Neutralizing antibody B826 of hepatitis b virus and application of neutralizing antibody B826

A hepatitis B virus and antibody technology, applied in applications, antibodies, antiviral agents, etc., can solve problems such as large side effects, low cost-effective treatment effect, and inability to completely remove hepatitis B virus.

Active Publication Date: 2020-06-05
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although hepatitis B vaccine has controlled the spread of hepatitis B virus, for a large number of patients who have been infected with hepatitis B virus, clinical treatment methods such as nucleoside drugs and interferon cannot cure hepatitis B virus infection
Nucleoside drugs can effectively control the DNA replication of hepatitis B virus, but they cannot completely eliminate hepatitis B virus, and it is difficult to reduce hepatitis B surface antigen. After hepatitis B patients stop taking drugs, hepatitis B virus will still recur
The interferon treatment cycle is long and the side effects are large. It can only be effective for a relatively small number of hepatitis B patients during the treatment period. The treatment effect is very cost-effective. At present, the treatment of hepatitis B infection is basically based on nucleoside drugs.

Method used

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  • Neutralizing antibody B826 of hepatitis b virus and application of neutralizing antibody B826
  • Neutralizing antibody B826 of hepatitis b virus and application of neutralizing antibody B826
  • Neutralizing antibody B826 of hepatitis b virus and application of neutralizing antibody B826

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the discovery of antibody

[0036] Using a single B cell clone, looking for monoclonal antibodies to hepatitis B virus from the blood of hepatitis B vaccine immunized persons, and then comparing the effects of each antibody obtained.

[0037] Antigen bait flow sorting method for cloning hepatitis B small membrane protein-specific B cell antibody gene: isolate peripheral blood lymphocyte PBMC from the peripheral blood of normal persons immunized with hepatitis B vaccine, sort memory B cells with magnetic beads, and use flow cytometry The cytometer sorts out the cells specifically bound to the hepatitis B small membrane protein from the memory B cells, lyses the cells, and obtains cDNA by RT-PCR, and then performs nested PCR with specific primers for the variable region of the antibody, and sequence the antibody gene. Further screening confirmed that the antibody was hepatitis B small membrane protein monoclonal antibody.

[0038] A monoclonal antibody (bin...

Embodiment 2

[0040] Embodiment 2, the preparation of monoclonal antibody B826

[0041] 1. Preparation of recombinant plasmids

[0042] Insert the DNA molecule shown in Sequence 2 of the sequence listing into pLB-simple Vector to obtain a recombinant plasmid. The recombinant plasmid has been verified by sequencing. This plasmid is also named heavy chain expression plasmid.

[0043] In Sequence 2 of the sequence listing, nucleotides 1-666 are the CMV promoter, nucleotides 915-971 are the signal peptide coding region, and nucleotides 972-1340 are the heavy chain variable region coding region 1341-2330 nucleotides are the coding region of the heavy chain constant region, 2331-2333 are stop codons, and 2392-2540 nucleotides are polyA terminators. The DNA molecule shown in Sequence 2 of the Sequence Listing expresses the heavy chain shown in Sequence 1 of the Sequence Listing.

[0044] Insert the DNA molecule shown in Sequence 4 of the sequence listing into pLB-simple Vector to obtain a reco...

Embodiment 3

[0055] Embodiment 3, the binding ability of antibody and hepatitis B virus

[0056] 1. ELISA detection

[0057] 1. Take the ELISA plate and add the original coating solution for coating (50ng original coating / well).

[0058] Coating was originally HBs antigen. A coating stock solution was prepared using PBS buffer as a solvent.

[0059] 2. After completing step 1, take the ELISA plate, add 200 μl of PBS buffer solution containing 2% BSA to each well, incubate at 37°C for 2 hours to block, then discard the supernatant and wash twice with PBST solution.

[0060] 3. After completing step 2, take the ELISA plate, add 200 μl of antibody diluent to each well, incubate at 37°C for 1 hour, then discard the supernatant and wash twice with PBST solution. The antibody diluent is obtained by diluting the B826 antibody solution or the HBIG solution or the MERS-27 antibody solution prepared in Example 2 with PBS buffer. Five replicate wells were set up for each antibody dilution.

[00...

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Abstract

The invention discloses a neutralizing antibody B826 of a hepatitis b virus and an application of the neutralizing antibody B826.The invention provides an IgG antibody which is named as a monoclonal antibody B826, and consists of a light chain and a heavy chain, wherein CDR1, CDR2 and CDR3 in a variable region of the heavy chain in the heavy chain are sequentially 45th-52th amino acid residues, 70th-77th amino acid residues and 116th-131st amino acid residues from the N tail end in the sequence 1 of a sequence table; and CDR1, CDR2 and CDR3 in a variable region of the light chain in the heavychain are sequentially 43rd-54th amino acid residues, 72nd-74th amino acid residues and 111st-119th amino acid residues from the N tail end in the sequence 3 of the sequence table. The neutralizing antibody B826 can be used for treating patients suffering from hepatitis B chronic infection, and can be used for clinically replacing serum purification polyclonal antibody to prevent mother-to-baby transmission. The neutralizing antibody B826 has important application prospects for treatment and prevention of hepatitis b virus infection.

Description

technical field [0001] The invention relates to a neutralizing antibody B826 of hepatitis B virus and its application. Background technique [0002] Hepatitis B Virus (HBV) infection is widespread all over the world, especially in China. Hepatitis B virus is a member of Hepadnaviridae, and its infection induces hepatitis. In China, about 80-100 million people are chronically infected with hepatitis B virus, and there are about 240 million chronic hepatitis B patients in the world. If considering the patients with acute HBV infection who have recovered, the cumulative number of HBV infection patients is more. Studies have shown that 35-62% of Chinese people have been infected with hepatitis B virus, and 56-98% of sub-Saharan Africans have been infected with hepatitis B virus. About 2 billion people in the world have been infected with hepatitis B virus. Hepatitis B virus infection is a global health problem. [0003] HBV induces immune inflammation during infection and dama...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13A61K39/42A61P1/16A61P31/20
CPCC07K16/082A61P1/16A61P31/20C07K2317/56C07K2317/76A61K2039/505
Inventor 张林琦贲海静史宣玲
Owner TSINGHUA UNIV
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