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Human Ly6d recombinant protein cell secretory expression method and application

A technology for recombinant protein and cell secretion, which is applied in the direction of animal/human protein, receptor/cell surface antigen/cell surface determinant, recombinant DNA technology, etc. It can solve the problems of LY6D protein secretion and expression, which are rarely studied, protein insoluble, expression non-secretion issues

Active Publication Date: 2022-04-01
WUHAN AIBO TAIKE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on the secretory expression of LY6D protein
The expression of membrane proteins is a difficult point in in vitro expression technology, because there will be problems of non-expression or insoluble protein expression, and there will also be difficulties in expressing and not secreting in cells, or only a very small amount of protein can be obtained, which cannot meet the requirements. Subsequent experimental requirements, it is difficult to exert its biological function

Method used

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  • Human Ly6d recombinant protein cell secretory expression method and application
  • Human Ly6d recombinant protein cell secretory expression method and application
  • Human Ly6d recombinant protein cell secretory expression method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 plasmid construction

[0033] 1. Gene Ly6D related sequence

[0034] (1) Sequence information: SEQ ID NO.1

[0035] ATGAGGACAGCATTGCTGCTCCTTGCAGCCCTGGCTGTGGCTACAGGGCCAGCCCTTACCCTGCGCTGCCACGTGTGCACCAGCTCCAGCAACTGCAAGCATTCTGTGGTCTGCCCGGCCAGCTCTCGCTTCTGCAAGACCACGAACACAGTGGAGCCTCTGAGGGGGAATCTGGTGAAGAAGGACTGTGCGGAGTCGTGCACACCCAGCTACACCCTGCAAGGCCAGGTCAGCAGCGGCACCAGCTCCACCCAGTGCTGCCAGGAGGACCTGTGCAATGAGAAGCTGCACAACGCTGCACCCACCCGCACCGCCCTCGCCCACAGTGCCCTCAGCCTGGGGCTGGCCCTGAGCCTCCTGGCCGTCATCTTAGCCCCCAGCCTGTGA

[0036] (2) Amino acid sequence: SEQ ID NO.2

[0037] MRTALLLLAALAVATGPALTLRCHVCTSSSNCKHSVVCPASSRFCKTTNTVEPLRGNLVKKDCAESCTPSYTLQGQVSSGTSSTQCCQEDLCNEKLHNAAPTRTALAHSALSLGLALLSLLAVILAPSL

[0038] 2. Gene cloning and construction

[0039] The full-length protein 1-20 is a signal peptide, and 21-98aa is a final stable form, which is constructed into a mammalian expression vector and expressed in a 293 mammalian cell eukaryotic system. The following expression pla...

Embodiment 2

[0083] Example 2 protein expression

[0084] Eukaryotic HEK293F cells are divided into transient transfection and stable transfection. The transient transfection plasmid DNA is not integrated into the chromosome after entering the cell, and the exogenous gene is expressed in a free state. The transient transfection cycle is short, and the target protein can be obtained quickly, which can be applied to the exploration of the research and development stage and large-scale high-throughput screening of proteins. EXPI293F (human kidney embryonic epithelial cells), as a high-density expression system for mammalian cells, is widely used in the research, development and large-scale production of biological products such as recombinant DNA proteins.

[0085] 1. Transient transfection

[0086] 1. Plasmid DNA list

[0087] serial number gene name signal peptide area Label Plasmid concentration (ng / ul) Plasmid 1 Ly6d A 21-98aa C-6His 1230 Plasmid 2 ...

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Abstract

The invention belongs to the technical field of mammal cell recombinant protein secretory expression, and particularly relates to a human Ly6d recombinant protein cell secretory expression method and application. The inventor of the application designs the combination and collocation of three signal peptides and five tags for human Ly6d recombinant protein, finally realizes the secretory expression of the protein under the coexistence condition of signal peptide B and AVI-C-6His tag, signal peptide B and rabbit FC tag, and signal peptide C and C-Mouse FC-6His, and the combination and collocation of signal peptide B and rabbit FC tag has better protein expression quantity. The Ly6d recombinant protein is successfully obtained through WB verification and affinity purification, the expression quantity is about 255.67 mg / 1000mL Cells, and a transfection expression process can be optimized subsequently, so that the higher expression quantity is realized.

Description

technical field [0001] The invention belongs to the technical field of mammalian cell recombinant protein secretion and expression, and in particular relates to a cell secretion and expression method and application of human Ly6d recombinant protein. Background technique [0002] Lymphocyte antigen-6 (LY6) complex is a group of alloantigens, which were first discovered in mouse lymphocytes 40 years ago, and its main members are LY6A / D / E / K / H, LY6 family members evolution is very conservative. According to reports, LY6 family members play an important role in cancer. LY6K can lead to breast cancer cell metastasis. Clinical analysis shows that LY6K is related to tumor differentiation, lymph node metastasis and TNM staging in patients with non-small cell lung cancer. As one of the most important members of the LY6 family, LY6D is often used to develop new therapeutic agents for various cancer types. [0003] The expression of LY6D gene is increased in many types of cancers, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K14/705C07K19/00
CPCY02A50/30
Inventor 余乐程威盛鑫龙刘佩佩
Owner WUHAN AIBO TAIKE BIOTECH CO LTD