Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP)

A technology of human papillomavirus and L1 protein, which is applied in the field of virus-like particles, can solve the problems of insufficient uniformity of VLP, poor uniformity of virus-like particles, and lower recovery rate of target proteins, etc., to achieve easy industrial production and long service life , cost-saving effect

Inactive Publication Date: 2016-08-31
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the commonly used systems for expressing HPV L1 include poxvirus expression system, insect baculovirus expression system, yeast expression system, Escherichia coli expression system, etc., but they all have the problems of cumbersome purification process and poor uniformity of virus-like particles to varying degrees. , US6,245,568B1, CN101153280A patents first depolymerize the target protein in the sample, and then replace the buff...

Method used

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  • Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP)
  • Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP)
  • Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP)

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Embodiment 1

[0035] 1) Preliminary purification

[0036] Bacterial cell disruption: Harvest the Pichia pastoris cells transformed by fermentation and culture and express HPV16L1VLP, wash the bacterial cells three times with physiological saline, remove the remaining components of the fermentation medium, and freeze the collected bacterial cells at -20°C. Take out the frozen bacterial cells, weigh them and add 0.2M NaCl, 5mM EDTA-2Na, 0.05% Tween-80, pH 8.050mM PB buffer solution pre-cooled to 0°C at a ratio of 1:10 to the bacterial liquid, stir and thaw Until it is completely melted, high-pressure homogenate is broken, and the operating pressure of 1200bar is circulated twice to break more than 95% of the bacterial cells. The lysate is stirred at 4°C for 1 hour to completely dissolve the target protein in the lysed suspension. At the same time, 20mM DTT was added to completely depolymerize the target protein in a less uniform form into a more uniform L1 protein pentamer.

[0037] Centrifu...

Embodiment 2

[0045] 1) Preliminary purification

[0046] Sample pretreatment: Harvest the Escherichia coli cells transformed by fermentation and culture and express HPV18L1VLP, wash the bacterial cells once with physiological saline to remove the remaining components of the fermentation medium, and freeze the collected bacterial cells at -20°C. Take out the frozen bacterial cells, weigh them and add 0.2M NaCl, 5mM EDTA-2Na, 0.05% Tween-80, pH 8.050mM PB buffer solution pre-cooled to 0°C at a ratio of 1:10 to the bacterial liquid, stir and thaw Until it is completely melted, high-pressure homogenate is broken, and the operating pressure of 700bar is circulated twice, which can break more than 95% of the bacterial cells. The lysate is stirred at 4°C for 1 hour to completely dissolve the target protein in the lysed suspension. At the same time, 20mM DTT was added to completely depolymerize the target protein in a less uniform form into a more uniform L1 protein pentamer. Centrifuge the lysed...

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Abstract

The invention relates to the field of biomedicine and discloses a method for fast acquiring recombinant HPV (human papilloma virus) L1 virus-like particles (VLP). The method is the optimization of a hydroxyapatite chromatography (HAP) method. The method includes: cells expressing the recombinant HPV L1 are broken to obtain coarse sample liquid, preliminary purification is performed, reducing agent is added to allow target protein components to depolymerize sufficiently and uniformly exist in a pentamer state, HAP is performed under a certain condition, and the target protein L1 can self-assemble into VLP during elution. By the method greatly contributive to the increasing of the yield of the target protein, target protein purity can be increased effectively, the virus-like particles can be obtained during chromatography, the special depolymerization and reassembling process is omitted, and the separation and purification process of the target protein L1 is allowed to be simple and easy to control and suitable for industrial production.

Description

technical field [0001] The invention relates to a method for rapidly and effectively obtaining high-purity viroid-like particles from prokaryotic organisms (especially Pichia pastoris) expressing human papillomavirus L1 protein. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) belongs to papillomavirus family (Papovaviridae) papillomavirus genus. HPV is a small (50-60 nm), non-enveloped, icosahedral DNA virus composed of 72 pentamers. The viral genome is a double-stranded closed-circle DNA of about 8 kb, with 8 open frames (ORFs), and the ORFs can partially or completely overlap. The early region of the genome encodes six non-structural proteins, E1, E2, E4, E5, E6, and E7; the late region encodes two structural proteins, L1 and L2, among which L1 is the main structural protein with a molecular weight of 55-60kDa, and the L1 and L2 proteins are in It is very conserved in different papillomaviruses, the two together constitute the capsid of HPV...

Claims

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Application Information

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IPC IPC(8): C12N7/02
CPCC12N7/00C12N2710/20051
Inventor 王振伟李胜超孙永川董军纪熊洋蒋鹏李志广林小鹊苏彦景李文佳
Owner SUNSHINE LAKE PHARM CO LTD
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