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Preparation method and application for monoclonal antibody against HPV16 L1 protein

A monoclonal antibody and protein technology, applied in antiviral immunoglobulins, immunoglobulins, chemical instruments and methods, etc., can solve the problem that the HPV virus antigen protein is difficult to apply, the detection technology of HPVL1 protein is lagging behind, and it is difficult to separate natural Virus and other problems, to achieve the effect of stabilizing antibody secretion capacity, extensive research application value and commercial use value, and improving specificity and affinity

Active Publication Date: 2018-09-28
ZHENGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the HPV gene exists in the body in the form of episomes or integrated into the host chromosome, there is no or only a small amount of coat protein in the above-mentioned forms, so it is difficult to isolate the natural virus of HPV in diseased tissues, so the HPV virus antigen Protein detection is difficult to be applied clinically
[0006] However, the research on HPVL1 protein detection technology is relatively lagging behind.

Method used

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  • Preparation method and application for monoclonal antibody against HPV16 L1 protein
  • Preparation method and application for monoclonal antibody against HPV16 L1 protein
  • Preparation method and application for monoclonal antibody against HPV16 L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of hybridoma cell lines secreting anti-HPV16 L1 protein monoclonal antibody

[0042] 1. Main Reagents and Materials

[0043]Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, PEG-1500, RPM1-1640 cell culture medium, fetal bovine serum were purchased from Gibco Company, HRP-labeled goat anti-mouse IgG was purchased from Sigma Company, liquid AEC enzyme The substrate kit was purchased from Zhongshan Jinqiao Company, the BCA protein concentration determination kit was purchased from Solarbio Company, the monoclonal antibody subtype determination kit was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; the commercial anti-HPV16 L1 monoclonal antibody was purchased from Abcam (Abcam, UK); plasmids p6SheLL, p16SheLL, p18SheLL, p31SheLL, p45SheLL, p52SheLL and p58SheLL containing the corresponding HPV L1 and L2 ORF region genes were purchased from Addgene, provided by Prof. John Schiller (Addgene, USA), BALB / c Mice were pu...

Embodiment 2

[0068] Example 2: Identification of hybridoma cell lines secreting anti-HPV16 L1 protein monoclonal antibody

[0069] 1. Screening, identification and subcloning of hybridoma cells

[0070] (1) ELISA screening

[0071] In the first round of ELISA screening, SUMO1-16 L1 recombinant protein (diluted with CBS 1:100) was used as the coating source, and 22 ELISA plates were coated to screen the hybridoma cell culture supernatant; the 96 strains that were initially screened were hybridized Tumor cell lines were transferred to two 48-well cell plates for further culture.

[0072] In the second round of ELISA screening, SUMO1-16 L1 purified protein (diluted with CBS 1:100) and supernatant of SUMO-tag recombinant protein sonicated (diluted with CBS 1:1000) were selected as the coating source, and SUMO-tag recombinant protein A total of 67 positive hybridoma cell lines were screened out after eliminating false positives.

[0073] In the third round of ELISA screening, the pre-prepare...

Embodiment 3

[0083] Example 3: Preparation and purification of anti-HPV16L1 protein monoclonal antibody ascites

[0084] 1. Preparation of anti-HPV16L1 protein monoclonal antibody ascitic fluid

[0085] The monoclonal hybridoma cell line 19H7 in Example 2 was expanded and cultured, and the titer of the culture supernatant was measured by ELISA to ensure the stability of the monoclonal cell line, and the cells were collected for large-scale preparation of cloned antibodies. The specific steps were as follows:

[0086] (1) Multiparous female BALB / c mice were selected, and 500 μl of sterilized paraffin was injected intraperitoneally to stimulate immune cells to promote the proliferation of hybridoma cells.

[0087] (2) Observe the state of the mice, and after 7-10 days, the 7 Inject the amount of cells into the monoclonal positive cells prepared in advance, and observe the state of the mice in time;

[0088] (3) After 10 days, extract ascites, centrifuge at 8000 r / min, 4°C for 20 min to rem...

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Abstract

The invention discloses a preparation method and an application for a monoclonal antibody against an HPV16 L1 protein. The invention also discloses the monoclonal antibody against the HPV16 L1 protein, and a DNA sequence and an amino acid sequence of the monoclonal antibody in a heavy chain variable region and a light chain variable region. The invention designs the preparation method for the monoclonal antibody against the HPV16 L1 protein. The monoclonal antibody against the HPV16 L1 protein is applied to immunological detection, antigen or antibody detection kits. The monoclonal antibody against the HPV16 L1 protein can specifically recognize a main epidemic subtype namely HPV16, has good reactivity with HPV16 pseudovirus and the HPV16 L1 protein, and is free of cross reactions with subtypes of L1 proteins. A good foundation is laid for further modification of the antibody variable region sequence of the monoclonal antibody to prepare genetically engineered antibodies with differentcombination forms and for clinical detection research on different subtypes of HPV pathogens.

Description

technical field [0001] The invention relates to the technical field of biological immunity, in particular to a preparation method and application of an anti-HPV16 L1 protein monoclonal antibody. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) is a non-enveloped, 20-hedron double-stranded closed circular DNA virus with strict species specificity. It mainly infects human skin and mucosal tissues, and can be transmitted through sexual contact and skin contact. and vaginal delivery microtrauma infection, causing hyperplastic lesions of the corresponding parts of the epithelial tissue. [0003] The HPV viral capsid consists of a major capsid protein (L1) and a minor capsid protein (L2). Among them, the L2 protein is a minor capsid protein and a highly variable nucleoprotein, reflecting the polymorphism of HPV antigens. The L1 protein is the main capsid protein of the HPV virus, accounting for about 80% of the total viral protein. It is a highly co...

Claims

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Application Information

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IPC IPC(8): C07K16/08G01N33/569G01N33/577
CPCC07K16/084C07K2317/56G01N33/56983G01N33/577G01N2333/025
Inventor 王爱萍陈玉梅张改平刘东民王彦伟刘运超栗宁蒋敏
Owner ZHENGZHOU UNIV
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