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Method for expressing human papilloma virus protein or preparing human cervical cancer vaccine by utilizing lettuce as host

A technology for human papillomavirus and human cervical cancer, applied in the direction of viruses, viral peptides, viruses/phages, etc., can solve problems such as hindering the development of plant exogenous protein drugs and increasing costs

Inactive Publication Date: 2020-07-10
王跃驹
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, tobacco has a high fiber content and potentially toxic compounds, such as the alkaloid nicotine, which significantly increase the cost in the downstream purification process, greatly hindering the further development of plant exogenous protein drugs

Method used

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  • Method for expressing human papilloma virus protein or preparing human cervical cancer vaccine by utilizing lettuce as host
  • Method for expressing human papilloma virus protein or preparing human cervical cancer vaccine by utilizing lettuce as host
  • Method for expressing human papilloma virus protein or preparing human cervical cancer vaccine by utilizing lettuce as host

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The construction of embodiment 1 plant transient expression vector

[0064] In order to provide high-efficiency expression of foreign aid proteins in plants, the present invention optimizes codons of human HPV16 L1 (variant 114K; EU118173) to plant-preferred codons, provided by GeneArt TM GeneOptimizer TM (ThermoFisher) and synthesized by GenScript. The Xbal restriction site was added to the 5' end of the optimized HPV16 L1 sequence, and the Sacl site was added to the 3' end, and cloned into the pUC57 vector by GenScript. The human HPV16 L1 gene fragment was isolated from pUC57-INS by Kpnl / Sacl and cloned into the binary plant vector pCam35S to generate the transient expression vector p35S-HPV16 L1 respectively. The plant expression constructs were individually transformed into Agrobacterium tumefaciens EHA105 by electroporation with a Multiporator (Eppendorf, Hamburg, Germany). The resulting bacterial strain was evenly spread on the selective YEP plate containing k...

Embodiment 2

[0065] Example 2 Agrobacterium-mediated vacuum infiltration

[0066] The invention optimizes the method of vacuum infiltration of Agrobacterium. The prepared Agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. The lettuce kept in this laboratory was turned upside down (core up) and gently swirled in the bacterial suspension, and the desiccator was sealed. The vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was seen in the leaf tissue. Maintain the pressure state for 30-60 seconds. The system is quickly opened to release the pressure and allow permeate to seep into the spaces within the tissue. It can be clearly seen that the permeate diffuses obviously in the lettuce tissue. The lettuce tissue was then gently removed from the permeate and rinsed three times consecutively with distilled water before being transferred to a container covered with plastic film. Keep the treated samples in the dark for 4 ...

Embodiment 3

[0068] Example 3 Protein Extraction and Separation

[0069] Lettuce samples vacuum-infiltrated with Agrobacterium were stirred with a stirrer and extracted with extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM at a ratio of 1:1 by volume). -Mercaptoethanol) high-speed homogenization in a blender for 1 to 2 minutes. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 min at 4°C to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%), and incubated with shaking on ice for 60 minutes. Centrifuge again (10,000 g) for 15 min at 4°C. The obtained supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, shaken and suspended on ice for 60 min, and centrifuged again at 10,000 g for 15 min at 4°C. Then, the supernatant was discarded, and the protein precipitated from the treated samples was dissolved in 5 mL of buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM ...

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Abstract

The invention relates to the technical field of biology, and particularly relates to a method for expressing human cervical cancer vaccine (Human Papillomaviruses, HPV) protein) by utilizing plants. The method utilizes the plants such as lettuce as an effective expression platform for producing recombinant protein, and utilizes a simple and effective agrobacterium-mediated vacuum infiltration method to express the human cervical cancer vaccine. The expression system determines that plant foreign protein can be collected after being infected by agrobacterium for 4 days. The SDS-PAGE method is utilized to determine that the recombinant human cervical cancer vaccine protein is successfully expressed. The expressed recombinant protein is self-assembled into virus-like particles and then used for immunizing New Zealand white rabbits, and the obtained serum proves that the human cervical cancer vaccine expressed by the plants has biological activity through an enzyme-linked immunosorbent assay (ELISA) and a pseudovirus particle neutralization test.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of lettuce as a host in expressing human papillomavirus protein or preparing human cervical cancer vaccine. Background technique [0002] Cervical cancer vaccine, also known as HPV vaccine, is a vaccine to prevent cervical cancer. Cervical cancer is mainly caused by infection with human papillomavirus (HPV). The vaccine can effectively prevent the occurrence of cervical cancer by preventing HPV infection, and can prevent human infection from the variation of human papillomavirus subtypes covered by the vaccine. Studies have found that 99.7% of cervical cancers are caused by infection with HPV, and HPV can also cause other relatively rare cancers, such as penile cancer, throat cancer, lung cancer and anal cancer. The main route of transmission of HPV is sexual or skin-to-skin contact. [0003] Human papillomavirus is a type of papillomavirus family, referred to as HP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025C12N15/82A61K39/12A61P35/00
CPCC07K14/005C12N15/8205A61K39/12A61P35/00C12N2710/20022C12N2710/20034
Inventor 王跃驹王海军庞小静
Owner 王跃驹
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