Construction method and application of peripheral blood B cell lines

A construction method and peripheral blood technology, applied in the field of construction of peripheral blood B cell lines, can solve the problems of lack of research and application of inflammatory diseases

Pending Publication Date: 2019-11-08
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, previous research on BCL usually focused on its ability to present tumor cell antigens or TT, and lacked application in the study of inflammatory diseases

Method used

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  • Construction method and application of peripheral blood B cell lines
  • Construction method and application of peripheral blood B cell lines
  • Construction method and application of peripheral blood B cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A method for constructing a peripheral blood B cell line

[0038] Collect heparin sodium anticoagulant blood from 200 cases of HBV infection, add 10mL peripheral blood to 10mL PBS (phosphate buffer saline) to dilute, add to 15mL centrifuge tube containing 5mL lymphocyte separation medium, 800g, 25min, acceleration and deceleration is 0, Normal temperature, density gradient centrifugation. Aspirate the buffy coat into a 50mL centrifuge tube, add PBS to 50mL, 1800rpm, 10min, centrifuge; break up the cells, add 5mL medium to resuspend, count; take 2million cells according to the counting results, and culture them with RPMI 1640 containing 15% FBS base (R15) to resuspend PBMC (peripheral blood mononuclear cells) to 0.5*10^ 6 ~1*10^ 6 / mL, centrifuge at 500g for 5min and discard the supernatant, 100μL EBV supernatant / 0.5*10^ 6～ 1*10^ 6 Cells were resuspended, and the suspension was transferred to a 96-well U-bottom plate. After incubating in an incubator (37°C, 5% carbon...

Embodiment 2

[0056] Example 2 BCL is used to screen dominant T cell immune response antigen epitopes

[0057] It has been proved in the above examples that after adding BCL loaded with HBV antigen peptide, the frequency of T cells capable of secreting IFN-γ was significantly increased, indicating that BCL still retains the antigen-presenting ability of B cells. However, there are differences in the reactivity of T cells from different patients to the same HBV antigen peptide (expressed as the difference in the frequency of T cells with the ability to secrete IFN-γ), and at the same time, the reactivity of T cells from the same patient to different HBV antigen peptides There are also differences, indicating that for different patients, the HBV antigen peptides that can induce the strongest T cell immune response are not the same. Therefore, BCL can be used to present antigenic peptides to T cells, and by detecting the frequency of T cells with the ability to secrete IFN-γ in T cells, the pa...

Embodiment 3B

[0060] Example 3 BCL is used to prepare high titer anti-HBsAg monoclonal antibody

[0061] After V(D)J gene rearrangement, antibody class switching, and high frequency mutation of somatic cells, B cells differentiate into plasma cells capable of secreting high-affinity antibodies. Due to allelic exclusion and isotype exclusion, each plasma cell can only secrete an antibody. In the present invention, it is found that BCL still has the ability to secrete antibodies, so it can be inferred that BCL contains "plasma cells", and such plasma cells have the characteristics of immortalization. By single cell sorting or limiting dilution, one or several plasma cell clones proliferate to form a monoclonal cell line that secretes monoclonal antibodies. Collect the culture supernatant of the monoclonal cell line, detect the antibody in the supernatant, and screen the BCL cell line that produces high-titer anti-HBsAg monoclonal antibody.

[0062] The specific method is as follows:

[006...

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PUM

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Abstract

The invention discloses a construction method and application of peripheral blood B cell lines (BCL). The BCL constructed in the invention have the characteristic of immortalized growth, the proportion of CD19<+> cells in the BCL is increased, the expression levels of CD138, CD38 and CD27 in the CD19<+> BCL are significantly higher than that of CD19<+> B cells before Epstein-Barr virus (EBV) transformation, and the capacities of antibody secretion and antigen presentation of the B cells are retained. The BCL disclosed by the invention can be used for screening dominant T cell immune response epitopes, or preparing a high-titer anti-HBsAg monoclonal antibody and inducing an HBV specific T cell line, and providing a new direction for removing hepatitis B.

Description

technical field [0001] The invention relates to a method for constructing a peripheral blood B cell line and its application, in particular to a method for constructing a peripheral blood B cell line capable of presenting HBV antigen peptides and its application. Background technique [0002] According to the Global Burden of Disease Study in 2013, hepatitis virus infection is the seventh leading cause of death in the world, of which hepatitis B virus (Hepatitis B virus, HBV) and hepatitis C virus infection accounted for 96% of deaths [1]. In HBV infection, the body's immunity against HBV can be divided into two parts, one is the innate immune response, which is the non-specific immune response of the immune system; the other is the adaptive immune response, which is the specificity of T cells and B cells against HBV antigens immune response. In chronic HBV infection, the functional state of HBV-specific T cells is closely related to the clearance of the virus. Among them,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0783C07K16/08C12R1/91
CPCC12N5/0635C12N5/0636C07K16/082
Inventor 侯金林李咏茵唐利波易璇古书琴温春花
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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