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Artificially synthesized signal sequence and application thereof

A signal peptide and mammalian technology, applied in peptides, using vectors to introduce foreign genetic material, cells modified by introducing foreign genetic material, etc., can solve the problems of high cost and low efficiency of exogenous protein expression in animal cells, and achieve expression The effect of increasing the level, increasing the expression of foreign proteins, and reducing production costs

Active Publication Date: 2014-09-24
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 sequence and application thereof
  • Artificially synthesized signal sequence and application thereof
  • Artificially synthesized signal sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The synthesis of embodiment 1 signal peptide

[0050] 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.

[0051] Signal peptide 1: TCGGAGCTAGCCACC ATGGATGTTCTTGCTTTTCTTCTGGGCTTGCTGCTTTTGTGGCTTCC

[0052] CGGGGTGAGGTGC GCCCCACCACG (SEQ ID NO: 11)

[0053] Signal peptide 2: TCGGAGCTAGCCACC ATGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTC

[0054] CGGAGTGCGTTGC GCCCCACCACG (SEQ ID NO: 12)

[0055] Signal peptide 3: TCGGAGCTAGCCACC ATGAGGGTTCTGCCTGAATTCCTGGGACTTTTGTTGTTGTGGATTTC

[0056] CGGCGTGCGATGT GCCCCACCACG (SEQ ID NO: 13)

[0057] Signal peptide 4: TCGGAGCTAGCCACC ATGGATGTACCACTTCAGCTTCTTGGCTTGTTGTTGCTTTGGCTTTC

[0058] TGGCGTGAGATGT GCCCCACCACG (SEQ ID NO: 14)

[0059] Signal peptide 5: TCGGAGCTAGCCACC ATGGATGTGCCTGCTGAACTTTT...

Embodiment 2

[0061] Embodiment 2 Amplification of the human EF-1α promoter

[0062] 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. F01: CATACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAG (SEQ ID NO: 16)

[0063] R01: ACGGCTAGCTCCGAGCTCGGTACCAAGCTTACCTAGCCA (SEQ ID NO: 17)

[0064] Table 1 PCR reaction conditions

[0065]

[0066] 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:

[0067] ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGT...

Embodiment 3

[0068] Embodiment 3 Amplification of the human EF1-HTLV promoter

[0069] 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.

[0070] F02: ACGACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGC (SEQ ID NO: 19)

[0071] R02: ATCGCTAGCGTAGGCGCCGGTCACAGCT (SEQ ID NO: 20)

[0072] 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:

[0073] ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCC...

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Abstract

The invention relates to an artificially synthesized signal sequence and an application thereof. The amino acid sequence of the signal sequence is SEQ ID NO:5. The signal sequence can be added into a mammalian cell expression vector for guiding secreting expression of a mammalian-derived protein, and can increase the expression quantity of an exogenous protein in animal cells.

Description

[0001] This case is a divisional application of the following patent application: [0002] Application number: 201210118230.9; [0003] Application date: April 20, 2012; [0004] Invention name: an artificially synthesized signal peptide and its application technical field [0005] 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 [0006] 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 synthe...

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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