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Mutant human plasminogen kringle5, preparation method and application thereof

A human plasminogen and mutant technology, applied in the biological field, can solve the problems of low expression yield, complex process, few exogenous amino acids, etc., and achieve the effects of improving biological activity, simplifying purification steps and low production cost

Active Publication Date: 2012-07-04
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although soluble and functional K5 can be obtained, the use of the Pichia pastoris expression system not only requires a huge investment, but also has problems such as methanol toxicity, difficulty in promoter regulation, too few screening markers, and differences in glycosylation from natural proteins. can't solve
In addition, there are some methods to obtain K5 using other eukaryotic cell lines, such as using recombinant baculovirus to express human plasminogen K5 in Spodoptera frugiperda ovary cells, but they have not been obtained due to low expression yield and complicated process. wide application
At present, K5 with high yield, few exogenous amino acids, and strong activity cannot be obtained in vitro by known methods, and the preclinical research and clinical trials of K5 are limited. Therefore, it is necessary to provide an improved K5 with a higher Method for producing human plasminogen K5 with high yield and simpler purification process

Method used

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  • Mutant human plasminogen kringle5, preparation method and application thereof
  • Mutant human plasminogen kringle5, preparation method and application thereof
  • Mutant human plasminogen kringle5, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Embodiment 1: be used for expressing the construction of the mK5 recombinant plasmid with GST mark

[0076] The nucleotide sequence of mK5 is as follows figure 2 shown. Synthetic primers were designed based on the K5 sequence, and the primers contained the nucleotide coding sequence of the N-terminal five neutral amino acid serines and restriction endonuclease enzyme cutting sites. Upstream primer: 5'-ACT GAATTC CCA TCT GTA TCG ACT CCT TCC TCA TCA TCC TGT ATG TTT GG-3', including the amino acid coding sequence of BamHI site and neutral amino acid (Ser) substitution mutation; downstream primer: 5'-TGCTGC CTCGAG TCA CGC ACA CTG AGG GAC ATC ACA GTA, which includes the stop codon followed by an XhoI site. Using human hepatocyte cDNA as a template, mK5 gene was obtained by PCR method. The PCR reaction conditions were: 95°C pre-denaturation for 5 minutes, 95°C for 45 seconds, 55°C for 60 seconds, 72°C for 60 seconds, a total of 30 cycles, and 72°C extension for 10 ...

Embodiment 2

[0078] Example 2: Expression and purification of GST-mK5 fusion protein and simple mK5 protein

[0079] Take the successfully transformed BL21 (DE3) strain for amplification, when the bacterial solution OD 600 When the concentration reaches about 0.6-0.8, add IPTG to a final concentration of 0.6 mmol / L, shake the bacteria at 25 °C and 220 rpm for 6 hours, and collect the bacteria by centrifugation. After the bacterial pellet was rinsed with distilled water, centrifuged, the pellet was resuspended in phosphate buffered saline (PBS) and then sonicated, centrifuged at 10,000 rpm for 30 min at 4°C, and the supernatant was filtered with a 0.45 μm filter membrane at 4°C for affinity chromatography. According to the operation manual of the product, pass the treated supernatant filtrate through the Glutathione Sepharose 4B affinity chromatography column, wash the affinity column with 300 ml washing buffer, add 20ml Elution buffer to incubate the Glutathione Sepharose 4B affinity chr...

Embodiment 3

[0081] Example 3: Identification of GST-K5 fusion protein and pure mK5 protein

[0082] The GST-mK5 protein and mK5 protein were analyzed by SDS-PAGE electrophoresis and identified by Western-blot.

[0083] 1. SDS-PAGE analysis: According to the method described in the literature (Sambrook et al, Molecular Cloning: A laboratory Manual, Cold Spring Harbour, 1989), take 20 μl of the above-mentioned eluted GST-mK5 protein and pure mK5 protein respectively, Add 4μl 6× loading buffer (7.2ml glycerol + 2.076g SDS + 1ml β-mercaptoethanol + 7ml 1M Tris PH6.8 + 0.012% (w / v) bromophenol blue, distilled water to 20ml) Afterwards, carry out boiling water bath 10min. The processed samples and 10 μl of commercial protein molecular weight standards were sequentially added to a 12% polyacrylamide gel for electrophoresis. After electrophoresis, stain the gel in Coomassie Brilliant Blue G250 solution (recipe: 0.25g G250, 90ml methanol:water = 1:1, 10ml glacial acetic acid) for 4 hours, and ...

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Abstract

The invention discloses a mutant human plasminogen kringle5 gene mK5 and an amplification primer of the mK5 gene, the mutant human plasminogen kringle5 gene modified by adding glutathione-S-transferase before the gene mK5, and a method for preparing protein mK5 recombinant protein of two gene codes, glutathioneS transferase (GST)-mK5 fusion protein and two proteins, wherein the mK5 gene and the GST-mK 5 gene can be applied to preparing medicaments for treating angiogenesis diseases. By the invention, the number of exogenous amino acid in the recombinant K5 protein molecules obtained by a geneengineering method is remarkably reduced, the K5 bioactivity of the obtained mK5 is improved, while the mK5 activity of the GST-mK5 fusion protein is maintained, the stability and water solubility ofthe protein are improved, the purification steps of an expressed product are simplified, and the purity of the product is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant human plasminogen kringle5 and its preparation method and application. Background technique [0002] Angiogenesis is the process of generating a large number of new blood vessels from the existing capillary network. Angiogenesis is involved in the occurrence and development of various diseases (Folkman J. Nat Rev Drug Discov. 6:273-286,2007). Angiogenesis is a disease that includes tumors (Folkman J.N Eng l J Med ,285:1182-1186,1971), diabetic eye disease (Campochiaro, et al. Expert Opin Biol Ther. 4:1395-1402, 2004), diabetic nephropathy (Yamamoto Y, et al. Diabetes. 53: 1831-1840. 2004), rheumatoid arthritis (Taylor P. C . et al. Curr Opin Rheumatol. 17:293-298, 2005), the main pathological features of more than 70 diseases . Worldwide, 500 million people suffer from diseases caused by angiogenesis or angiogenesis disorders. In the field of tumor therapy, Folkman first...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/58C12N9/68C07H21/04C07K19/00C12N15/09C12P21/02A61K48/00A61P9/10A61P35/02A61P3/10A61P29/00A61P27/02A61P13/12
Inventor 高国全杨霞蔡卫斌李朝阳
Owner SUN YAT SEN UNIV
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