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A kind of human-mouse chimeric anti-hev whole molecule igg and its application

A molecular and nucleic acid molecule technology, which is applied in the field of biopharmaceuticals and can solve problems such as humanized antibodies yet

Active Publication Date: 2019-11-08
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The antibody prepared by the present invention can effectively combine with HEV, block the infection of the virus, and shows good neutralization properties, and there is no patent report of a humanized antibody with related functions

Method used

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  • A kind of human-mouse chimeric anti-hev whole molecule igg and its application
  • A kind of human-mouse chimeric anti-hev whole molecule igg and its application
  • A kind of human-mouse chimeric anti-hev whole molecule igg and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1. The cultivation and preparation of murine-derived anti-HEV capsid protein hybridoma cells

[0032] According to the gene sequence of type I-IV HEV, the corresponding sequence of ORF2 was synthesized and cloned into the prokaryotic expression vector, and the corresponding antigen was expressed and purified in prokaryotic.

[0033] The recombinant protein was used as the immunogen to subcutaneously inject 200 μg each time into the abdomen of pure-line BALB / c mice for five times in total, and intraperitoneal booster immunization was carried out seven days before the last immunization was cell fusion. On the day of fusion, the mouse spleen was taken, and a single-cell suspension was prepared with DMEM medium (GIBCO, USA). In the presence of 50% PEG (PH 8.0), the spleen cells and SP2 / 0 mouse myeloma cells were fused, and used HAT selective medium (DMEM medium 98ml, HT stock solution 1ml, A stock solution 1ml) was cultivated for 7 days, and replaced with HT medi...

Embodiment 2

[0034] Example 2. Screening, preparation and identification of mouse-derived anti-HEV antibodies

[0035] The enzyme-linked immunoassay (ELISA) detection and screening was carried out according to the growth status of the hybridoma cells, and the specific method is as follows. The cells in the positive wells were cloned and cultured again. After 3 times of subcloning, when the supernatant of the monoclonal cells in all the wells was positive for anti-HEV, several wells were taken for expansion and cultured and some of them were frozen.

[0036] The specific steps for screening anti-HEV positive monoclonal antibodies by ELISA method are as follows:

[0037] (1) Purify the prokaryotic expressed antigen, coat the 96-well plate with ELISA, dilute to 2 μg / ml with coating solution (0.1M carbonate buffer, pH9.6), add 100 μl to each well, and overnight at 4°C;

[0038] (2) Wash 5 times with PBST washing solution (PBS containing 0.05% Tween), add 3% BSA (200 μl / well) to block, incubat...

Embodiment 3

[0048] Example 3. Preparation, expression and purification of human-mouse chimeric anti-HEV whole molecule IgG

[0049] The hybridoma cells were recovered, and the total RNA was extracted according to the instructions of the Trizol Reagent Kit, and the cDNA was obtained by RT-PCR. According to the statistical data of the IgG BLAST database, 19 VH forward and 17 Vκforward primers, 4 VH reverse and 3 Vκreverse primers were designed respectively. The primer sequences are as follows:

[0050] Vκforward primers

[0051] Vκ-1: 5'-GGGCCCAGGCGGCCGAGCTCGAYATCCAGCTGACTCAGCC-3'

[0052] Vκ-2: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTTCTCWCCCAGTC-3'

[0053] Vκ-3: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTGMTMACTCAGTC-3'

[0054] Vκ-4: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTGYTRACACAGTC-3'

[0055] Vκ-5: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTRATGACMCAGTC-3'

[0056] Vκ-6: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTMAGATRAMCCAGTC-3'

[0057] Vκ-7: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTCAGATGAYDCAGTC-3'

[0058] Vκ-8: 5'-GGGCCCAGGCGGCCGAGCTC...

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Abstract

The invention belongs to the field of biological pharmacy, and discloses a human-mouse chimeric anti-HEV whole molecule IgG. The human-mouse chimeric anti-HEV whole molecule IgG particularly comprisesa light chain variable region and a heavy chain variable region, wherein the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO. 1, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2. The invention further discloses a DNA molecule, expression vector, host cell and application of the whole molecule IgG antibody. By mainly utilizinga mouse immune to HEV capsid protein produced through prokaryotic expression recombination, capsid protein expressed by a purified open reading frame (ORF2) and a virus blocking screening method areadopted for multi-time screening, and the recombination human-mouse chimeric anti-HEV protection whole molecule IgG is prepared. The chimeric antibody can effectively recognize HEV and is effectivelyused for detecting the HEV and making passive protection cell strains immune to infection of the HEV.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a human-mouse chimeric anti-HEV capsid full-molecule IgG antibody and its application, and also relates to the DNA molecule, expression vector and host cell of the above-mentioned full-molecule IgG antibody. Background technique [0002] Monoclonal antibody technology has played a huge role in the diagnosis and treatment of diseases and has greatly improved the level of medical technology. However, the conventional mouse or other animal-derived antibodies can cause strong heterogeneous immune responses due to their origin. Very restrictive. Although a vaccine for HEV immune protection has been successfully developed in China, there is still no antibody that can be used for immune passive protection. HEV is a typical zoonotic pathogen, and there is still a high infection rate and persistent infection in both developed and developing countries. Therefore, in addition to the active...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
Inventor 杨永林冯振卿李冰唐奇张明顺
Owner NANJING MEDICAL UNIV
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