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Expression system of in vitro culturing skeletal muscle

An expression system and in vitro culture technology, applied in the field of establishment of skeletal muscle expression system, can solve problems such as purification effects, and achieve the effect of increasing secretion

Inactive Publication Date: 2006-01-25
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although QM7 cells can differentiate into myotubes and survive for a long time in a medium containing 0.5% serum, such a concentration of serum can still have an impact on subsequent purification and future drug development. The screening of suitable serum-free medium is to important

Method used

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  • Expression system of in vitro culturing skeletal muscle
  • Expression system of in vitro culturing skeletal muscle
  • Expression system of in vitro culturing skeletal muscle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 and Implementation Effect Examples 1-4 will illustrate the specific implementation of the in vitro cultured skeletal muscle expression system involved in the present invention.

[0023] Example 1

[0024] 1. Construction of the carrier

[0025] 1. Construction of prGH / PEX / hismyc expression vector

[0026] pIg / PEX / hismyc and prGH / PEX / hismyc are chicken PEX expression vectors constructed on the basis of pcDNA3.0. The coding sequence of myc10 peptide antigen tag and 6×his purification tag was fused at the C-terminal XhoI site of PEX, and the mouse immunoglobulin Ig light chain and rat growth hormone (rGH) were inserted between the N-terminal HindIII and BamHI sites, respectively. ) signal sequence.

[0027]Rat growth hormone (rGH) signal peptide sequence and chicken MMP-2 C-terminal fragment PEX sequence were amplified from rat pituitary gland and 11-day-old chicken embryo by RT-PCR. Rat Growth Hormone Primer: Sense: 5'-GGCAAGCTT(Hind III)ACAGATCACTGAGTGG-3',...

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Abstract

The invention relates to a skeletal muscle expression system cultivated in vitro that includes building the expression gene of the aim gene, expressing carrier transferring host cell group and filtering, expressing the aim gene in host cell and separating and purifying the expression product. The feature is that the expression carrier is constructed on the base of pcDAN3.0 and carrying myc10 testing mark and 6*his purified mark, and inserting 9(MMP-9) signal sequence into expression carrier. QM7 cell is adopted as host cell expression albumen. Non-serum separating culture medium suited to QM7 cell separating is filtered. The cost of culture medium is low and is benefit for the late purifying stage, and lays a strong foundation for large scale manufacturing medicine that uses PEX as rebuilding albumen.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the establishment of an in vitro cultured skeletal muscle expression system. Background technique [0002] With the continuous development of genetic engineering technology, a variety of expression systems can be used to produce human or animal-derived proteins with research or medical value. Although prokaryotic expression systems, yeast expression systems, and insect expression systems have high expression levels, their applications in drug production are greatly limited due to the lack of effective processing and modification of their products. Therefore, most of the therapeutic protein biological drugs that have been put on the market and are undergoing clinical trials come from mammalian cell expression systems. [0003] Although the proteins expressed by mammalian expression systems are competitive in quality and safety, the yields are lower and the costs are h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/09C12N15/63C12N15/65C12N15/79
Inventor 张志谦宋琳
Owner BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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