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HCV RNA having novel sequence

A hepatitis C virus and gene technology, applied in the direction of viral peptide, virus/phage, recombinant DNA technology, etc., can solve the problem that the hypothesis cannot be verified, and it is not used to prove the HCV infection and proliferation model.

Inactive Publication Date: 2007-05-30
ADVANCED LIFE SCI INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, various proteins of HCV are expressed in cultured cells through gene recombination, and then many hypotheses have been made about the mechanism of disease manifestation and deterioration by analyzing the state of the expressed cells. However, because there are no suitable HCV infection and proliferation models to prove these hypotheses, Therefore the hypothesis cannot be tested

Method used

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  • HCV RNA having novel sequence
  • HCV RNA having novel sequence
  • HCV RNA having novel sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1. Separation and decoding of shortened sequence

[0111] Slice the patient’s liver with BP207 (0.5mm×1mm) in 100μl of RIPA buffer (20mM TriS-HCl [pH7.5], 150mM NaCl, 1% NP40, 0.1% deoxycholic acid, complete protease inhibitor mixture [Roche diagnostics Corporation]), centrifuged at 10krpm for 5 minutes, and then recovered the supernatant. Use a high-purity viral nucleic acid kit (Roche diagnostics corporation) to purify nucleic acid from the extract according to the method recommended by the manufacturer. HC9405R-1b primer was added to the purified nucleic acid, and MMLV reverse transcriptase (Invitrogen) was used to perform reverse transcription reaction at 42°C for 1 hour under the conditions recommended by the manufacturer to obtain cDNA.

[0112] RNaseH (Invitrogen) was added to the reaction solution and reacted at 37°C for 30 minutes to decompose RNA. Using a part of the reaction solution, in the presence of HC-LongAl primers and T7-HC9313R primers, use Klen...

Embodiment 2

[0145] Example 2. Isolation and analysis of cDNA of TF HCV genomic RNA from patients with Lyceum code

[0146] Next, 23 specimens from liver biopsy of liver with chronic active hepatitis and tissue specimens BP1, BP2, BP3 of liver cancer patients during surgery, and 3 specimens were tried to detect HCV-RNA of TF. Out.

[0147] RNA extraction

[0148] The RNA was extracted based on the rules of "Isolation of RNA from Micro Samples" of ISOGEN (Japan GENE Co., Ltd.), which is a test drug for RNA extraction. Add 0.8ml of ISOGEN to the patient's liver slice with a size of about 0.5mm×1mm, use a 1ml needle tip to decompose the tissue piece, and then pulverize by suction. Leave it in the greenhouse for 5 minutes, add 0.2ml of chloroform, stir vigorously for 30 seconds, and leave it at 4°C for 5 minutes. Centrifuge at 12,000 g in a small refrigerated centrifuge for 15 minutes at 4°C to recover the water phase.

[0149] Add about 5ug of yeast tRNA, add 0.8ml of isoproterenol, and pla...

Embodiment 3

[0213] Example 3. Preparation of HCV RNA replicon

[0214]By inserting a linking fragment obtained by heat-treating XhoX-Xba-s oligomer and XhoX-Xba-as oligomer between the XhoI and XbaI restriction sites of pBluescriptIISK(+), pBSIISK(+)△XX was constructed. In addition, pLV207-0007 was provided by PCR with Sbf_H1 primer and Cla_as primer, and a fragment of about 0.7kb was amplified, and then cloned by pGEM-T Easy to obtain pLVC-0007Sbf. By inserting NotI and ClaI between the NotI and XbaI restriction sites of pBSIISK(+)△XX, the fragment of about 0.7kb can be obtained by digesting pLVC-0007Sbf with ClaI and XbaI, and pLVC-ClaXba7.2K can be digested with ClaI and XbaI. The resulting fragment of about 7.2 kb yielded pSbf-LV207TF.

[0215] On the other hand, T7-HC9313b primer was added to HCV antibody-positive serum and RNA purified from G14, and cDNA was synthesized by SuperscriptII reverse transcriptase (Invitrogen) using the method recommended by the manufacturer. Using a part o...

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Abstract

A truncated form of hepatitis C virus gene obtained by deleting a portion of a region of a gene encoding NS2 protein from the core protein of hepatitis C virus while sustaining its translation frame. In particular, a gene as described above wherein the above-described portion of a region of the gene is located in a region encoding E1 protein and E2 protein.

Description

Technical field [0001] The present invention relates to the genome of chronic hepatitis C virus (hereinafter referred to as HCV). Background technique [0002] Hepatitis C virus (HCV) is the cause of chronic hepatitis C. According to statistics from the World Health Organization (WHO), there are 170 million infected people worldwide. HCV is different from other viral hepatitis in that it only causes relatively mild symptoms at the initial stage of infection, but the chronicity of the infected person is high, and after a certain period of asymptomatic period, chronic hepatitis develops. And as the infection progresses for a long time, the symptoms further worsen, transforming into cirrhosis of the liver, leading to a high frequency of liver cancer. 95% of liver cancers are related to hepatitis virus, and most of them (80%) are caused by HCV infection. [0003] HCV is infected through blood, blood components, and a few bodily fluid components. The HCV test method has been introduce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/18C07K16/10C12N5/10C12N7/00C12Q1/68
Inventor 八木慎太郎山口健次郎森健一田中荣司清泽研道槙升
Owner ADVANCED LIFE SCI INST
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