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Nanometer antibody for resisting hepatitis B virus core antigen and application of nanometer antibody

A technology of hepatitis B virus and nano-antibody, which is applied in the direction of antiviral agents, antiviral immunoglobulins, antitumor drugs, etc., can solve the problems of heavy economic burden, serious side effects, and long treatment cycle of patients, and achieve the reduction of HBeAg level, significant hepatitis B, significant therapeutic effect

Inactive Publication Date: 2020-02-07
SHENZHEN IMMUNOTHERAPY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, liver diseases such as chronic hepatitis, cirrhosis and primary liver cancer caused by HBV infection, especially chronic HBV infection, bring a heavy economic burden to patients.
The currently approved drugs for the clinical treatment of chronic hepatitis B include interferon and nucleoside analogues. Although they have positive effects on inhibiting virus replication and delaying disease progression, they still have long treatment cycles, low response rates, and Serious side effects (interferons) and potential drug resistance (nucleoside analogs) and other issues, therefore, there is an urgent need to develop anti-HBV drugs based on new targets or new mechanisms

Method used

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  • Nanometer antibody for resisting hepatitis B virus core antigen and application of nanometer antibody
  • Nanometer antibody for resisting hepatitis B virus core antigen and application of nanometer antibody
  • Nanometer antibody for resisting hepatitis B virus core antigen and application of nanometer antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of HBcAg antigen

[0040] In this experiment, the immunogen used for alpaca is hepatitis B core antigen HBcAg, and the E.coli prokaryotic expression system is used, the bacteria are crushed and centrifuged, and the nickel column is used for separation and purification. Purified proteins were quantified using 12% polyacrylamide gels. Then the purified protein was subpackaged and freeze-dried and stored at -80°C (to avoid protein degradation caused by repeated freezing and thawing).

Embodiment 2

[0041] Example 2: Alpaca immunization and blood collection

[0042] We immunized the alpaca four times in total. The alpaca was immunized with HBcAg. The injection site was the cervical lymph nodes of the alpaca, and half of the samples were injected in the left and right lymph nodes of the alpaca neck. During the operation, attention should be paid to the whole process Preserve proteins and adjuvants at low temperature to avoid protein degradation caused by repeated freezing and thawing. The initial immunization dose is 200ug mixed with complete Freund's adjuvant, boosted with 100ug HBcAg mixed with incomplete Freund's adjuvant at the 2nd week, boosted with 100ug HBcAg mixed with incomplete Freund's adjuvant at the 10th week For booster immunization, mix 100ugHBcAg with incomplete Freund's adjuvant. Before each immunization, 10ml of blood was collected, anticoagulated with EDTA, and plasma and PBMC were separated.

[0043] After the fourth immunization, 40 ml of blood was t...

Embodiment 3

[0044] Example 3: Isolation of alpaca lymphocyte PBMC

[0045] Fresh blood samples should be stored at 18-20°C, and the separation of lymphocytes should be completed as soon as possible within 2-6 hours (preferably within 2 hours) after blood collection. The steps of lymphocyte separation are as follows: 1) Take out the Histopaque-1077 lymphocyte separation medium from the refrigerator at 4°C one day in advance, and place it at room temperature. 2) Take 5ml of Histopaque-1077 Lymphocyte Separation Medium at room temperature into a 15ml centrifuge tube, take 8ml of whole blood and add carefully and slowly above the liquid level of Histopaque-1077 Lymphocyte Separation Medium. 3) Centrifuge at 400g, 20°C, with a ramp-up rate of 0 and a ramp-down rate of 0, for 30 minutes. The centrifuged sample should be divided into four layers, which are serum, lymphocytes, dextran in lymphocyte separation medium and red blood cells from top to bottom. Carefully aspirate the monocytes in the...

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Abstract

The invention discloses a nanometer antibody for resisting a hepatitis-B-virus core antigen (HBcAg). HBcAg is used for immunization of alpacas, and through screening, the nanometer antibody for resisting the HBcAg is obtained. The invention further relates to a complete antibody consisting of antibody fragments in fusion with Fc. The antibody is used for treatment of hepatitis-B-viruses and livercancer, and the invention provides a method for preparing the antibody.

Description

technical field [0001] The invention relates to the technical field of antibody medicines, in particular to nanobody sequences against hepatitis B virus core antigen (HBcAg). The invention is used for diagnosing or treating hepatitis B disease and liver cancer. Background technique [0002] The invention relates to a nanobody against hepatitis B virus core antigen (HBcAg), which is used for diagnosing or treating hepatitis B disease and liver cancer. [0003] Hepatitis B virus (HBV) infection is a serious public health problem faced by the whole world. Statistics show that more than 2 billion people worldwide have been infected with HBV, and about 350 million of them are chronic HBV infection (Chronic hepatitis B, CHB). Because the infection rate of HBV infection is extremely high, and the mortality rate of liver cancer developed by patients with chronic hepatitis B ranks second in the total cancer mortality rate in my country, hepatitis B has caused a serious economic bur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/569G01N33/574A61P31/20A61P35/00
CPCA61P31/20A61P35/00C07K16/082C07K2317/22C07K2317/565C07K2317/569G01N33/56983G01N33/57438G01N2333/02
Inventor 张军方
Owner SHENZHEN IMMUNOTHERAPY BIOTECH CO LTD
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