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HBV (Hepatitis B Virus) preS1 B cell epitope peptide, hybridoma cell strain prepared by same and application

A technology of hybridoma cell lines and preparations, applied in the direction of microorganism-based methods, biochemical equipment and methods, instruments, etc., can solve the problems of complex antibody production process, difficulty in increasing production, and few successes, so as to reduce false negatives The effect of improving detection results, improving detection rate, and improving detection specificity

Inactive Publication Date: 2012-11-07
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, domestic laboratories mainly use domestic ELSIA kits, but the raw material antibody production process used in these kits is still relatively complicated, and it is difficult to increase the output, and some antibodies from abroad are relatively expensive
Domestic preS1 monoclonal antibodies are rarely successful, and their potency and specificity need to be further improved

Method used

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  • HBV (Hepatitis B Virus) preS1 B cell epitope peptide, hybridoma cell strain prepared by same and application
  • HBV (Hepatitis B Virus) preS1 B cell epitope peptide, hybridoma cell strain prepared by same and application
  • HBV (Hepatitis B Virus) preS1 B cell epitope peptide, hybridoma cell strain prepared by same and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Bioinformatics analysis and determination of detection targets

[0058] 1) Find the amino acid sequence of the HBV adr serotype preS1 protein from GENEBANK, which consists of 119 amino acid residues.

[0059] 2) Analyze the potential B cell epitope region in the preS1 protein online, comprehensively consider the secondary structure, antigenicity, hydrophilicity, accessibility and flexibility of the protein, analyze and score each segment, and select the two with the highest score The region is used as a candidate B cell epitope region, which are 27aa-59aa (peptide 1, the amino acid sequence is represented by SEQ ID NO: 1: DHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG) and 83aa-111aa (peptide 2, the amino acid sequence is represented by SEQ ID NO: 2: GILTTVPTAPPPASTNRQSGRQPTPISPP) . Among them, peptide 1 is a widely used B cell epitope region, and the application of peptide 2 has not been reported yet.

Embodiment 2

[0060] Embodiment 2. Preparation of HBV preS1 recombinant protein

[0061] 1) Whole gene synthesis of preS1 coding sequence

[0062] The coding sequence of human HBV adr subtype preS1 was obtained from GenBANK, and the coding sequence of human HBV adr subtype preS1 after codon bias modification was obtained by synonymous substitution with codons favored by Escherichia coli ( SEQ ID NO: 3):

[0063] catgccatgg gtggttggtc ttccaaacct cgccaaggca tgggcacgaa tctgtctgtt 60

[0064] ccgaatcctc tgggtttctt tccggatcac cagctggacc ctgcgttcgg tgccaacagc 120

[0065] aacaatccag attgggactt caacccgaac aaggatcact ggccagaggc aaatcaggta 180

[0066] ggtgcgggtg cattcggccc aggtttcacc ccaccacacg gcggtctgct gggctggagc 240

[0067] cctcaggctc agggcatcct gaccaccgtg ccaaccgcac ctcctcctgc ctccaccaat 300

[0068] cgtcagagcg gtcgtcagcc tactccaatc tctccacctc tgcgtgacag ccatcctcag 360

[0069] gcctaatagg tcgacgcct 379

[0070] Entrusted Shanghai Sangon Bioengineering Co., Ltd. to carry out the whole g...

Embodiment 3

[0094] Example 3. Preparation of anti-HBV preS1 monoclonal antibody

[0095] 1) Synthesis and crosslinking of epitope peptides

[0096] Chemically synthesized HBV preS1B cell epitope peptides, including peptide 1 and peptide 2, were purified and cross-linked with BSA to obtain preS1-peptide 1-BSA and preS1-peptide 2-BSA

[0097] 2) PreS1 epitope peptide immunization of Balb / c mice

[0098] The cross-linked product of the preS1 epitope peptide and BSA synthesized was taken out from the -80°C refrigerator, dissolved and filtered, and the protein content was determined by the Lowry method. It was diluted to 0.5 mg / ml with 20 mmol / L PBS at pH 5.8. Fifteen female Balb / c mice aged 6 weeks and weighing about 20 g were selected and divided into three groups A, B and C for immunization. Antigen emulsification adopts double syringe mutual pushing method. For the first immunization, the preS1 epitope peptide-BSA was emulsified and mixed with an equal volume of Freund's complete adjuva...

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Abstract

The invention relates to application of a peptide segment shown as SEQ ID NO:2 in preparing a hepatitis B virus (HVB) diagnose preparation, an anti-HBV hybridoma cell strain CCTCC (China Center for Type Culture Collection) NO:C201012 prepared by using the peptide segment shown as the SEQ ID NO:2 and an anti-HBV presS1 monoclonal antibody secreted by the hybridoma cell strain, wherein the secretedanti-HBV presS1 monoclonal antibody belongs to IgG1, and a light chain is of a kappa type. The specific monoclonal antibody is used as a capture antibody, which can obviously improve the detectable rate of HBV LHBs and effectively avoid a false negative result in detection.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a hybridoma cell strain of anti-HBV preS1 monoclonal antibody. Background technique [0002] Viral hepatitis is one of the most prominent public health problems in my country, and is one of the four most serious infectious diseases alongside AIDS, tuberculosis and schistosomiasis. Among various types of viral hepatitis, human hepatitis B is one of the most prevalent viral infectious diseases in the world, and hepatitis B virus (HBV) is its main cause. The treatment of hepatitis B virus infection has not yet had breakthrough progress, and the public health problems caused by HBV are far from being solved. HBV can cause acute and chronic hepatitis. Most acute infections in adults can be cured by themselves, but chronic infections can lead to serious consequences, such as liver cirrhosis, hepatocellular carcinoma, and chronic liver failure. Hepatitis B virus is a s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C12N5/20G01N33/577C12R1/91
Inventor 胡川闽易维京李新军陈安
Owner ARMY MEDICAL UNIV