Preparation method of recombinant protein

A technology of recombinant protein and ascites, applied in the biological field, can solve the problems of high technical requirements, poor flexibility, and high cost, and achieve the effect of good natural activity, good applicability, and low cost

Inactive Publication Date: 2012-10-10
ZHEJIANG CHINESE MEDICAL UNIVERSITY
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Vitro is the method of cell culture. Although new cell culture reactors are being developed continuously, the cost is still high and the yield is low.
In Vivo ready-to-use transgenic animals, ...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Method for preparing recombinant human hepatocyte growth factor (hHGF) in mouse peritoneal transgenic bioreactor

[0010] The recombinant human hepatocyte growth factor (hHGF) was cloned into the reading frame of the His tag of the eukaryotic expression vector pSP-His, and transfected into the mouse liver cancer cell line H22; several stably transfected transgenes were cloned after Zeocin screening After the cell line H22 / hHGF, use WB and ELISA to detect the protein expression level of the target gene in each clone line; select a high-expression clone cell line, and inject it into the mouse; 7-14 days later, extract the ascites, and use His affinity Ni column to separate and purify The target protein hHGF, the detected and calculated yield was 0.87mg / 100ml.

Embodiment 2

[0012] Method for preparing recombinant human leukemia inhibitory factor (hLIF) in rat peritoneal transgenic bioreactor

[0013] The recombinant human recombinant human leukemia inhibitory factor (hLIF) was cloned into the reading frame of the His tag of the eukaryotic expression vector pSP-His, and transfected into the rat ascites cell line Walker 256; multiple stable transfectants were cloned after Zeocin screening. After transfection of the transgenic cell line Walker 256 / hLIF, detect the protein expression level of the target gene in each clone with WB and ELISA; select a high-expression clone cell line and inject it into the rat; 10-18 days later, extract the ascites, and use His affinity Ni The target protein hLIF was separated and purified by the column, and the detected and calculated yield was 0.53mg / 100ml.

Embodiment 3

[0015] Method for Preparing Recombinant Coagulation Factor VIII (FVIII) in Mouse Peritoneal Transgenic Bioreactor

[0016] The recombinant human blood coagulation factor VIII was cloned into the reading frame of the His tag of the eukaryotic expression vector pSP-His, and transfected into the mouse liver cancer cell line H22; several stably transfected transgenic cell lines H22 / After FⅧ, use WB and ELISA to detect the protein expression level of the target gene in each clone line; select a high-expression clone cell line and inject it into the mouse; 7-14 days later, extract the ascites, and use His affinity Ni column to separate and purify the target protein coagulation factor VIII, the detection and calculation yield was 0.92mg / 100ml.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a preparation method of recombinant protein. In existing methods, some methods are high in cost and low in output, some methods are both high in technical requirements and cost, and preparing new protein needs long period, and is high in risk and poor in flexibility. The invention is characterized by cloning a recombinant protein gene into a eukaryotic expression vector and transfecting the vector into a mice ascites cell line, building a stabilized transfection transgene cell line through screening in vitro and cloning, and injecting the cell line into a mice abdominal cavity; and using the mice abdominal cavity as a transgene bioreactor for the transgene cell line to express the recombinant protein, extracting ascites after the ascites is formed and extracting and purifying the recombinant protein from the ascites. According to the method, the preparation of the recombinant protein is not only simple, highly efficient, quick and well applicable, but also low in cost, high in yield and good in natural activity of the recombinant protein.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for preparing recombinant protein using a mouse peritoneal transgenic bioreactor. Background technique [0002] The preparation of recombinant proteins is an important part of the modern biological industry chain. At present, many recombinant proteins are produced through prokaryotic expression. Although this method can be produced quickly and in large quantities, most of the proteins in eukaryotic cells have post-translational modifications, such as disulfide bonds and glycosylation, which are not modified in prokaryotic expression, so the product activity is greatly reduced. There are currently two types of eukaryotic expression systems: in vitro (In Vitro) and in vivo (In Vivo). In Vitro is the method of using cell culture. Although new cell culture reactors are constantly being developed, the cost is still high and the yield is low. In Vivo ready-to-use transgenic animals, s...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/85C12N5/10C07K14/00
Inventor 范春雷
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap