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Preparation method and application of recombinant VEGFB

A purification method and technology of equilibration liquid, applied in the biological field, can solve the problems of unsatisfactory recovery rate and purity, many steps, complicated process, etc.

Pending Publication Date: 2021-11-05
沈阳百发科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method has many steps, and the process is complicated, and the recovery rate and purity are not ideal

Method used

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  • Preparation method and application of recombinant VEGFB
  • Preparation method and application of recombinant VEGFB
  • Preparation method and application of recombinant VEGFB

Examples

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preparation example Construction

[0072] The present invention provides a preparation method and application of recombinant VEGFB, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize it. What needs to be pointed out in particular is that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.

[0073] The test materials used in the present invention are all common commercially available products, which can be purchased in the market.

Embodiment 1

[0076] 1. By bioinformatics methods, the VEGFB nucleic acid sequence obtained by searching in the NCBI database is as follows.

[0077] >VEGFB-544bp

[0078] gaattcATGCCGGTGTCTCAGCCGGATGCGCCGGGTCACCAACGTAAA GTGGTTAGCTGGATCGATGTTTACACCCGTGCGACCTGCCAGCCGCGTG AGGTGGTTGTGCCGCTGACCGTGGAACTGATGGGCACCGTTGCGAAGC AGCTGGTGCCGAGCTGCGTTACCGTGCAACGTTGCGGTGGCTGCTGCCC GGATGATGGTCTGGAGTGCGTTCCGACCGGCCAGCACCAAGTGCGTATG CAGATCCTGATGATTCGTTATCCGAGCAGCCAACTGGGCGAGATGAGCCTGGAGGAACACAGCCAGTGCGAATGCCGTCCGAAGAAAAAGGACAGC GCGGTGAAGCCGGATAGCCCGCGTCCGCTGTGCCCGCGTTGCACCCAGC ATCATCAGCGTCCGGACCCGCGTACCTGCCGCTGCCGCTGCCGTCGTCG TAGCTTCCTGCGTTGCCAAGGTCGTGGCCTGGAACTGAACCCGGATACC TGCCGTTGCCGTAAGCTGCGTCGTGGCAGCCACCACCACCACCACCACT AAaagctt

[0079] According to the nucleic acid sequence information, the DNA of VEGFB is artificially synthesized.

[0080] 2. Using the DNA of the artificially synthesized VEGFB as a template to carry out PCR amplification.

[0081] Using the DNA of the artificially synthesized VEGFB ...

Embodiment 2

[0106] 1. Inclusion body dissolution

[0107] Dissolve 25 g of inclusion bodies (prepared in Example 1) in 250 ml of inclusion body solution, stir magnetically at room temperature until the solution is clear, centrifuge the solution at 10,000 rpm, 20 min, and 4°C, separate insoluble substances, and collect the supernatant liquid.

[0108] Inclusion body solution: Prepare 100ml according to the following proportions, dissolve in 80ml water, adjust pH to 8.5 and dilute to 100ml, ready for immediate use.

[0109] Tris 121mg NaH 2 PO 4 .2H 2 O(0.1M)

1.56g DTT (20mM) 0.309g Guanidine Hydrochloride (6M) 57.31g

[0110] 2 Affinity chromatography purification

[0111] ready to be fixed with Ni 2+ The chelating Sepharose Fast Flow column. Equilibrate the column with equilibration solution 1 and 3-5 volumes respectively, and the flow rate is 5-50ml / min. Load the dissolved sample at a flow rate of 5-50ml / min. Balance solution 2, balance sol...

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Abstract

The invention relates to the technical field of biology, in particular to a preparation method and application of recombinant VEGFB (vascular endothelial growth factor B). The preparation method of the recombinant VEGFB mainly comprises a purification method of the recombinant VEGFB and comprises the steps of inclusion body dissolution, affinity chromatography purification, renaturation and gel filtration chromatography, and the recombinant VEGFB obtained through purification not only has good yield and purity, but also has good physiological activity and has a good protection effect on retinal pigment epithelial cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of recombinant VEGFB. Background technique [0002] Vascular endothelial growth factor B (VEGF-B) is one of the members of human vascular endothelial growth factor (VEGF). VEGF-B gene is located on chromosome 11 q13, the full length of the gene is 4000bp, including 8 exons and 6 introns; VEGF-B is divided into two isoforms, VEGF-B167 contains more than 80% of the transcription information , contains a C-terminal binding domain that enables it to sequester on the cell surface; another isoform, VEGF-B186, also has a hydrophobic C-terminus that allows it to dissolve freely. VEGF-B contains the VEGF family logo sequence PXCVXXX-RCXGCC, and has 47% amino acid identity with VEGF-A, but studies have shown that VEGF-B does not have the property of promoting angiogenesis. Although VEGFB has no direct role in angiogenesis, it is associated with increases ...

Claims

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Application Information

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IPC IPC(8): C07K14/475C07K1/22C07K1/18C12N1/21C12N15/12C12N15/70A61K38/18A61P27/02C12R1/19
CPCC07K14/475C12N15/70A61K38/1866A61P27/02
Inventor 张朔李雪娇冯铃刘辉赵航吴骁康慧颖张鹏赵洪礼何伟
Owner 沈阳百发科技有限公司
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