Solid-phase synthesis method of vMIP-II (viral macrophage inflammatory protein-II) protein

A solid-phase synthesis and protein technology, which is applied to peptide preparation methods, chemical instruments and methods, viral peptides, etc., can solve the problems of long purification cycle, industrial production and product application limitations, and high preparation cost, and achieve shortened production cycle, Improve the purity and synthesis efficiency of the target peptide, and improve the effect of purity

Inactive Publication Date: 2011-11-16
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The applicant has successfully used the E. coli prokaryotic expression system to express vMIP-II, but due to the need to go through the process of vMIP-II inclusion body denaturation and renaturation, the purification cycle is long, the preparation cost is also high, and industrial production and product application are limited.

Method used

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  • Solid-phase synthesis method of vMIP-II (viral macrophage inflammatory protein-II) protein
  • Solid-phase synthesis method of vMIP-II (viral macrophage inflammatory protein-II) protein
  • Solid-phase synthesis method of vMIP-II (viral macrophage inflammatory protein-II) protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 vMIP- II Peptide Synthesis and Purification

[0020] (1) Synthesis of vMIP-II polypeptide

[0021] Polypeptides were synthesized on a peptide synthesizer by Fmoc-solid-phase synthesis. After the reaction bottle was fully cleaned with 100% ethanol, about 40ml of 5% dimethyldichlorosilane was added and placed in a fume hood overnight to silanize the reaction bottle. After roughly washing with ethanol, place the reaction bottle in a -80°C refrigerator for 30 minutes to cool, wash with ethanol again, and dry it for later use; weigh 0.5mmol Wang's resin (Wang's resin) 0.7130g into the reaction bottle, add 3 times the volume of the resin DCM (dichloromethane) solution, swell for 30min and drain the liquid. Then, on the polypeptide synthesizer, it is carried out from the C-terminal to the N-terminal according to the cycle of coupling amino acid, ninhydrin reaction detection and α-amino deprotection until all amino acid residues are coupled. After the coupling i...

Embodiment 2

[0026] Example 2 Determination of polypeptide activity - vMIP-II to HIV-1 ⅢB Inhibition of induced cellular syncytia formation

[0027] Logarithmic growth phase MT 4 Cells (provided by the Guangdong Provincial Center for Disease Control and Prevention) were adjusted to 8×10 per ml with RPMI-1640 medium 6 Take 1ml centrifuge to discard the supernatant, add 500μl cell culture medium to resuspend the cells. Add 80μl MT to each well of a 96-well plate 4 Cell suspension, add HIV-1 ⅢB Viruses (Human HIV-1 ⅢB type) (provided by the Guangdong Provincial Center for Disease Control and Prevention) [approximately 1000 TCID 50 / (10 6 cell)] 20 μl, add 100 μl of different concentrations of vMIP-II (600, 60, 6 ng / ml) in different wells, and set 3 replicate wells for each concentration (the wells with vMIP-II are experimental wells).

[0028] At the same time, a blank control group and a positive control group were set up. Only normal MT was added to the blank control group 4 ...

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Abstract

The invention discloses a solid-phase synthesis method of vMIP-II (viral macrophage inflammatory protein-II) protein, comprising the following steps of: adding dimethyldichlorosilane into a reaction flask, ventilating and staying overnight to silanize the reaction flask, coarsely washing with ethanol and cooling at the temperature of minus 80 DEG C, washing and drying again, weighing resin and placing the resin into the reaction flask, adding three times volume of the resin of DCM (dichloromethane) solution, swelling for 30min and evacuating liquid, and then carrying out amino acid coupling, ninhydrin reaction detection and alpha-amino group deprotection circularly from a terminal C to a terminal N on a polypeptide synthesizer until all the amino acid residues are coupled; after the coupling is completed, taking down the reaction flask and cleaning with the DCM, draining the resin and then cutting with precooled cutting fluid, and purifying the cut polypeptide by adopting HPLC (High Performance Liquid Chromatography), wherein the polypeptide is purified on a Waters600E high pressure liquid phase chromatograph; and carrying out high pressure reversed phase chromatography by adopting a Kromasil100-5C18 pillar, and collecting target peak polypeptide, namely the vMIP-II protein.

Description

technical field [0001] The invention relates to a chemical synthesis method of polypeptide, in particular to a solid-phase synthesis method of protein vMIP-II. Background technique [0002] Viral macrophage inflammatory protein-II (vMIP-II) is a viral gene product encoded by Kaposi's sarcoma virus (also known as human herpesvirus 8). The whole vMIP-II gene encodes 94 amino acids, and the mature protein is 74 amino acids, which has a homology with human MIP as high as 41%, of which 8 non-polar amino acids account for 40%, and have strong hydrophobicity. [0003] vMIP-II protein can not only play a role in the pathological response of immune inflammation by binding to chemokine receptors, but also can act on chemokine receptors on immune cells to regulate the function of immune cells by agonizing or antagonizing receptors , has more research and application value in HIV infection / AIDS and transplant rejection. [0004] The applicant has successfully used the E. coli prokaryo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/03C07K1/06C07K1/04
Inventor 孙晗笑莫雪梅李秀英张光
Owner JINAN UNIVERSITY
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