Screening and validation of human papillomavirus type 16 tripeptide vaccine and construction of tumor animal models that continuously express hpv16 E5, E6, E7

A HPV16E5, vaccine technology, applied in the direction of anti-tumor drugs, viral antigen components, peptides, etc., to achieve significant therapeutic and preventive effects, inhibition of tumor formation, inhibition of invasion effects

Active Publication Date: 2016-04-27
马丁
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Based on the above technical background and major medical needs, the present invention will disclose a polypeptide vaccine prepared for the three targets of type 16 HPVE5, E6, and E7. We hope that this vaccine with both preventive and therapeutic effects can effectively solve the current problems. Difficulties and shortcomings in the treatment of HPV16-positive tumors, and at the same time open up a broad road to immunotherapy for malignant tumors

Method used

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  • Screening and validation of human papillomavirus type 16 tripeptide vaccine and construction of tumor animal models that continuously express hpv16 E5, E6, E7
  • Screening and validation of human papillomavirus type 16 tripeptide vaccine and construction of tumor animal models that continuously express hpv16 E5, E6, E7
  • Screening and validation of human papillomavirus type 16 tripeptide vaccine and construction of tumor animal models that continuously express hpv16 E5, E6, E7

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0021] Example 1: Screening of HPV16E5, E6, E7 polypeptide fragments

[0022] The full-length sequences of HPV16E5, E6 and E7 were analyzed through the National Institute of Health Bioinformatics and Molecular Structure Analysis System, Antigen Epitope Prediction System and TAP Prediction System, and the corresponding fragments were obtained. We selected higher scores, strong affinity, and immunogenicity. And target fragments with better antigenicity are used as candidate fragments for the next step of laboratory screening. For details, see (http: / / bimas.dcrt.nih.gov / cgi-bin / molbio / ken_parker_comboform)[1](http: / / www.syfpeithi.de / )[2](http: / / www.imtech .res.in / cgibin / tappred / )[3] See the results Figure 1-3

[0023] 1. Parker, K.C., M.A. Bednarek, and J.E. Coligan. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 1994; 152: 163-175.

[0024] 2. Rammensee, H.G., J. Bachmann, N.P.N. Emmerich, et a...

example 2

[0026] Example 2: Construction of tumor animal models that continuously express HPV16E5, E6, and E7

[0027] 1) Construction of AAV-E5 destination vector

[0028] pEGFP-C1-HPV16E5 and pAAV-MCS were digested with restriction endonucleases EcoRI and BAMHI, respectively, to construct AAV-E5 recombinant plasmids.

[0029] ① Enzyme digestion system

[0030]

[0031] Mix well, 37 ℃, 2-4h or overnight, electrophoresis analysis and identification.

[0032] ② Gel purification: (U-gene kit, see Part 2.2.23 for specific operation)

[0033] ③Connection:

[0034]

[0035] Mix well, overnight at 4°C, and identify by electrophoresis analysis.

[0036] ④ Transformation screening: transform the competent bacteria DH5a with the ligation product. Coat the plate (kanamycin / IPTG / X-gel), carry out the blue and white spot screening, and select the white spot.

[0037] ⑤ Enzyme digestion identification: After extracting the plasmid DNA (Plasmid mini-extraction kit from Biotech, the operat...

example 3

[0079] Example 3: Further screening and identification of HPV16 E5, E6, E7 polypeptide fragments by in vivo and in vitro tests.

[0080] (1) Tumor growth

[0081] First, the mice were randomly divided into 9 groups, and rTC-1 cells in the logarithmic growth phase were taken from each group to prepare single-cell suspensions (1×10 6 / 100μl), inoculate 200μl at the root of the left thigh of each mouse, and give the corresponding peptide preparation to the root of the left thigh of the mouse one week later, CpG and saline, a total of 9 groups, after that, give the corresponding peptide preparation once a week, or CpG, or saline. Once every 1-2 days, observe the formation of tumor masses. After a tumor grows, observe and measure the tumor size 2-3 times a week (vertically measure the longest diameter of the tumor a, b) until the tumor is formed or 3-4 weeks after treatment, calculated by the formula 1 / 2*a*b tumor volume.

[0082] (2) ELISA method to detect the expression of im...

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Abstract

The invention discloses screening and verifying of a human papilloma virus (HPV) 16 tripeptide vaccine and a construction scheme of a tumor animal model for continually expressing HPV16 E5, E6 and E7. Polypeptide fragments with high scores are screened with a method for analyzing polypeptide fragments specific to a transporter associated with antigen processing (TAP) in combination with bioinformatics, a tripeptide vaccine core consists of three fragments with strongest immune effects obtained by performing in-vivo and in-vitro tests, and an HPV16 vaccine with preventing and treating functions is constructed in combination with a CPG adjuvant. Simultaneously, a tumor animal model for continually expressing HPV16 E5, E6 and E7 is constructed. The invention is technically characterized in that: by stably integrating HPV16E5 into a TC-1 cell through an adeno-associated virus expression system and injecting the modified TC-1 cell into a C57BL / 6 mouse body, a tumor can be formed successfully.

Description

1. Technical field [0001] The invention relates to the screening and verification of a human papillomavirus (Humanpappilomavirus, HPV) type 16 tripeptide vaccine and the construction of a tumor animal model continuously expressing HPV16 E5, E6 and E7. Its technical features are: designing the major histocompatibility complex I (MHC-I) antigen-binding epitopes of HPV16E5, E6, and E7 proteins for antigen processing-related transporters (transporter associated with antigen processing, TAP), and using bioinformatics analysis platform to screen out HPV16 tripeptides with high consistency, specificity and strong affinity were used as research objects to prepare HPV16 peptide vaccines, so as to obtain vaccines with dual functions of prevention and treatment for HPV16-positive tumors; at the same time, through the expression of adeno-associated virus The system stably integrates HPV16E5 into TC-1 cells, and then injects the modified TC-1 cells into C57BL / 6 mice, which can successfully...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K49/00A61K39/12A61K48/00G01N33/569C07K7/06C12N15/864A01K67/027A61P35/00
Inventor 马丁王世宣卢运萍廖书杰韩志强蒋学锋
Owner 马丁
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