Separation and purification of recombinant human blood vessel endothelia cell growth factor, chemical labeling in vitro and use thereof

A technology of biotin labeling and cells, which is applied in the field of recombinant VEGF protein and biology, can solve the problems of no separation and purification of recombinant VEGF protein method or data, cumbersome process, and limitations of industrial scale production and application

Inactive Publication Date: 2009-04-29
SUZHOU STAINWEI BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although the above documents all report the expression of human vascular endothelial cell growth factor gene by the Pichia pastoris system, and obtain the expression products with biological activity (i.e. the recombinant VEGF165 protein in document 1, and the VEGF121 protein in document 2), the two Both of them also have their own limitations: as in document 1, only the recombinant VEGF165 protein detected in the yeast cell supernatant was detected, bu

Method used

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  • Separation and purification of recombinant human blood vessel endothelia cell growth factor, chemical labeling in vitro and use thereof
  • Separation and purification of recombinant human blood vessel endothelia cell growth factor, chemical labeling in vitro and use thereof
  • Separation and purification of recombinant human blood vessel endothelia cell growth factor, chemical labeling in vitro and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Construction of VEGF165 expression vector.

[0085] 1.1 PCR amplification of VEGF165 gene

[0086] Forward upstream primer primer (VEGF-F-EcoR I): ggg tag aat tc g cac cca tgg cag aag gag ga

[0087] Reverse Downstream Primer Primer (v165-R-Not I): gga ttc gcg gcc gcc c gc ctc ggc ttg tca ca

[0088] PCR amplification system:

[0089]

[0090] PCR amplification conditions: 94°C for 5 minutes / 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 40 seconds, a total of 20 cycles.

[0091] After PCR amplification of VEGF165 gene was completed, a small amount was taken and analyzed by agarose gel electrophoresis. There was an amplified band at about 500 bp, and the size of the fragment was in line with the expectation.

[0092] 1.2 Construction and identification of VEGF165-pPic9K plasmid

[0093] The VEGF165 gene PCR amplified fragment and the pPic9K plasmid were digested with EcoR I and Not I respectively, and then separated and recovered by agarose gel...

Embodiment 2

[0094] Example 2: Expression Vector Transformation of GS115 Host Bacteria

[0095] 2.1 The VEGF165-pPic9K plasmid was prepared in 100ml bacterial liquid by alkaline lysis-PEG purification method. For specific steps, refer to the relevant chapters of "Molecular Cloning III".

[0096] 2.2 Take two tubes of about 15ug VEGF165-pPic9K plasmids and linearize them thoroughly with 30U Sal I and 30U Bgl II respectively. After digestion, the plasmid was precipitated with ethanol and redissolved with 5ul ultrapure sterile deionized water.

[0097] 2.3 Preparation of GS115 Competent Cells

[0098] (1) Inoculate a single clone in 3ml YPD medium and culture overnight at 30°C

[0099] (2) Put 300ul of the above-mentioned fresh bacterial solution in 300ml of YPD medium and culture at 30°C for 18 hours. At this time, the OD value is about 1.3.

[0100] (3) Bacteria were collected by centrifugation at 1500 g for 5 minutes, and then resuspended with 300 ml of ice-cold sterile deionized wate...

Embodiment 3

[0123] Example 3: Detection of VEGF target protein

[0124] The target protein was detected by methods such as SDS-PAGE and Wester-blot. The correct expression plasmid identified by DNA sequencing was transformed into GS115 yeast host bacteria by electroshock transformation method, and thousands of recombinant clones were obtained. Subsequently, screening was performed with gradient concentrations of G418, and dozens of colonies that grew well on YPD plates containing 4 mg / ml G418 were obtained. Three clones were picked for induced expression. After 96 hours of induction, the expression supernatant was collected by centrifugation for SDS-PAGE and Western-blot analysis, and the expression supernatant of the plasmid pPic9K transformed clone was used as a negative control. The result is as figure 1 : Compared with the negative group (lane3), after VEGF165-pPic9K-GS115 expression supernatant was reduced by DTT, specific bands (lane2, 4 and 5) can be seen at about 22-25KD; the ...

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Abstract

The invention relates to the field of biotechnology. A vascular endothelial cell grow factor (VEGF) plays an important function in the regeneration and hyperplasia of blood vessels; but the VEGF in the body has low content and a short half life; therefore, sufficient endogenous VEGF protein is hardly extracted to meet the ever growing experimental study and clinical requirements. The invention discloses a method for economically and conveniently preparing, extracting, purifying and recombining the VEGF protein of a human body through a recombination gene engineering and immunity affinity column method. The invention also discloses a method for carrying out the in-vitro non-radioactive chemical coupling label processing of the purified VEGF protein. The VEGF protein (including the protein having no label or having the chemical coupling label) can be widely applied to experimental study and clinical diagnosis and treatment.

Description

technical field [0001] The present invention belongs to the field of biotechnology. The invention relates to the preparation, separation and purification of biologically active recombinant VEGF protein by means of recombinant genetic engineering and affinity chromatography column technology; the invention also relates to a method for in vitro non-radioactive chemical coupling labeling treatment of the purified recombinant VEGF protein. The VEGF protein of the present invention (including chemically coupled labeled or unlabeled) has a wide range of applications in experimental research and clinical practice. Background technique [0002] Angiogenesis or hyperplasia (angiogenesis) biologically refers to the process in which existing blood vessels (such as capillaries and venules) in the body produce new blood vessels by sprouting or dividing. Angiogenesis is beneficial and necessary to maintain many normal physiological processes of the body, such as tissue embryonic developm...

Claims

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Application Information

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IPC IPC(8): C07K14/475C07K1/14C12N15/09C12N15/12G01N33/68G01N33/53C12N5/08C12N5/071
Inventor 周群敏罗师平周青陶飞龙胡红群谢凤娟
Owner SUZHOU STAINWEI BIOTECH INC
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