Human papilloma virus (HPV) capsid protein L1 polypeptide and preparation and application thereof

A technology of capsid protein and papilloma, applied in the field of peptides, can solve the problems of aggravating the psychological and economic burden of patients and overtreatment

Inactive Publication Date: 2010-12-15
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it often leads to overtreatment and related complications, and increases the psychological and economi...

Method used

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  • Human papilloma virus (HPV) capsid protein L1 polypeptide and preparation and application thereof
  • Human papilloma virus (HPV) capsid protein L1 polypeptide and preparation and application thereof
  • Human papilloma virus (HPV) capsid protein L1 polypeptide and preparation and application thereof

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Experimental program
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Effect test

Embodiment 1

[0035] 1. Screening and peptide synthesis of conserved epitopes of 18 low-risk and high-risk HPV subtypes:

[0036] Through computer simulation, L1 protein epitope prediction and multiple sequences of 18 HPV subtypes (6, 11, 16, 18, 52, 58, etc.) Alignment, and using the naccess program to calculate the liquid-phase accessible area of ​​the corresponding amino acid residues, we screened a very conservative peptide located on the surface of the protein (N---Cys Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His--- C), and entrusted Beijing Saibaisheng Biotechnology Co., Ltd. to synthesize the peptide and its cross-linked product (Cys Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His--KLH), with a purity of >90%.

[0037] 2. Preparation of antiserum:

[0038] In the primary immunization experiment group, Freund's complete adjuvant was mixed with the synthetic immunogen Cys Thr Leu Thr Ala Asp Val Met Thr Tyr Ile His-KLH protein in equal amounts, and stirred on a magnetic stirrer for 30 minu...

Embodiment 2

[0040] Immunocytochemical detection of the reactivity of anti-polypeptide antiserum to cells infected with HPV virus: HeLa (cervical adenocarcinoma cell line infected with HPV18), Siha (cervical squamous cell line infected with HPV16) in logarithmic growth phase After cells were digested with trypsin, the cell suspension was seeded on coverslips placed in 24-well plates, cultured overnight at 37°C in a CO2 incubator, and fixed with 4% paraformaldehyde for 15 minutes when the cells covered about 70% of the slides. Wash with PBS for 5min×3 times, 3%H 2 o 2 Eliminate endogenous peroxidase for 30 min, PBS 5 min x 3 times, punch with 0.1% Triton for 15 min, PBS 5 min x 3 times, block with goat serum blocking solution for 30 min, add 1:500 diluted rabbit serum at 4°C overnight, wash with PBS 5min×3 times, then drop goat anti-rabbit secondary antibody working solution for 15min, wash with PBS for 5min×3 times, add horseradish-labeled streptavidin working solution, wash with PBS for ...

Embodiment 3

[0043]Immunohistochemical detection of the reactivity of anti-polypeptide antiserum to HPV-positive clinical specimens: tissue specimens identified as condyloma acuminata, CIN, cervicitis and HPV-infected positive by the clinical pathology department were fixed in 10% formaldehyde, embedded in paraffin, sliced, After deparaffinization and hydration, repair with 0.01mol / lpH6.0 citrate buffer at 100°C for 20min, cool to room temperature naturally, wash with PBS for 5min×3 times, 3%H 2 o 2 Eliminate endogenous peroxidase for 30 min, PBS 5 min x 3 times, block with goat serum blocking solution for 30 min, add 1:500 diluted rabbit serum overnight at 4°C, wash with PBS for 5 min x 3 times, then add goat anti-rabbit II dropwise Anti-working solution for 15 minutes, wash with PBS for 5 minutes×3 times, add horseradish-labeled streptavidin working solution, wash with PBS for 5 minutes×3 times, develop color with DAB for 3 minutes, stop the reaction with tap water, counterstain with hem...

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Abstract

The invention relates to a human papilloma virus capsid protein L1 (HPVL1) peptide and preparation and application thereof. The HPVL1 peptide is a peptide segment of which the amino acid of the sequence is N end-Cys Thr Leu Thr Ala Asp Val Met listed Thr Tyr Ile His-C end, or is a peptide segment comprising the amino acid of the sequence and with a length less than 50 amino acids. In the invention, the peptide which is a very conservative peptide segment located on the surface of protein is sieved out by performing epitope forecasting and multi-sequencing comparison on the L1 proteins of 18 low-risk types and high-risk type HPV subtypes (6, 11, 16, 18, 52, 58 and the like); the antibody generated from induction can react with multiple types of HPVL1, and can be used for the detection of the multiple types of HPVL1, in particular to the application of precancerous lesions detection of human cervical carcinoma; and the antibody aiming at the peptide segment also can be used for purifying and preparing the multiple types of HPVL1. In addition, the invention also provides the applications of the peptide segment used as multiple types of HPV antiserum and detected matters of monoclonal antibody.

Description

Technical field: [0001] The invention relates to a polypeptide, in particular to a human papilloma virus (HPV) capsid protein L1 polypeptide and its preparation and application. technical background: [0002] Cervical cancer is a disease that seriously affects women's health. Globally, cervical cancer is the second most common cancer among women, and even ranks first in some developing countries, with nearly 500,000 new cases each year (Valdespino VM, Valdespino VE..Curr Op in Obstet Gynecol, 2006, 18: 35- 40.). In recent years, human papillomavirus (HPV) infection is considered to be the main cause of cervical cancer. In 1999, Warlboomers et al. found that the detection rate of human papillomavirus (Human papillomavirus, HPV) was 99.7% in the study of 1035 cases of cervical cancer specimens in 22 countries. Since the first case of human papillomavirus (HPV) was discovered in 1933, more than 200 subtypes of human papillomavirus have been identified so far. According to t...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K16/08C07K16/06G01N33/577G01N33/569C12N7/02
Inventor 胡丽娜王霞刘宝林勇陈恒禧张林甘晓玲
Owner SICHUAN UNIV
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