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Anti-HPV16 E6 protein monoclonal antibody and application thereof

A technology of HPV16E6 and monoclonal antibody, applied in the field of biological detection, can solve the problems of low specificity and no provision

Active Publication Date: 2018-11-13
BEIJING SOLARBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few studies on the detection of HPV16E6 antigen and antibody. Therefore, expressing HPV16E6 antigen and preparing antibodies specific to HPV16 E6 are of great significance for the screening and diagnosis of cervical cancer, and can also be used for human reproductive system infectious diseases and tumors research, as well as research on malignant tumors such as esophageal cancer and breast cancer
Currently, there is no specific mouse antibody against HPV16 E6 protein on the market, and the only mouse monoclonal antibody sold on the market is sc-460 from Santa Cruz, USA, which is an HPV16 E6 / 18 E6 antibody that can simultaneously detect HPV16 E6 and HPV18 E6 Two proteins, but the specificity is not high; and the other is the rabbit monoclonal antibody 251401 of Abbiotec Company in the United States. Their company only provides a picture of IHC, and does not provide relevant pictures applied to wb

Method used

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  • Anti-HPV16 E6 protein monoclonal antibody and application thereof
  • Anti-HPV16 E6 protein monoclonal antibody and application thereof
  • Anti-HPV16 E6 protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Preparation and purification of the monoclonal antibody of embodiment 1 anti-HPV16 E6 protein

[0046] In this example, the monoclonal antibody secreted by the hybridoma cell line with the deposit number CCTCC NO: C2018152 was prepared by hybridoma technology, and a large amount of it was prepared and purified.

[0047] 1. Antigen preparation

[0048] 1.1 Polypeptide antigen screening The full-length sequence of HPV16 E6 protein is analyzed by AbDesigner and DNASTAR software, and the amino acid secondary structure, hydrophilicity, and antigenicity of the protein are analyzed; the three-dimensional structure of the protein is further analyzed, and blasted in the database By comparing the specificity of these sequences, the excellent polypeptide epitope sequence (SEQ ID NO: 1) of HPV16 E6 protein was finally screened out

[0049] SEQ ID NO: 1: DPQERPRKLPQLC.

[0050] 1.2 Synthetic peptide antigen

[0051] The selected polypeptide sequence is synthesized by the Fmoc-pro...

Embodiment 2

[0080] Example 2 Characterization of the monoclonal antibody against HPV16 E6 protein

[0081] In this example, the subtype of the monoclonal antibody prepared in the present invention was identified and its affinity constant for binding to HPV16 E6 protein was tested.

[0082] 1 Subtype identification: Dilute the coated goat anti-mouse IgG (BD Company, NO.KW09JL0510) with 100 mM PBS (pHT.4), and operate according to the product manual. The results show that the monoclonal antibody of the present invention is an IgG1 type mouse monoclonal antibody.

[0083] 2 Determination of affinity constant:

[0084] Synthetic polypeptide (SEQ ID NO: 1) was coated with a coating concentration of 1 μg / ml, 100 μL / well, coated overnight at 4°C, and washed 3 times with PBS-T. Add 200 μL of blocking solution to each well to block at 37°C for 2 hours, and wash 3 times with PBS-T. The monoclonal antibody purified in the above step 7 was serially diluted 2 times from 1:200, leaving a blank contr...

Embodiment 3

[0087] The reaction specificity of the monoclonal antibody of embodiment 3 anti-HPV16 E6 antigens

[0088] In this example, the immunoblotting method was used to test the reaction specificity of the monoclonal antibody prepared by the present invention to the HPV16 E6 protein.

[0089] Caski, Hela cell lysate protein and prokaryotic recombinant protein were selected, and the recognition specificity of the monoclonal antibody of the present invention was detected by immunoblotting. Gel electrophoresis. Gel protein bands were transferred to PVDF membranes in a Bio-Rad electrotransfer system as usual. Place the membrane in TBS-T blocking solution containing 5% skim milk powder at 4°C overnight. Add monoclonal antibody (about 1 mg / ml, diluted 1:1000) and incubate overnight at 4°C. After washing the membrane with TBS-T solution, a 1:5000 dilution of goat anti-mouse secondary antibody (Beijing Suo Laibao Technology Co., Ltd., No. SE131) was added and incubated at room temperature...

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Abstract

The invention discloses an anti-HPV16 E6 protein monoclonal antibody, which is characterized by having heavy chain variable regions with the amino acid sequences shown as SEQ ID NO:4, SEQ ID NO:6 andSEQ ID NO:8 and light chain variable regions with the amino acid sequences shown as SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14. The immunoblot experiments show that the antibody can be used for specifically recognizing the recombinant HPV16 E6 protein and cervical cancer tissues expressing HPV16 E6 protein. The antibody can be used for detecting the expression level of the HPV16 E6 protein in thecells in artificial or automatic modes through immunohistochemical staining (IHC), enzyme-linked immunosorbent assay (ELISA) or western blot manners; the HPV16 viruses can be specifically diagnosed;the novel measure is provided for detecting HPV16 viruses on the protein level; the antibody can be used or the study of human reproductive system infectious diseases and tumors, and can also be usedfor the study of relevant malignant tumor such as esophagus cancer, breast cancer and anal carcinoma.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a monoclonal antibody capable of recognizing human HPV16 E6 protein molecules and a hybridoma cell line capable of secreting the antibody. Background technique [0002] Cervical cancer is the second most common female malignancy. About 500,000 women worldwide are diagnosed with cervical cancer every year, and more than half of them die from it. The early symptoms of cervical cancer are not obvious, and there is a long and reversible precancerous lesion period in the development process. According to statistics, about 20% of low-grade cervical injuries will turn into high-grade injuries, and if not treated in time, 30% of them will further turn into malignant tumors. It takes about 10 years for the general cervical precancerous lesions to develop into cervical cancer. During this period, early diagnosis of precancerous lesions can prevent the occurrence of cancer. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/574G01N33/577G01N33/569C12N5/20
CPCC07K16/084C07K2317/55C07K2317/56C07K2317/569C07K2317/622C07K2317/92G01N33/56983G01N33/57415G01N33/57442G01N33/577
Inventor 张波石海军赵佳王萌萌姚元生王珍珍
Owner BEIJING SOLARBIO TECH CO LTD
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