Antibody capable of realizing specific binding with norovirus GI.1 gene type VP1 protein and/or VLP, and preparation method and applications thereof

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

Active Publication Date: 2019-01-11
NAT VACCINE & SERUM INST +1
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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 the VP1 protein of the norovirus GI.1 genotype, we are looking for such an antibody and applying it to the diagnosis and detection of norovirus GI.1 genotype infection very necessary

Method used

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  • Antibody capable of realizing specific binding with norovirus GI.1 gene type VP1 protein and/or VLP, and preparation method and applications thereof
  • Antibody capable of realizing specific binding with norovirus GI.1 gene type VP1 protein and/or VLP, and preparation method and applications thereof
  • Antibody capable of realizing specific binding with norovirus GI.1 gene type VP1 protein and/or VLP, and preparation method and applications thereof

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 GI.1VP1 protein is the Norovirus GI.1VP1-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. The norovirus GI.1VP1-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 inject mice subcutaneously at multiple points on the back, and immunize 3 mice at 0.2 mg / mouse, immunizing 4 times in total, with an interval ...

Embodiment 2

[0134] Example 2 Identification of 4H4-E6(401) 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 4H4-E6 (401) 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. The protein A affinity chromatography was used for purification to finally obtain the monoclonal antibody 4H4-E6 (401) of the GI.1VP1 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 r...

Embodiment 34

[0150] Example 3 Identification of 4G2-D1-B10(101) Antibody

[0151] 1. Antibody acquisition

[0152] Except for inoculating 4G2-D1-B10(101) 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 4G2-D1-B10 (101) to the GI.1VP1 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 heavy chain variable region gene sequence

[0158] Extract the mRNA of 4G2-D1-B10(101) hybridoma cells, reverse transcribe it into cDNA, perform high-fidelity PCR amplification with universal primers for the variable region, and insert the PCR product fragment into the T vector for DNA sequence determination, 4G2- The variable region gene sequence of D1-B10(101): the heavy chain is shown in SEQ ID...

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Abstract

The invention discloses an antibody capable of realizing specific binding with norovirus GI.1 gene type VP1 protein and/or VLP, and a preparation method and applications thereof. The antibody containsCDR1, CDR2 and CDR3 zones containing heavy chain variable regions represented by SEQ ID NO.1-3, and/or CDR1, CDR2 and CDR3 zones containing light chain variable regions represented by SEQ ID NO.4-6.No cross reaction of the norovirus GI.1 gene type VP1 protein and/or VLP antibody or an antigen binding part with GII.4 gene type is caused; the specificity is high; the antibody is capable of blocking binding of norovirus GI.1 gene type VP1 protein and/or VLP with HBGA; the endophilicity is high; the antibody can be used for research and development of kits used for detecting existing or contentof norovirus GI.1 gene type VP1 protein and/or VLP in samples, and treatment on patients and passive immunity on susceptible population, and is promising in 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 GI.1 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 seafood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63G01N33/68
CPCC07K16/10C07K2317/24C07K2317/54C07K2317/55C12N15/63G01N33/68G01N2333/08
Inventor 李启明唐芳张靖马智静陈实靳玉琴邵帅雷泽华张学峰梁宇侯俊伟韩子泊郑凡
Owner NAT VACCINE & SERUM INST
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