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Application of plants as host in M7842 expression

A plant and host technology, applied in the direction of microorganisms, animal/human proteins, antibody mimics/scaffolds, etc., can solve problems such as product safety hazards, infection of human diseases and insect pests, time-consuming expression systems, etc., to improve product safety and eliminate Effects of genetic pollution and elimination of potential pests and diseases

Pending Publication Date: 2019-09-10
王跃驹
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current expression system takes a long time, the purification process is complicated, there are problems such as gene contamination, potential pests and diseases that infect the human body, and the production cost is high, and there are hidden dangers of product safety

Method used

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  • Application of plants as host in M7842 expression
  • Application of plants as host in M7842 expression
  • Application of plants as host in M7842 expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The construction of embodiment 1 plant transient expression vector

[0072]In order to efficiently express foreign proteins in plants, the M7842 heavy chain, light chain, and amino acid sequences were obtained by backtranslation software (https: / / www.ebi.ac.uk / Tools / st / emboss_backtranseq / ) to obtain nucleotides sequence, and its codons were optimized to plant-preferred codons, which were synthesized by GenScript (Nanjing, China). An Xbal restriction site was added to the 5' end of the optimized M7842 heavy chain sequence, and a SacI site was added to the 3' end. Xbal restriction sites were added to the 5' end of the M7842 light chain sequence, and Sacl sites were added to the 3' end. and cloned into the pUC57 vector by GenScript Company to obtain pM7842-H and pM7842-L cloning vectors respectively ( Figure 1A , B), the gene fragments are separated from the cloning vector by Xbal / Sacl respectively, and cloned into the binary plant vector, pCam35S, to produce the plant ...

Embodiment 2

[0073] Example 2 Agrobacterium-mediated vacuum infiltration

[0074] Mix the prepared Agrobacteria containing p35S-M7842-H and p35S-M7842-L in equal amounts until the O.D.600 is 0.5. The 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. Hold the pressure for 30-60 seconds. The system is quickly opened to release the pressure and allow permeate to seep into the spaces within the tissue. This process was repeated 2 to 3 times until the penetration of the permeate into the lettuce tissue was clearly visible. 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. ...

Embodiment 3

[0075] Example 3 Protein Extraction and Separation

[0076] Lettuce samples vacuum-infiltrated by Agrobacterium were stirred with a stirrer, and homogenized at high speed in a blender with extraction buffer (100mM KPi, pH7.8; 5mM EDTA; 10mM β-mercaptoethanol) at a ratio of 1:1 by volume for 1-2 minute. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 minutes 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 (10,000g) for 15 minutes at 4°C. The obtained supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended by shaking on ice for 60 minutes, and centrifuged again at 10,000 g at 4°C for 15 minutes. 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 β-merc...

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Abstract

The invention relates to the technical field of biology, in particular to application of plants as a host in M7842 expression. Plants such as lettuce are used as an effective expression platform for recombination protein production, and the simple and efficient agrobacterium mediate vacuum infiltration method is adopted for expressing the M7842. The expression system determines that plant foreignproteins can be collected four days after infection of agrobacterium. The Western Blot protein hybridization method is adopted for determining successful expression of M7842, the AKTA protein purifying system is used for successfully separating out M7842. The activity testing result shows that the tumor growth restraining effect of M7842 on the mouse model is remarkable.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of plants as hosts in expressing M7842. Background technique [0002] At present, cancer has become the leading cause of death among Chinese residents. Doctors have been experimenting with so-called cancer immunotherapies since the 1850s. Because German doctors discovered that if a tumor becomes infected and triggers the patient's immune response, it sometimes shrinks. Now, some 160 years later, the idea of ​​mobilizing the body's natural defenses against cancer is finally having its moment. Pharmaceutical companies led by Merck & Co. and Bristol-Myers Squibb have developed a range of treatments designed to remove the barriers cancer cells use to evade the immune system. By releasing the body's disease-fighting T cells into tumors, the drugs have shown the potential to greatly extend the lives of some patients with advanced melanoma. In recent years, immunotherapy...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/82
CPCC07K16/2827C07K14/71C12N15/8205C12N15/8257C07K2319/00C12N2800/22
Inventor 王跃驹马磊张月恒
Owner 王跃驹
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