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Establishment of HPV gene yeast external translating system

A technology of translation system and yeast, which is applied in the biological field to achieve high-efficiency response, low cost and ideal translation effect

Inactive Publication Date: 2011-04-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the deficiencies of existing in vitro translation systems, the problem to be solved in the present invention is to provide a method for establishing an HPV gene yeast in vitro translation system and the HPV gene yeast in vitro translation system established by this method

Method used

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  • Establishment of HPV gene yeast external translating system
  • Establishment of HPV gene yeast external translating system
  • Establishment of HPV gene yeast external translating system

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Embodiment 1

[0052] 1. Yeast cell culture

[0053] Brewer's yeast strains Y303 and YPH500 were inoculated with standard YPED liquid medium, and cultured under aerobic conditions at 28°C-30°C at 200rpm / min for 24 hours until the cells grew to 10 8 cells / ml, centrifuge at 2500 rpm for 10 minutes to collect the cells, and discard the supernatant for later use.

[0054] 2. Preparation of yeast cell protoplasts

[0055] Resuspend the yeast cell pellet with sterile water, centrifuge at 2500rpm for 10 minutes, discard the supernatant; then resuspend the pellet with 1M sorbitol, centrifuge at 2500rpm for 10 minutes, discard the supernatant; then resuspend the pellet in 20000U containing Lysozyme and 0.2 mM β-mercaptoethanol in SCE buffer (1 M sorbitol, 0.1 M sodium citrate, 10 mM EDTA, pH 6.8). The suspension was slightly shaken at 28°C-30°C in the dark for 3 hours, and checked under a microscope at any time to determine whether the yeast cell wall was fully digested.

[0056] After most of the...

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Abstract

The invention discloses a buildup method of an HPV gene yeast in vitro translating system. The improved yeast in vitro translating system built up by the method of the invention is particularly applicable to in vitro expression of coat protein of human papilloma virus (HPV), providing a convenient and rapid method for researching the coat protein expression system of the HPV. The system is also applicable to in vitro expression of other eukaryotic proteins at the same time. The buildup of the in vitro translating system of the invention provides an effective tool for the research of VLP forming and DNA package after terminal protein expression of the HPV and is hopeful to assist the research of HPV prevention and therapeutic vaccine. The method of the invention can also be used for preparing lysate of various yeast mutants researching the various factors that influence the protein translation after the transcription of eukaryotic mRNA and the reciprocity system of related molecular.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the establishment of an HPV gene yeast in vitro translation system. The improved yeast in vitro translation system established by the present invention is particularly suitable for the in vitro expression of human papillomavirus (HPV) capsid protein, and provides a convenient and quick method for studying the expression mechanism of HPV capsid protein; the system can also be applied simultaneously In vitro expression of other eukaryotic proteins. Background technique [0002] Human papillomavirus (Human papillomavirus, HPV) is a double-stranded DNA tumor virus with a size of about 8kb. HPV infection can lead to a variety of benign and malignant proliferative diseases of human skin and mucous membranes. High-risk HPV (type 16, 18, 45, 58) infection is closely related to the occurrence of malignant tumors such as cervical cancer. type) infection causes venereal genital wa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/37
Inventor 赵蔚明王晓于修平王红
Owner SHANDONG UNIV
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