Artificially synthesized signal peptide and application thereof

A signal peptide, mammalian technology, applied in the direction of peptides, introduction of foreign genetic material using a carrier, and cells modified by introduction of foreign genetic material, etc., can solve the problems of low expression efficiency and high cost of foreign protein expression in animal cells, and achieve the The effect of increasing protein expression, increasing expression level and reducing production cost

Active Publication Date: 2010-12-22
SHANGHAI CHEMO WANBANG BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression efficiency of exogenous protein in animal cells is low and the cost is high. Therefore, improving the expression efficiency of exogenous protein in animal cells and reducing production costs are the top priorities in current work

Method used

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  • Artificially synthesized signal peptide and application thereof
  • Artificially synthesized signal peptide and application thereof
  • Artificially synthesized signal peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Synthesis of Signal Peptide

[0045] Because the signal peptide is short, the signal peptide is synthesized according to the primer. The NheI restriction site and Kozak sequence are added to the 5' end, and the target gene sequence is added to the 3' end, so as to add the signal peptide on the target gene.

[0046] Signal peptide 1: TCGGAGCTAGCCACC ATGGATGTTCTTGCTTTTCTTCTGGGCTTGCTGCTTTTGTGGCTTCCCGGGGTGAGGTGC GCCCCACCACG (SEQ ID NO: 11)

[0047] Signal peptide 2: TCGGAGCTAGCCACC ATGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTCCGGAGTGCGTTGC GCCCCACCACG (SEQ ID NO: 12)

[0048] Signal peptide 3: TCGGAGCTAGCCACC ATGAGGGTTCTGCCTGAATTCCTGGGACTTTTGTTGTTGTGGATTTCCGGCGTGCGATGT GCCCCACCACG (SEQ ID NO: 13)

[0049] Signal peptide 4: TCGGAGCTAGCCACC ATGGATGTACCACTTCAGCTTCTTGGCTTGTTGTTGCTTTGGCTTTCTGGCGTGAGATGT GCCCCACCACG (SEQ ID NO: 14)

[0050] Signal peptide 5: TCGGAGCTAGCCACC ATGGATGTGCCTGCTGAACTTTTGGGCCTTCTTTTGTTGTGGATATCAGGAGTACGATGC GCCCCACCACG (...

Embodiment 2

[0051] Embodiment 2 Amplification of the human EF-1α promoter

[0052] Using the plasmid pEF6 / V5-HisA (purchased from Invitrogen) as a template and the human EF-1α promoter sequence as a reference, primers F01 / R01 were designed and polymerase chain reaction was performed to amplify the human EF-1α promoter sequence, The reaction conditions are shown in Table 1.

[0053] F01: CATACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAG (SEQ ID NO: 16)

[0054] R01: ACGGCTAGCTCCGAGCTCGGTACCAAGCTTACCTAGCCA (SEQ ID NO: 17)

[0055] Table 1 PCR reaction conditions

[0056]

[0057] The resulting PCR product was ligated with SmaI-treated pUC57 (purchased from Fermentas), and sequenced for identification. The results showed that the sequence was as follows:

[0058] ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAA...

Embodiment 3

[0059] Embodiment 3 Amplification of the human EF1-HTLV promoter

[0060] Using the plasmid pFUSE-CHIg-hG3 (purchased from InvivoGen) as a template and the human EF1-HTLV promoter sequence as a reference, primers F02 / R02 were designed and polymerase chain reaction was performed to amplify the human EF1-HTLV promoter sequence, The reaction conditions are shown in Table 1.

[0061] F02: ACGACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGC (SEQ ID NO: 19)

[0062] R02: ATCGCTAGCGTAGGCGCCGGTCACAGCT (SEQ ID NO: 20)

[0063] The resulting PCR product was ligated with SmaI-treated pUC57 (purchased from Fermentas), and sequenced for identification. The results showed that the sequence was as follows:

[0064] ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCC...

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Abstract

The invention relates to an artificially synthesized signal peptide and application thereof. An amino acid sequence of the signal peptide is selected from any one of SEQIDNO: 1-5. The signal peptide can be added in a mammal zooblast expression vector for guiding a mammal source protein to secrete and express, and can improve the exogenous protein expression in a zooblast by 2-36 times.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a signal peptide used to guide the secretion and expression of a mammalian protein in a mammalian cell and an application thereof. technical background [0002] High-efficiency expression of foreign genes in host cells is a prerequisite for protein structure and function analysis, protein or polypeptide drug research and development. The expression systems used to express recombinant proteins include microorganisms, plants, yeast, insect cells, and animal cells. Mammalian cells are the best hosts for expressing naturally active proteins, and their advantages lie in the ability to correctly and effectively recognize the synthesis, processing and secretion signals of eukaryotic proteins, identify and remove introns in genes, and then undergo shearing and processing into mature cells. The mRNA can accurately complete glycosylation, phosphorylation, formation of intra-chain and inter-ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11C12N15/85C12N5/10C12P21/00
Inventor 周永春张玉晶厉颖
Owner SHANGHAI CHEMO WANBANG BIOPHARMA
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