Small interfering ribonucleic acid (siRNA) of targeted human protein disulfide isomerase (PDI) gene and application thereof

A technology of disulfide bond isomerase and human protein, which is applied in the field of molecular biology and biomedicine, can solve the problems that detection methods are difficult to detect, and there is no vaccine for hepatitis C

Active Publication Date: 2015-03-04
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hepatitis C is relatively hidden, and it is difficult to detect it with general detection methods
Also, unlike hepatitis A and B, hepatitis C has no effective vaccine to date, and limited clinical treatments have been successful in only a minority of patients

Method used

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  • Small interfering ribonucleic acid (siRNA) of targeted human protein disulfide isomerase (PDI) gene and application thereof
  • Small interfering ribonucleic acid (siRNA) of targeted human protein disulfide isomerase (PDI) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of the RNA interference plasmid vector for the PDI gene

[0028] 1.1 Design and synthesis of oligonucleotide sequences expressing PDI gene shRNA

[0029]Log in to Genebank, retrieve the full-length mRNA coding sequence of PDI gene P4HB, the gene sequence number is GI: 121256637, enter the sequence into the online database siRNA Wizard v3.1 to find the target site of siRNA, design the interference hairpin sequence and control sequence, The BamH I and EcoR I restriction sites were introduced at the ends, respectively, and submitted to Shanghai Shenggong Company for synthesis. Three pairs of shRNAs were tentatively named pLVX-shRNA1-1, pLVX-shRNA1-2, and pLVX-shRNA1-3.

[0030] 1.2 Annealing of oligonucleotide sequences

[0031] Centrifuge the dry powder of the synthesized RNA interference oligonucleotide fragment at 12,000 rpm for 30 s, add an appropriate amount of sterile ultrapure water to dissolve, and further dilute with TE solution to make th...

Embodiment 2

[0046] Example 2: Detection of the expression level of PDI in cells co-transfected with the PDI expression plasmid and the RNA interference plasmid of the PDI gene by immunoblotting to verify the effect of RNA interference

[0047] 2.1 Cloning of PDI gene and construction of eukaryotic expression vector

[0048] Retrieve human PDI gene sequence from NCBI database, use Primer Premier 5.0 software, design primers, introduce Hind III and EcoR I restriction site. Human 293T cells were lysed with Trizol, cellular RNA was extracted, and then reverse-transcribed with random primer Oligo(dT) to obtain cDNA. The PDI gene was amplified by PCR with synthetic primers SEQ ID NO: 10 and SEQ ID NO: 11. The amplification program was: 94°C 3min; 94°C 30s, 62°C 50s, 72°C 30s, 30 cycles ; 72 ? C extension 7min. After the PCR product of the PDI gene was recovered from the gel, it was simultaneously injected with the pCMV-Flag plasmid Hind III and EcoR I double enzyme digestion, the l...

Embodiment 3

[0053] Example 3: Effect of PDI Gene Interference on HCV Replication

[0054] 3.1 RNA interference analysis

[0055] Huh7.5.1 cells were cultured in DMEM containing 10% fetal bovine serum, and the cells were inoculated into six-well cell culture plates in DMEM medium without double antibody one day in advance, so that the cell density reached 10 6 . The next day, the extracted endotoxin-free ultrapure plasmid pLVX-shRNA1-PDI was transfected into Huh7.5.1 cells with liposome Lipofectamine 2000 Reagent. For specific steps, refer to the Invitrogen product instructions. After 6 hours of transfection, the HCV virus was inoculated at a ratio of MOI value = 1, cultured for 72 hours, the culture supernatant was collected, and the virus titer in the supernatant was determined by the TCID50 method.

[0056] 3.2 Determination of virus TCID50

[0057] Virus titers were measured using 96-well cell culture plates. Centrifuge the culture supernatant of infected cells at 8000 rpm for ...

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Abstract

The invention discloses a small interfering ribonucleic acid (siRNA) of targeted human protein disulfide isomerase (PDI) gene, of which the nucleotide sequence is selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. By using the protein coded gene sequence of the human PDI as the target gene, three 21nt target sequences are selected, and a complementary DNA (deoxyribonucleic acid) sequence comprising all the target sequences and a nonsense interfering DNA chain are designed and synthesized; and after annealing, a pLVX-shRNA1 expression vector is inserted to construct three interfering plasmids for PDI. The experiment indicates that the RNA interfering expression vectors have very high inhibiting actions on the expression of the disulfide isomerase protein and can generate certain influence on replication of HCV (hepatitis C virus). The siRNA has huge application value and prospects in research and / or identification of gene functions, and preparation of gene drugs for resisting infections of HCV and other viruses by using the host gene as the target spot.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and biomedicine, and relates to interference targets used for RNA interference on the human protein disulfide bond isomerase gene sequence, and small interfering RNA molecules obtained in various ways for these targets and Its application in the preparation of medicaments against HCV infection. Background technique [0002] Hepatitis C is a blood-borne disease with high incidence and difficult treatment, and is a serious social and public health problem. About 150 million people worldwide are chronically infected with hepatitis C virus (HCV), and more than 350,000 people die from HCV-related diseases every year. In my country, the chronic infection rate of HCV is 3.2%, which is one of the countries with a relatively high infection rate (WHO 2012). Hepatitis C is relatively hidden, and it is difficult to detect it with general detection methods. Moreover, unlike hepatitis A and B, ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85A61K31/7088A61K31/713A61P31/18
Inventor 张金阳叶承金宋玉竹韩芹芹夏雪山
Owner KUNMING UNIV OF SCI & TECH
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