12 peptide ZB for inhibiting hepatitis c virus to infect human cell and preparation method and application

A technology of E2-GST and BL21DE3, applied in antiviral agents, peptides, pharmaceutical formulations, etc., can solve the problems of individual patient differences, adverse reactions, and limited drug doses

Inactive Publication Date: 2009-06-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chronic hepatitis C treatment can have serious adverse effects, limiting doses and individual patient variability

Method used

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  • 12 peptide ZB for inhibiting hepatitis c virus to infect human cell and preparation method and application
  • 12 peptide ZB for inhibiting hepatitis c virus to infect human cell and preparation method and application
  • 12 peptide ZB for inhibiting hepatitis c virus to infect human cell and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0166] 1. The steps for preparing E2-GST fusion protein are as follows:

[0167] A. Pick Escherichia coli BL21DE3 containing pGEX-KG-E2 (purchased from U.S. Invitrogen Company) and cultivate it in 3ml of LB medium containing ampicillin (50g / ml), and control groups were inoculated with pGEX-KG (GST protein) Prokaryotic expression vector, purchased from Amersham Biosciences), cultured at 37°C for 12-16 hours.

[0168] B. Transfer the bacterial solution in the small test tube to 200ml LB liquid medium containing ampicillin (50g / ml) respectively (LB liquid medium is: 10g tryptone, 5g yeast extract, 10g sodium chloride, dissolved to 1000ml double-distilled water) at 37°C until the OD600 is 1-2.

[0169] C. Add 100 mM isopropyl-β-D-thiogalactoside (IPTG) so that the final concentration is 0.1 mM, and induce at 30° C. for 4 to 5 hours.

[0170] D. Divide the induced bacterial solution into 250ml centrifuge tubes, centrifuge at 7700g at 4°C for 5min, and discard the supernatant.

...

Embodiment 2

[0292] The steps of surface plasmon resonance (SPR) detection of affinity between polypeptide and E2 protein are as follows:

[0293] A. Let the test instrument Biocore (USA) pass through a period of HBS buffer until the baseline is stable.

[0294] B. Inject 40 μl of amino coupling reagent (containing 0.02M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.05M N-hydroxysuccinimide (NHS) ), activate the carboxyl group on the GM5 sensor chip.

[0295] C. Dilute the E2 protein to 5 mg / ml with an acetate buffer solution with a pH value of 5.0, and inject 40 μl of the solution.

[0296] D. On-machine detection, the flow rate is 20 μl / min.

[0297] E. Dilute the polypeptides PE2A, PE2B, PE2C, and PE2D to different concentrations, and react with the E2 protein on the CM5 sensor chip at 2 μl / min for 7 minutes.

[0298] F. Inject 10 μl of HCl regeneration solution at a flow rate of 2 μl / min to restore the baseline, and collect response signals in real time.

[0299] G. Per...

Embodiment 3

[0303] The steps for flow cytometry to detect the combination of polypeptides and target molecules are as follows:

[0304] A. Culture human liver cancer cells Huh7.5 (see reference Zhong, et al., Robust hepatitis C virus infection in vitro, 2005, PNAS, 102: 9294-9299) and DC-Sign-NIH3T3 (with DC-Sign molecules on the surface Mouse fibroblast) cells (see reference Wu, et al., Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGNinteractions with ICAM-3 do not promote human immunodeficiency virustype I transmission.J.virol.2002, 76 :5905-5914). The culture conditions were all DMEM medium (purchased by Gibco) with 10% fetal bovine serum, cultured at 37° C. in an incubator containing 5% carbon dioxide.

[0305] B. Mix polypeptides ZB and ZC with E2-GST protein respectively (GST protein is added to the control tube), the total volume is 50 μl, wherein the concentration of polypeptide is 500 μM, and the concentration of E2-GST protein and GST protein is 20 μg / ml, ...

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PUM

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Abstract

The invention discloses a dodecapeptide ZB capable of inhibiting the hepatitis C virus from infecting human body cells and a preparation method and application thereof. The preparation method comprises the following steps: firstly preparing an E2-GST fusion protein; then preparing an E2-GST fusion protein antibody; thirdly establishing a random DNA library; fourthly preparing a ribosome library; and fifthly screening the polypeptide bonded with the hepatitis C virus E2 glycoprotein through the ribosome display library so as to obtain the polypeptide specially bonded with the E2 protein. Due to the high affinity of the polypeptide ZB with the hepatitis C virus E2 glycoprotein, the polypeptide ZB can remarkably alleviate the intrusion of HCV into the human body cells, inhibit the binding force of HCV with human body hepatocytes, have a high inhibition capacity, and competitively inhibit the integration of a virus HCVE2 and a receptor CD81 after having an action with the E2 protein. The dodecapeptide is used for preparing the medicines capable of treating and preventing hepatitis C virus infection. In addition, the polypeptide is applied to a kit for detecting the hepatitis C virus envelope antigen.

Description

technical field [0001] The invention belongs to the field of molecular biology of viruses. More specifically, it relates to a 12-peptide ZB that inhibits hepatitis C virus from infecting human cells, and also relates to inhibiting the binding of hepatitis C virus to human hepatocytes and DC-SIGN + The preparation method of the polypeptide ZB of the target cell of the present invention also relates to a small molecule 12 peptide capable of targeting the envelope protein E2 of hepatitis C virus, that is, the use of the polypeptide ZB in the preparation of drugs for treating or preventing hepatitis C virus from infecting human cells , the polypeptide is also used as a kit for detecting hepatitis C virus envelope antigen. Background technique [0002] Hepatitis C virus (hepatitis C for short) is an inflammatory disease of the liver caused by hepatitis C virus (Hepatitis C Virus, HCV). HCV infection has a global distribution and is mainly transmitted through blood. According t...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P31/12A61P1/16G01N33/576
Inventor 章晓联
Owner WUHAN UNIV
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