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Novel protein expression system U2-OS and application thereof

A protein expression and U2-OS technology, applied in the field of bioengineering, can solve the problems of low yield and low activity, and achieve the effects of simple preparation method, efficient post-translational modification system and high efficiency

Inactive Publication Date: 2018-03-20
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Established a new human cancer cell hBMP-7 protein expression system, which overcomes the problems of low yield and low activity of the dimer glycoprotein hBMP-7 during the expression process

Method used

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  • Novel protein expression system U2-OS and application thereof
  • Novel protein expression system U2-OS and application thereof
  • Novel protein expression system U2-OS and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Expression of hBMP-7 in human osteosarcoma U2-OS cells

[0051] The self-signal peptide gene sequence of hBMP-7 described in this implementation:

[0052] SEQ ID NO:1

[0053] ATGCACGTGCGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGGCACCCCTGTTCCTGCTGCGCTCCGCCCTGGCC

[0054] The self-signal peptide amino acid sequence of hBMP-7 described in this implementation

[0055] SEQ ID NO:2

[0056] MHVRSLRAAAAPHSFVALWAPLFLLRSALA

[0057] The hBMP-7 mature peptide gene sequence described in this implementation

[0058] SEQ ID NO:3

[0059] TCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCCAAGAACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGCAGCAGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGAGACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCCGCCTACTACTGTGAGGGGGAGTGTGCCTTCCCTCTGAACTCCTACATGAACGCCACCAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAACCCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAATACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCAC

[0060] The hBMP...

Embodiment 2

[0095] Example 2 Expression of hBMP-7 recombinant adenovirus vector in human osteosarcoma U2-OS cells

[0096] Add its own signal peptide gene sequence at the 5' end of the hBMP-7 mature peptide gene sequence, and add a Not I restriction site at the 5' end of the signal peptide gene sequence, and add 6XHis tag and Hind at the 3' end of the hBMP-7 mature peptide gene sequence III restriction site. The designed human hBMP-7 gene (rhB7-hBMP-7) sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The synthesized gene sequence was digested with Not I and Hind III and inserted into the pShuttle-CMV shuttle vector to construct the recombinant adenovirus shuttle plasmid pShuttle-hBMP7. Eco RI digested pShuttle-hBMP7, and combined with pAdEasy TM DNA electroporation co-transformed Bj5183 competent cells, extracted the recombinant AdBMP7 plasmid, digested with Pac I to linearize the recombinant plasmid AdBMP7, transfected Ad293 cells for virus packaging and amplificati...

Embodiment 3

[0097] Example 3 Expression of hBMP-2 in human osteosarcoma U2-OS cells

[0098] Replace the amino acid sequence of hBMP-7 with the amino acid sequence of hBMP-2, add the signal peptide of hBMP-7 to the amino terminal of hBMP-2, and add a Hind III restriction site at the 5' end of the signal peptide gene sequence, add 6ΧHis tag and Bam HI restriction site. Both the recombinant expression vector rhB7-hBMP-2-pcDNA3.1 and the recombinant adenovirus expression vector AdBMP2 constructed separately can be expressed in human osteosarcoma U2-OS cells, and the expression products are glycosylated and have high ALP activity Recombinant protein activity obtained from mammalian or microbial protein expression systems.

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Abstract

The invention provides a cancer cell expression system for high-secretory dipolymer glycoprotein hBMP-7, which expresses human bone morphogenetic protein hBMP-7 by utilizing human-derived cancer cellvia a gene engineering technology by utilizing the characteristics of a cancer cell U2-OS rapid proliferation and galactosylated modification system. According to the invention, a recombinant expression vector with an amino terminal containing different signal peptides is constructed, and U2-OS cells capable of realizing efficient secretion and expression of hBMP-7 are screened; and the glycosylation state of recombinant hBMP-7 and biological activity thereof are purified and detected. According to the invention, a new human-derived cancer cell hBMP-7 protein expression system is established,thus laying a foundation for large-scale expression and preparation of cell factors, humanized antibodies, vaccines and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a U2-OS expression system for expressing glycoprotein with high secretion. Background technique [0002] Cancer cells are mutated cells that have the characteristic of infinite proliferation. In the past, the first thing people thought of when mentioning cancer cells was to eliminate or inhibit them. The present invention does the opposite, utilizes the characteristics of rapid proliferation of cancer cells, unlimited passage, easy in vitro culture, and complete glycosylation modification system, and uses human cancer cells to express and produce clinically needed important protein molecules. The method of the invention can greatly improve the gene engineering expression efficiency of the protein, and has broad application prospects. [0003] At present, protein expression systems mainly include prokaryotic expression system, yeast expression system, plant expression sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/12C07K14/51
CPCC07K14/51C12N5/0693
Inventor 付加芳王世立曹广祥宗工理薛俊英
Owner SHANDONG MEDICAL BIO TECH RES CENT
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