Hepatitis c virus NS3/4A protease, gene and method for detecting activity of inhibitor

A hepatitis C virus and protease inhibitor technology, which is applied in the field of anti-hepatitis C virus drug screening, can solve the problems of cumbersome and time-consuming cell models, difficult safety issues, difficult preparations, etc., and achieve high screening efficiency, low cost, avoid cumbersome effects

Active Publication Date: 2016-10-05
SHANGHAI INST OF PHARMA IND +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is aimed at the shortcomings of the complicated and time-consuming cell model used in the screening of hepatitis C virus drugs in the prior art, and the difficulty in ensuring safety issues, as well as the lengthy sequence and difficult preparation of the existing NS3 / 4A protease expression system. Deficiency, providing a method for hepatitis C virus NS3 / 4A protease and its gene and detection inhibitor activity

Method used

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  • Hepatitis c virus NS3/4A protease, gene and method for detecting activity of inhibitor
  • Hepatitis c virus NS3/4A protease, gene and method for detecting activity of inhibitor
  • Hepatitis c virus NS3/4A protease, gene and method for detecting activity of inhibitor

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Embodiment 1

[0038] Construction of embodiment 1 recombinant plasmid pET28a-NS3

[0039] 1) A total of 164 complete gene sequences of all reported hepatitis C subtype 1b subtypes were obtained from the NCBI website, and cluster analysis was performed using http: / / www.drive5.com / uclust software, and the ones with a similarity of more than 90% were selected. The most representative sequence was used as the research object, namely the sequence HCV-1b / US / BID-V130 / 1994 (GenBank: EU155330.2). The entire sequence encoding NS3 / 4A is selected, the full length of the nucleic acid of the sequence is 2055bp, encoding 685 amino acids. In order to increase the in vitro activity and stability of the NS3 / 4A protease, the gene sequence was codon-optimized, and the nucleotide sequence of the optimized NS3 / 4A protease gene is shown in SEQ ID No.1 in the sequence table. The optimized gene was synthesized by chemical synthesis (synthesized by Shanghai Jierui Bioengineering Co., Ltd.), and the synthesized full...

Embodiment 2

[0044] Example 2 Construction, induced expression and protein purification of recombinant strains.

[0045] 1) The recombinant plasmid pET28a-NS3 was transformed into the expression host Escherichia coli BL21(DE3), spread on an LB agar plate containing 30 μg / mL kanamycin, and cultured at 37° C. for 12 hours. Pick a single colony grown on the resistant plate and inoculate it in 5 mL of LB liquid medium containing 30 μg / ml kanamycin, culture overnight at 200 rpm at 37°C, extract the plasmid, and carry out Bam HI and NdeI double enzyme digestion identification to obtain recombinant strains. Save the verified correct strain.

[0046] 2) Inoculate 50 μL of the correct single colony verified in step 1) into 5 mL of LB, and shake overnight at 200 rpm at 37° C. to obtain an overnight culture. Take 500μL overnight culture in 50mL LB, shake at 200rpm at 37℃ and culture to OD 600 When it is 0.6, add 0.5mM IPTG to induce, 18°C, 200rpm to induce culture for 22h, then collect the bacteri...

Embodiment 3

[0048] Activity experiment of embodiment 3NS3 / 4A protease

[0049] React 100 μg / mL NS3 / 4A protease with substrate P06221, add NS3 / 4A protease and substrate to the reaction system, and incubate. The reaction system is: NS3 / 4A protease solution 95 μL, 5 μM substrate 2.5 μL, incubated at 30°C for 60 minutes, set up 5 parallel controls, and adjusted the pH to 6.5, 7, 7.5, 8 and 8.5, respectively. Fluorescence-pH curves were measured under different pH conditions. The result shows that when measuring 60min, the fluorescence intensity is the highest when showing pH value 7.5 ( Figure 4 ).

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Abstract

The invention discloses hepatitis c virus NS3/4A protease, a gene and a method for detecting activity of an inhibitor. The nucleotide sequence of the gene is a nucleotide sequence shown as SEQ ID No.1 in a sequence table or the encoded amino acid sequence of the gene is the nucleotide sequence of protein shown as SEQ ID No.2 in the sequence table. According to the method for detecting the activity of the inhibitor, the defects that when a common cell screening model is used for screening medicine, the process is complex, and cost is high are avoided, and the advantages of being clear in medicine acting mechanism, low in cost, high in efficiency, high in sensitiveness and capable of achieving high-throughput screening are achieved.

Description

technical field [0001] The invention belongs to the field of screening anti-hepatitis C virus drugs, and in particular relates to a method for detecting hepatitis C virus NS3 / 4A protease and its gene and inhibitor activity. Background technique [0002] Hepatitis C is a common blood-borne infectious disease, and its etiology is the infection of hepatitis C virus (Hapatitis CVirus, HCV). HCV infection of human hepatocytes can lead to chronic hepatitis, liver fibrosis, liver cirrhosis and liver cancer. Clinically, interferon combined with ribavirin is used to treat hepatitis C. However, the patient's compliance with this therapy is poor, and it will cause side effects and easy recurrence. Therefore, it is urgent to develop new drugs for the treatment of hepatitis C. [0003] HCV virus particles cannot replicate in vitro, so it is difficult to establish cell culture models and animal culture models in vitro, which greatly limits the development of specific HCV drugs. In recen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/70C12N1/21C12Q1/37C12R1/93
Inventor 朱宝泉杨丽鸳林军胡海峰周斌
Owner SHANGHAI INST OF PHARMA IND
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