Anti-HPV53 L1 protein monoclonal antibody as well as preparation and application thereof

A monoclonal antibody and protein technology, applied in antiviral immunoglobulin, antibody, immunoglobulin and other directions, can solve the problems of lag, HPV virus antigen protein is difficult to apply, and it is difficult to separate HPV natural virus, etc. Specificity, Enhanced Specificity and Affinity, High Sensitivity Effect

Active Publication Date: 2022-03-25
ZHENGZHOU UNIV
View PDF6 Cites 0 Cited by
  • 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; therefore, it is difficult to isolate the natural virus of HPV in diseased tissues. Therefore, the detection of HPV virus antigen protein is difficult to be applied clinically.
[0006] However, the research on HPV L1 protein detection technology is relatively lagging behind.
No monoclonal antibody against HPV 53L1 protein has been reported so far

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Anti-HPV53 L1 protein monoclonal antibody as well as preparation and application thereof
  • Anti-HPV53 L1 protein monoclonal antibody as well as preparation and application thereof
  • Anti-HPV53 L1 protein monoclonal antibody as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of hybridoma cell lines secreting anti-HPV53 L1 protein monoclonal antibody

[0039] 1. Main Reagents and Materials

[0040]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-HPV53 L1 monoclonal antibody was purchased from Abcam (Abcam, UK); plasmids 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 purchased from Z...

Embodiment 2

[0062] Example 2: Identification of hybridoma cell lines secreting anti-HPV5 L1 protein monoclonal antibody

[0063] 1. Screening, identification and subcloning of hybridoma cells

[0064] (1) ELISA screening

[0065] In the first round of ELISA screening, SUMO1-53 L1 recombinant protein (diluted with CBS 1:100) was used as the coating source, and 22 ELISA plates were coated to screen the supernatant of hybridoma cell culture; the 8 strains that were initially screened were hybridized Tumor cell lines were transferred to a 24-well cell plate for further culture.

[0066] In the second round of ELISA screening, SUMO1-53 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 4 positive hybridoma cell lines were screened out after excluding false positives.

[0067] In the third round of ELISA screening, HPV53 PsV prepared i...

Embodiment 3

[0077] Example 3: Preparation and purification of anti-HPV53 L1 protein monoclonal antibody ascites

[0078] 1. Preparation of anti-HPV53 L1 protein monoclonal antibody ascitic fluid

[0079] The monoclonal hybridoma cell line 1A10 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 the cloned antibody. The specific steps were as follows:

[0080] (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.

[0081] (2) Observe the state of the mice. 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;

[0082] (3) After 10 days, extract ascites, centrifuge at 8000 r / min, 4°C for 20 min to rem...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses an anti-HPV53 L1 protein monoclonal antibody as well as preparation and application thereof, and aims to solve the problem that an HPV53 subtype is difficult to specifically recognize. A heavy chain variable region of the antibody contains a DNA sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2, and a light chain variable region of the antibody contains a DNA sequence as shown in SEQ ID NO.3 and an amino acid sequence as shown in SEQ ID NO.4; the antibody can be applied to immunological detection or vaccine preparation, and also can be applied to an antigen or antibody detection kit. The anti-HPV53 L1 protein monoclonal antibody is prepared by immunizing a BALB / c mouse through an immunological method, the antibody can specifically recognize an HPV53 high-risk (HR) subtype, is extremely high in sensitivity, has good reactivity with an HPV53 pseudovirus and an HPV53 L1 protein, and does not have cross reaction with L1 proteins of other subtypes. The invention lays a good foundation for further modifying the sequence of the variable region of the antibody to prepare genetic engineering antibodies in different combination forms and for clinical detection and research on different subtype pathogens of HPV (human papillomavirus).

Description

technical field [0001] The invention relates to the technical field of biological immunity, in particular to an anti-HPV53 L1 protein monoclonal antibody and its preparation and application. Background technique [0002] Human Papillomavirus ( Human Papillomavirus , HPV) is a non-enveloped, 20-hedral double-stranded closed circular DNA virus with strict species specificity, mainly infects human skin and mucous membrane tissues, and can be transmitted through sexual contact, skin contact and microtrauma of vaginal delivery Infection causes proliferative lesions of the epithelial tissue in the corresponding parts. [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 ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K16/08C12N5/20G01N33/569G01N33/577A61K39/42A61P31/20
CPCC07K16/084G01N33/56983G01N33/577A61P31/20C07K2317/56G01N2333/025A61K2039/505Y02A50/30
Inventor 王爱萍张改平张雨晴周景明刘红亮陈玉梅丁培阳朱习芳祁艳华李永欣
Owner ZHENGZHOU UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap