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AFP recombinant protein, and in vitro high-efficiency recombination expression method

A recombinant protein and in vitro expression technology, applied in chemical instruments and methods, recombinant DNA technology, medical preparations of non-active ingredients, etc., can solve problems such as dysfunction and reduced ability of allogeneic lymphocyte proliferation

Active Publication Date: 2014-04-30
SHANGHAI CLAISON BIOTECH
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AI Technical Summary

Problems solved by technology

Basic research has shown that DC is the most functional professional antigen-presenting cell in the body, but because most DCs in patients with liver cancer are in an immature state, their function is impaired, and the ability to induce allogeneic lymphocyte proliferation is reduced

Method used

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  • AFP recombinant protein, and in vitro high-efficiency recombination expression method
  • AFP recombinant protein, and in vitro high-efficiency recombination expression method
  • AFP recombinant protein, and in vitro high-efficiency recombination expression method

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Embodiment 1

[0038] 1. Research materials: The recombinant plasmid pUC57-AFP was provided by an outsourcing service company; DNA markers, restriction endonucleases, T4 ligase, etc. were all purchased from Takara; pcDNA3.1+, CHO-S and Ni-NTA resins were all from Invitrogen The company's products; CHO-S cell culture medium FreeStyle CHO are all products of Gibco. PEI is a product of Polysciences.

[0039] 2. Workflow:

[0040](1) Optimize the DNA expression sequence of recombinant AFP in CHO: mainly optimize in three aspects: transcription, translation and protein folding. Transcription efficiency optimization: optimize GC content; SD sequence; TATA box; termination signal; CpG dinucleotide content. Translation efficiency optimization: optimize codon preference in CHO; mRNA secondary structure; PolyA early signal; repression site; RNA instability motif. Protein folding efficiency optimization: optimize RNA secondary structure; codon context correlation; codon-anticodon interaction.

[00...

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Abstract

The invention relates to an AFP recombinant protein, and an in vitro high-efficiency recombination expression method, and specifically relates to a recombinant protein of artificial signal peptide-AFP-609aa-6his, and an in vitro high-efficiency recombination expression method of mammal transient expression system. DNA sequence of the AFP recombinant protein is represented by SEQ ID No.1. The in vitro high-efficiency recombination expression method comprises following steps: a, DNA sequence of the AFP recombinant protein is optimized; b, gene of the AFP recombinant protein is cloned into pcDNA3.1+ carrier via double enzyme digestion, pcDNA3.1+-AFP expression plasmid is constructed, and plasmid extraction is performed; c, transfection of pcDNA3.1+-AFP plasmid, which is obtained via extraction, into CHO-S suspension cells is realized using PEI, and recombinant protein expression is preformed; and d, 7 days later, the AFP recombinant protein is obtained via separation and purification. Activity of the AFP recombinant protein is close to that of natural AFP protein, wherein the artificially synthesized AFP recombinant protein with 6HIS labels is prepared via large-scaled transient suspension CHO-S expression system; purity is relatively high; endotoxin content is low; no pollution is caused by animal-based protein; and it is possible for the AFP recombinant protein to be used as a tumor antigen vaccine in liver cancer immunotherapy.

Description

[technical field] [0001] The invention relates to an AFP recombinant protein and a method for high-efficiency recombinant expression in vitro, in particular to an artificial signal peptide-AFP-609aa-6his recombinant protein and a mammalian transient expression system in vitro recombinant expression method. [Background technique] [0002] Primary hepatocellular carcinoma (PHC, referred to as liver cancer) is one of the common malignant tumors, with an annual incidence of about 1.2 million worldwide. At present, the overall curative effect of liver cancer treatment is still far from ideal, and clinical treatment methods for liver cancer mainly include surgical resection and liver transplantation. Surgically resected patients have a high recurrence rate, with a recurrence rate of 50% at 3 years and 70% at 5 years. Liver transplantation, especially living donor liver transplantation, has made some progress, but it faces problems such as lack of donor sources. Therefore, there ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/00A61K47/48A61P35/00A61P1/16A61K47/62
Inventor 陆玲玲钱妮苏国新叶永清
Owner SHANGHAI CLAISON BIOTECH
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