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Preparation method for human papillomaviral empty capsid particles, and products thereof

A technology of human papillomavirus and empty capsid particles, which is applied in the field of preparation of human papillomavirus empty capsid particles, and can solve the problems of high production cost, high energy consumption, and poor safety

Active Publication Date: 2012-01-18
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the defects of high production cost, high energy consumption and poor safety in the existing method for preparing human papillomavirus empty capsid particles, and provide a new human papillomavirus empty capsid particle preparation method. A method for preparing capsid particles, which uses a baculovirus expression system to safely and efficiently express an antigen or human papillomavirus empty capsid particles composed of two antigens in insects, which has the advantages of safety, high efficiency, low energy consumption, Low cost and many other advantages

Method used

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  • Preparation method for human papillomaviral empty capsid particles, and products thereof
  • Preparation method for human papillomaviral empty capsid particles, and products thereof
  • Preparation method for human papillomaviral empty capsid particles, and products thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Expression and immune experiment of embodiment 1 human papillomavirus (HPV) type 16 main capsid protein gene L1 in silkworm bioreactor

[0033] 1 Experimental method

[0034] 1.1. About the configuration of solution and medium

[0035] Solution I: 50mmol / L glucose, 25mmol / L Tris-HCl (pH8.0), 10mmol / L EDTA.

[0036] Solution II: 0.2mol / L NaOH, 1% SDS (prepared and used immediately).

[0037] Solution III: 100mL system, 5mol / L potassium acetate 80mL, glacial acetic acid 12mL, ddH 2 O8mL.

[0038] TAE (50×): 242g Tris base, 57.1mL glacial acetic acid, 100mL 0.5mol / L EDTA (pH8.0), dilute to 1000mL with sterile water.

[0039] TER solution: Pancreatic RNAse (RNAse A) was dissolved in 10mM Tris-HCl, 15mMNaCl, made into a 10mg / mL storage solution and stored at -20°C, then diluted with 1×TE buf to a 20μg / mL working solution and stored at 4°C .

[0040] PPt Buffer: isopropanol 22mL; 5mol / mL KAc 1mL; ddH 2 02mL.

[0041]PEG solution: Weigh a certain amount of NaCl to prep...

Embodiment 2

[0167] Embodiment Two papillomavirus (HPV) type 16 capsid protein gene L1-L2 combined expression and immune experiment in silkworm bioreactor

[0168] 1 Experimental method

[0169] 1.1. About the configuration of solution and medium

[0170] The routine reagent preparation of this embodiment is the same as embodiment one.

[0171] 1.2. Acquisition of the main capsid protein gene L2 and IRES gene of human papillomavirus (HPV) type 16.

[0172] 1.21 DNAiso (TaKaRa company) reagent method to extract genomic DNA

[0173] The preparation of human papillomavirus DNA in this example is the same as in Example 1.

[0174] 1.2.2 Design and synthesis of primers for the target gene

[0175] Primers were designed to amplify the human papillomavirus type 16 minor capsid protein L2 gene (SEQ ID NO: 3) by PCR. The amplification primers of the designed human papillomavirus type 16 minor capsid protein L2 gene are:

[0176] HPV16L2 upstream:

[0177] 5′-GG AGATCT TCAACATGCGACACAAACGTT...

Embodiment 3

[0322] Embodiment three human papillomavirus (HPV) type 18 main capsid protein gene L1 expression in the silkworm bioreactor and immune experiment

[0323] 1 Experimental method

[0324] 1.1. About the configuration of solution and medium

[0325] The routine reagent preparation of this embodiment is the same as embodiment one.

[0326] 1.2. Acquisition of the main capsid protein gene L1 of human papillomavirus (HPV) type 18.

[0327] 1.2.1 DNAiso (TaKaRa company) reagent method to extract genomic DNA

[0328] The preparation of human papillomavirus DNA in this example is the same as in Example 1.

[0329] 1.2.2 Design and synthesis of primers for the target gene

[0330] Primers were designed to amplify the human papillomavirus type 18 main capsid protein L1 gene (SEQ ID NO: 5) by PCR. The amplification primers of the designed human papillomavirus type 18 main capsid protein L1 gene are:

[0331] HPV18L1 upstream:

[0332] 5′-CGCC GGATCC AACATGTGCCTGTATACACGGGTCCTG-3...

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Abstract

The present invention discloses a preparation method for human papillomaviral (HPV) empty capsid particles, and products thereof. The preparation method comprises: (1) inserting main capsid protein gene of the human HPV empty capsid particles or a combination coexpressed from the main capsid protein gene of the human HPV empty capsid particles and the secondary capsid protein gene of the human HPV empty capsid particles into baculovirus genome to construct recombinant baculovirus; (2) transfecting an insect host or insect cells through the constructed recombinant baculovirus; (3) culturing the transfected insect host or the transfected insect cells, collecting and purifying the expressed antigen to obtain the human HPV empty capsid particles. According to the preparation method, the baculovirus expression system is adopted for safely and efficiently producing the human HPV empty capsid particles in a bombyx mori bioreactor; with adopting the method provided by the present invention, the production cost for the human HPV empty capsid particles can be significantly reduced; the method has advantages of safety, high performance, less energy consumption, low cost, and the like.

Description

technical field [0001] The invention relates to a method for preparing human papillomavirus empty capsid particles, in particular to a method for expressing human papillomavirus (HPV) type 16 and type 18 capsid protein genes in insects by using recombinant baculovirus and the method prepared by the method The product belongs to the field of preparation of human papillomavirus empty capsid particles. Background technique [0002] Cervical cancer is a relatively common gynecological malignancy in China. It is a serious threat to women's life and health. There are about 500,000 new cases of cervical cancer every year in the world, and about half of them die. In recent years, cervical cancer has been increasing year by year, especially among younger urban women. Studies in the past few decades have proved that high-risk HPV infection is an important cause of cervical cancer. HPV16 and / or HPV18 can be detected in 80% to 90% of cervical cancer tissues. HPV16 and HPV18 are consid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/37C12N15/62C12N15/866C12N7/04C12N7/01A61K39/12A61P31/20A61P35/00C12R1/93
Inventor 张志芳李轶女边大勇易咏竹杨标王国增沈桂芳舒惠国
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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