristic of blocking binding between Norovirus GII.4 genotype VP1 protein and/or VLPand HBGA, is high in affinity, can be used for detecting the presence or level of Norovirus GII.4 genotype VP1 protein and/or VLP in a sample developing kits, as well as treating patients and performing passive immunization on susceptible populations, and has good application prospect.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, the present invention relates to an antibody specifically binding to Norovirus GII.4 genotype VP1 protein and / or VLP, its preparation method and application. Background technique [0002] Norovirus is a globally important foodborne virus that can infect humans and animals and cause acute gastroenteritis. Whether in developed or developing countries, norovirus epidemic infection involves people of all ages and can cause explosive epidemics, which has become a non-negligible problem affecting human daily health. The first case of norovirus infection was reported in my country in 1995. For more than 20 years, there have been many outbreaks of group and sporadic norovirus infectious diarrhea all over the country, especially in semi-closed environments such as kindergartens, schools, the army, and hospitals. , causing certain social panic. Norovirus mainly comes from seafoo...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/63G01N33/68
CPCC07K16/10C07K2317/24C07K2317/54C07K2317/55C12N15/63G01N33/68G01N2333/08
Inventor 李启明梁宇侯俊伟张学峰杜丽芳张靖刘兆明栗子谦张浩唐芳侯亚楠郑凡
Owner NAT VACCINE & SERUM INST
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