Neutralizing antibody B430 of hepatitis B virus (HBV) and application of neutralizing antibody B430

A hepatitis B virus and antibody technology, applied in applications, antibodies, antiviral agents, etc., can solve problems such as difficulty in reducing hepatitis B surface antigen, long interferon treatment cycle, and large side effects.

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 B430 of hepatitis B virus (HBV) and application of neutralizing antibody B430
  • Neutralizing antibody B430 of hepatitis B virus (HBV) and application of neutralizing antibody B430
  • Neutralizing antibody B430 of hepatitis B virus (HBV) and application of neutralizing antibody B430

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the discovery of antibody

[0035] Using the culture of human memory B cells, 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.

[0036] B cell culture screening method for cloning hepatitis B small membrane protein-specific B cell antibody gene: isolate peripheral blood lymphocyte PBMC from the peripheral blood of normal people immunized with hepatitis B vaccine, and sort out memory B cells from PBMC by flow cytometry. Co-culture memory B cells and supporting cells 3T3-CD40L for 10 days, use Elisa to detect whether the secreted cell supernatant contains hepatitis B virus small membrane protein HBs antibody, antibody-positive holes are lysed to reverse transcribe cDNA from B cells, and use variable antibody Region-specific primers were used to clone all antibody variable region genes of positive wells, and a pair of hepatitis B small memb...

Embodiment 2

[0039] Embodiment 2, the preparation of monoclonal antibody B430

[0040] 1. Preparation of recombinant plasmids

[0041] 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.

[0042] 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 972-1343 are the heavy chain variable region coding region , 1344-2333 nucleotides are the heavy chain constant region coding region, 2334-2336 are stop codons, and 2395-2543 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.

[0043] Insert the DNA molecule shown in Sequence 4 of the sequence listing into pLB-simple Vector to obtain a recombina...

Embodiment 3

[0054] Embodiment 3, the binding ability of antibody and hepatitis B virus

[0055] 1. ELISA detection

[0056] 1. Take the ELISA plate and add the original coating solution for coating (50ng original coating / well).

[0057] Coating was originally HBs antigen. A coating stock solution was prepared using PBS buffer as a solvent.

[0058] 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.

[0059] 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 B430 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 B430 of hepatitis B virus (HBV) and application of the neutralizing antibody B430. The invention provides an IgG antibody named a monoclonal antibody B430 consisting of a light chain and a heavy chain. CDR1, CDR2 and CDR3 in a heavy chain variable region in the heavy chain are sequentially a (45-52)th amino acid residue, a (70-77)th amino acid residue and a (116-132)th amino acid residue from an end N of a sequence 1 in a sequence listing; and CDR1, CDR2 and CDR3 in a light chain variable region in the light chain are sequentially a (43-48)th amino acid residue, a (66-68)th amino acid residue and a (105-113)th amino acid residue from an end N of a sequence 3 in the sequence listing. The neutralizing antibody B430 can be used for treating HBVchronic infection patients and can clinically replace serum purification polyclonal antibodies to prevent mother-to-child transmission. The neutralizing antibody B430 has important application prospects for treating and preventing HBV infection.

Description

technical field [0001] The invention relates to a neutralizing antibody B430 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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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13A61K39/42A61P31/20A61P1/16
CPCC07K16/082A61P31/20A61P1/16C07K2317/565C07K2317/92C07K2317/76
Inventor 张林琦贲海静史宣玲
Owner TSINGHUA UNIV
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