Poultry and porcine hepatitis E virus common antigen, monoclonal antibody, preparation method and application

A monoclonal antibody, antibody technology, applied in the field of biology

Active Publication Date: 2019-11-12
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still no relevant reports on the accurate identification of the common epitope and the application of the mimic peptide of the common antigenic epitope in the serological diagnosis of poultry and porcine HEV

Method used

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  • Poultry and porcine hepatitis E virus common antigen, monoclonal antibody, preparation method and application
  • Poultry and porcine hepatitis E virus common antigen, monoclonal antibody, preparation method and application
  • Poultry and porcine hepatitis E virus common antigen, monoclonal antibody, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Mouse Immunization and Preparation of Monoclonal Antibody

[0053] (1) Expression and purification of 268 amino acids at the C-terminus of avian HEV ORF2 protein

[0054] According to the Chinese isolate of avian hepatitis E virus (Hepatitis E virus, HEV, CaHEV, Genbanknumber GU954430), the primer ORF2-268-F with BamHI and XholI restriction sites was designed: CAC GGATCC CAGTATATGTACGGC (SEQ ID No: 3); ORF2-268-R: GAA CTCGAG TTAGGGTGGTGAGGGGAAT (SEQ ID No: 4), using CaHEV suspension as a template, the target gene sequence was obtained by RT-PCR method, and then the target gene and pET-28a(+) vector were digested with BamHI and XholI at the same time, T4 ligase Connected to construct the recombinant expression plasmid pET-ORF2-268. After the recombinant plasmid was identified by enzyme digestion, 804bp of the target gene fragment and 5300bp of the vector fragment were obtained. After the recombinant plasmid was sequenced, its homology with the CaHEV sequenc...

Embodiment 2

[0071] Example 2 Identification of the amino acid sequence of the antigenic epitope recognized by the monoclonal antibody

[0072] In order to further identify the amino acid sequence of the antigenic epitope recognized by monoclonal antibodies 3E8 and 1B5, the present invention utilizes a phage random ring heptapeptide display kit (NEW ENGLAND BioLab, U.S.A.), and then adopts a biological screening method to screen and pan with monoclonal antibody 3E8 and 1B5 can specifically bind to the phages, the phages are concentrated and identified, sequenced, and the obtained nucleotide sequences are deduced into amino acid sequences, and compared, the amino acid with the highest probability of occurrence may be the epitope recognized by the monoclonal antibody The amino acid sequence of the antigenic epitope is further accurately positioned by synthesizing the polypeptide.

[0073] (1) Phage titer determination

[0074] The titer of the phages from each round of panning was determine...

Embodiment 3

[0092] Example 3 The application of poultry and pig HEV common epitope mimetic peptide

[0093] In order to analyze the application of the above-identified monoclonal antibodies 3E8 and 1B5 to recognize the amino sequence of the consensus epitope, this patent synthesized and coupled the mimic peptide of the consensus epitope of hemocyanin (KLH) through Nanjing KingScript Biotechnology Co., Ltd. IPHD (3E8) and KLYMSV (1B5), and then the mimic peptide was coated with 0.01M PBS (pH7.2) buffer solution on 96-well ELISA plate (100ng / well), incubated overnight at 4°C with 0.5% Tween-20 Wash the plate 5 times with PBS washing solution (PBST) (300 μl / well), then block with blocking solution (PBST containing 2.5% skimmed milk powder) for 1 hour, after washing 5 times with PBST, add 100 μl poultry and pig HEV antibody positive and negative chicken and Pig serum (1:100), incubate at room temperature for 1 h, wash with PBST 5 times, add 100 μl (1:4000) of HRP-labeled goat anti-chicken and...

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Abstract

The invention relates to two monoclonal antibody hybridoma cell lines which secrete avian and porcine hepatitis e virus (HEV) shared epitope by virtue of a conventional hybridoma technique, wherein the lines are named 3E8 and 1B5 and the cell preservation numbers are respectively CCTCC No: C201395 and CCTCC No: C201396. The amino acid sequences of the shared epitope of the two lines identified by two monoclonal antibodies are identified by using a phage random 7 peptide library kit. The amino acid sequences are respectively V / IPHD (SEQ ID No: 1) and VKLYM (SEQ ID No: 2). Moreover, application of two mimic peptides of epitope in avian and porcine HEV serological diagnosis is analyzed. The two mimic peptides of the shared epitope of avian and porcine HEV capsid proteins disclosed by the invention can be used for rapid diagnosis of avian and porcine HEV and monitor of prevalence of disease. In addition, the two lines disclosed identify the monoclonal antibodies of the shared epitope and can be further used for rapidly diagnosing avian and porcine HEV.

Description

technical field [0001] The present invention relates to the amino acid sequence of the common antigenic epitope of two poultry and porcine hepatitis E viruses, and the monoclonal antibody capable of recognizing the above-mentioned common antigenic epitope, and also relates to the application of the mimetic peptide of the epitope and the monoclonal antibody in in vitro detection , belonging to the field of biology. Background technique [0002] Hepatitis E is an acute viral hepatitis caused by Hepatitis E virus (HEV) infection. It is mainly prevalent in developing countries such as Asia, Africa, and Latin America, with occasional cases reported in developed countries. Hepatitis E accounts for about 20% of acute viral hepatitis in my country. Hepatitis E is an important public health problem as more and more studies identify it as a zoonotic disease. [0003] Studies have found that HEV can infect animals such as pigs, chickens, sheep, mice and cattle. Meng et al. isolated ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/08C07K5/103C07K16/10A61K39/29A61P31/14G01N33/577G01N33/569
Inventor 赵钦周恩民王鑫杰孙亚妮李爽
Owner NORTHWEST A & F UNIV
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