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Recombinant polypeptide having islets beta- cell protection function

A technology for recombinant polypeptides and protective effects, applied in the field of biomedicine, can solve problems such as cheap, laborious, and time-consuming

Inactive Publication Date: 2008-04-16
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The Escherichia coli expression system is widely used for the expression of recombinant proteins. One of the main obstacles to the production of recombinant proteins using the E. coli system is that the recombinant proteins often form insoluble and inactive inclusion bodies. Although some renaturation processes have been developed, but Time-consuming, labor-intensive, inexpensive, and the effect is uncertain

Method used

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  • Recombinant polypeptide having islets beta- cell protection function
  • Recombinant polypeptide having islets beta- cell protection function

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Experimental program
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Effect test

Embodiment 1

[0030] Cloning of embodiment 1 recombinant cSHAP gene

[0031] (1) Extraction of total RNA from shark (Chiloscyllium Plagiosum) liver

[0032] (2) Wu Wutong's laboratory obtained the N-terminal sequence LVGPIGAVGP of the natural shark liver active peptide in the previous work, and designed a merger primer: 5'-AA(C)TIGTIGGGICCIATC(T)GGIGCIG-3' as the upstream primer, and the downstream primer: oligodT18;

[0033] (3) Using shark liver cDNA as template, by PCR method (94 DEG C for 45s, 56 DEG C for 50s, 72 DEG C for 1min, 30 cycles; 72 DEG C for 10 min to amplify the SHAP coding sequence (accompanying drawing 1); obtain two gene fragments , the sizes are 350bp and 610bp respectively, the molecular weight of the 350bp gene partial code protein is similar to the natural active protein, so we choose the 350bp gene sequence as the coding sequence of the recombinant protein;

[0034] (4) The PCR product is cloned into the vector pGEM-T-easy to obtain the vector pT-SHAP containing th...

Embodiment 2

[0042] Example 2 Establishment of cSHAP secretion expression, separation and purification process

[0043] (1) Screening of cSHAP secretion and expression conditions

[0044] The cloned bacteria were transferred into LB medium, and the expression was induced at different culture temperatures (37°C, 28°C) with different inducer IPTG concentrations (1mM, 0.5mM, 0.1mM); Proteoproteins, as well as cytoplasmic proteins, were analyzed by 15% SDS-PAGE for protein expression; SHAF was secreted and expressed at 0.5mM and cultured at 28°C (accompanying drawing 3);

[0045] (2) Separation and purification of cSHAP

[0046] A. Extraction of periplasmic protein: Induced expression bacterial solution, centrifuged at 4°C, 12000r / min for 5 minutes, collected bacterial cells; suspended bacterial cells in ice-cold sucrose buffer solution with 1 / 10 volume of original bacterial liquid, stirred slowly in ice bath 15min. Then centrifuge at 12000r / min at 4°C for 5min to collect the bacteria; susp...

Embodiment 3

[0051] The mouse insulinoma β-cell strain NIT-1 was used as the target cell, and streptozotocin (STZ) had a damaging effect on the target cell. Take the NIT-1 cells in the logarithmic growth phase, digest them with trypsin, and use 5×10 4 Inoculate 6-well plates at a concentration of 3ml per well, culture for 24 hours, and add STZ solution to each test well at a final concentration of 5mM / L after the cells have adhered to the wall. After 1 hour, add the final concentration to each well of the test group at the same time 45 μg / ml, 15 μg / ml, 0 μg / m1 of the recombinant protein cSHAP dissolved in DMEM high-glucose medium (containing 0.5% fetal bovine serum), cultured at 37°C for 24 hours in a 5% CO2 incubator, digested and collected each Group cells, wash cells with pre-cooled PBS, fix cells with ice-cold 70% ethanol overnight, collect cells, suspend cells in each group with pre-cooled PBS, add RNaseA, act at 37°C for half an hour, add stain PI and place at room temperature for 20...

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Abstract

The present invention provides a recombinant polypeptide with protective function on pancreatic islet beta-cells, wherein the encoding sequence of the recombinant polypeptide is clarified through PCR, based on N-sequence of LVGPIGAVGP of shark hepatic active peptide, with shark hepatic cDNA as the template, while the secreted expression vector of the recombinant polypeptide is constructed, the acquired recombinant polypeptide is capable of suppressing the STZ-induced apoptosis of pancreatic islet cells NIT-1 in vitro.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant polypeptide with protective effect on pancreatic islet β-cells, as well as the cloning, expression and purification of the polypeptide. Background technique [0002] The liver is not only the largest digestive gland in the body, but also an important immune organ. It has very complex functions, including metabolism, synthesis and storage, as well as the function of regulating the internal environment of the body, water and electrolyte balance, blood volume, and blood sugar levels. The liver can synthesize a variety of active substances, among which the research on active peptides and proteins has attracted widespread attention from scholars at home and abroad. Scholars at home and abroad have isolated some proteins or peptides with different biological activities from the livers of various animals and humans (LaBrecque DR. Hepatic stimulator substance discovery, character...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/17A61P43/00A61P3/10
Inventor 吴梧桐吕立力欧瑜
Owner CHINA PHARM UNIV
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