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Monoclonal antibody capable of specific identification of multiple antigen epitopes of HBsAg and application thereof

A monoclonal antibody and specific technology, applied in the field of genetic engineering, can solve the problems of insufficient and lack of immunogenicity of HBV mutants, and difficulties in HBV prevention and treatment, so as to improve the protein expression efficiency and overcome the weak immunogenicity. Effect

Active Publication Date: 2015-12-09
NANJING RED CROSS BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, virus mutation has brought great difficulties to HBV prevention and treatment
The immunogenicity of HBV variants is insufficiently studied due to the lack of specific antibodies that can specifically recognize HBsAg space epitopes

Method used

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  • Monoclonal antibody capable of specific identification of multiple antigen epitopes of HBsAg and application thereof
  • Monoclonal antibody capable of specific identification of multiple antigen epitopes of HBsAg and application thereof
  • Monoclonal antibody capable of specific identification of multiple antigen epitopes of HBsAg and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] According to the codon preference of mammals, use http: / / www.jcat.de online software to carry out codon optimization to the coding sequence (HBsAg-Wt) of the HBsAg amino acid sequence shown in SEQIDNO.1, obtain the optimized HBsAg gene sequence SEQIDNO .2, and clone it into the HindIII / BamHI restriction site of the eukaryotic expression vector pJW4303 to obtain the HBsAg DNA vaccine HBsAg-Opt, which is applied to mice for in vivo immunization and in vitro transfection to collect antigens for monoclonal antibody screening .

[0037] At the same time, using the HBsAg-Opt sequence as a template, site-directed mutagenesis was carried out by the PCR method, and the mutant sequence was cloned into the HindIII / BamHI restriction site of the eukaryotic expression vector pJW4303 to obtain the HBsAg gene variant expression plasmid HBsAg-Mut (Table 1: primers); further expressed in 293T cells in vitro and collected antigens for screening monoclonal antibodies to specific epitopes o...

Embodiment 2

[0041] Example 2 Production of Antigens for Monoclonal Antibody Screening

[0042] 1) Inoculate 293 cells 6x10 the day before transfection 5 Into a six-well plate, 3ml of complete medium per well.

[0043] 2) At 37°C and 5% CO 2 The cells were cultured in the incubator, and the cell density in the plate was between 40-80% on the day of transfection.

[0044] 3) Dilute 2 μg of the plasmids to be transfected (HBsAg-Wt, HBsAg-Opt and HBsAg-Mut) into 100 μl of culture medium (without serum, protein, or antibiotics), and mix well by inverting.

[0045] 4) Add 20 μl PolyFect transfection reagent to the plasmid solution in the previous step, and pipette 5 times.

[0046] 5) Incubate the mixture of transfection reagent and plasmid at room temperature for 5-10 minutes.

[0047] 6) During the incubation interval, the medium in the culture plate was removed, and 1.5 ml of fresh medium was added.

[0048] 7) Add 0.6ml of culture medium until the mixed species are transfected, and pip...

Embodiment 3

[0050] The preparation of embodiment 3 hybridomas

[0051] (1) The source of bone marrow cells and the animal sources for maintaining culture and immunization;

[0052] SP2 / 0 was donated by the Department of Infectious Diseases, Jiangsu Provincial People's Hospital, and was routinely preserved and frozen.

[0053]Selection of immunized mice: the type of immunization: Balb / c, female, 8 weeks old: body weight: 20g; purchased from the Animal Center of Yangzhou University. 6 rats were immunized each time, and 2 were blank.

[0054] (2) Immunization process;

[0055] Immunization scheme: extract HBsAg-Opt plasmid, adjust the concentration to 1mg / ml after quantification, and immunize by instantaneous tail vein injection of 10μg / mice, a total of 5 times with an interval of 4 weeks, and take splenocyte fusion two weeks after the last immunization. One week after each immunization, the antigen expression level was monitored, and it can be seen that the HBsAg antibody titer of mice i...

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Abstract

The invention belongs to the field of genetic engineering, and relates to a monoclonal antibody capable of specific identification of multiple antigen epitopes of HBsAg and an application thereof. A strain of hybridoma cell is preserved in China center for type culture collection with the preservation date of September 16, 2014 and the preservation number of CCTCC NO:C2014151. The monoclonal antibody 5H2 capable of specific identification of the spatial epitopes consisting of 120th, 122th, 126th, 141th, 147th and 155th loci of HBsAg is secreted by the hybridoma cell having the preservation number of CCTCC NO:C2014151. The antibody can identify the spatial epitopes consisting of the multiple loci, and can be applied in preparation of reagents for detection of HBsAg virus.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a monoclonal antibody capable of specifically recognizing HBsAg space epitope and application thereof. Background technique [0002] Hepatitis B virus (HepatitisBvirus, HBV) is one of a kind of contagious virus that is widely distributed and is more harmful. Nearly 2 billion people in the world have been infected with HBV, and the number of people with persistent virus infection has reached 300 to 400 million people. my country is a big country with HBV infection, and the number of persistently infected patients has reached more than 100 million. With the application of preventive immunization and high-titer immunoglobulin, the proportion of HBV variants in the population is getting higher and higher under selection pressure. HBV variants may reduce the sensitivity of antibody detection and the effectiveness of vaccination. Therefore, virus mutation has brought great difficulti...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/08A61P31/20G01N33/577G01N33/569C12R1/91
Inventor 杨永林蔡杰许纪玲汪林娣蔡旭兵
Owner NANJING RED CROSS BLOOD CENT
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