Construction method of recombinant adeno-associated virus vector of targeting interference SOCS1 gene and application of construction method

A technology of SOCS1 and viral vectors, applied in the fields of molecular biology and biomedicine, can solve the problems of short duration, small molecule RNA is easily degraded by RNase, and great difference in transfection efficiency

Inactive Publication Date: 2013-06-26
吕成伟
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the application of RNAi technology, the use of chemically synthesized siRNA has the disadvantages of small molecule RNA being easily degraded by RNase in vitro, low transfection efficiency, and short duration; plasmid-mediated RNAi also has limitations, and transient transfection in different The transfection efficiency of host cell plasmids varies greatly, especially for primary cells, stem cells and other systems that are difficult to meet the experimental requirements, and adenovirus and retrovirus are biologically toxic and have great potential related risks, which limits their clinical application

Method used

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  • Construction method of recombinant adeno-associated virus vector of targeting interference SOCS1 gene and application of construction method
  • Construction method of recombinant adeno-associated virus vector of targeting interference SOCS1 gene and application of construction method
  • Construction method of recombinant adeno-associated virus vector of targeting interference SOCS1 gene and application of construction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Obtaining of target gene fragments

[0025] The primers and DNA sequences used were synthesized by Shanghai Sangon Biotechnology Company, and the DNA sequencing was completed by Huada Gene Company.

[0026] hU6 promoter primer sequences: U6F: 5'-ATATGCATCCAAGGTCGGG CAGGAAGAGGGCCTAT-3' and U6R: 5'-ATCTCGAGATCGATGC GGCCGCCA TATGGA-3'.

[0027] According to the human SOCS1 gene sequence (NM_003745) registered in NCBI Genebank and the shRNA design principles, the RNAi Design software of Ambion Company was used to design and synthesize an effective RNAi sequence for the SOCS1 gene:

[0028] 5′-CAGCTTAACTGTATCTGGA-3′

[0029] The DNA sequence used to transcribe the shRNA is:

[0030] Sense strand: 5′-TCGAC CAGCTTAACTGTATCTGGA AAGC TCCAGATACAGTTAAGCTGTTTTTT-3′;

[0031] Antisense strand: 5′-CTAGAAAAAA CAGCTTAACTGTATCTGGA GCTT TCCAGATACAGTTAAGCTG G-3′;

Embodiment 2

[0032] Embodiment 2 pAAV2-SOCS1-shRNA vector construction and identification

[0033] A. Using PCR amplification technology to amplify the hU6 promoter

[0034] Using primer U6F: 5′-ATATGCATCCAAGGTCGGGCAGGAAGAGGG CCTAT-3′

[0035] and U6R: 5′-ATCTCGAGATCGATGCGGCCGCCA TATGGA-3′,

[0036] The sequence fragment containing the hU6 promoter was amplified from the PAVU6+27 plasmid by PCR reaction and identified by agarose gel electrophoresis ( figure 1 ), gel recovery and purification of the product.

[0037] B. After the amplified fragment was digested by SalI and XbaI, it was ligated with the SOCS1-shRNA interference sequence, identified by agarose gel electrophoresis, and the purified product was recovered from the gel to obtain the complete hU6 promoter and interference sequence, and sequenced in parallel Identification.

[0038] C. Digest pAAV2 and hU6 promoters and interference sequences with XhoI and NsiI, recover large fragments respectively, insert hU6 promoters and int...

Embodiment 3

[0043] Example 3 Detection of recombinant adeno-associated virus transfection efficiency

[0044]Aseptically extract 50 mL of peripheral blood from healthy volunteers, anticoagulate with heparin, separate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient method, culture them in 6-well plates, adhere to the wall for 5 hours, and gently wash away the suspension Lymphocytes, the remaining adherent cells are DC precursor cells, add 2.5ml of AIM-V medium containing GM-CSF (800U / mL) to each well, change half of the medium every other day, and add from the second day of culture IL-4 (1000U / mL). DCs were infected with recombinant adeno-associated virus (rAAV / shRNA) on the third day of culture, and the experiment was divided into interference group, negative control group (NC) and blank control group (Mock). The multiplicity of infection (MOI) was 100. After 8 hours, the virus-containing medium was removed and replaced with fresh AIM-V medium, and TNF-a (20ng / mL) wa...

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Abstract

The invention belongs to the biomedical field and relates to a construction method of a shRNA (short hairpin RNA, shRNA) recombinant adeno-associated virus vector for specially restraining SOCS1 gene expression and an application of the construction method. The shRNA recombinant adeno-associated virus vector is transferred to a monocyte-macrophage-dendritic cell system for lowering the SOCS1 gene expression and strengthening the anti-tumor activity of CTL (Cytotoxic T Lymphocyte). Therefore, the construction method can play an important role in the biomedical field and the research of gene function, can be used for preparing anti-tumor dugs and is high in clinical application value.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, in particular to shRNA recombinant adeno-associated virus vector and its construction method and its application in the preparation of antitumor drugs and vaccines. Background technique [0002] Suppressor of cytokine signaling (SOCS) is a family of negative regulatory molecules of the signal transduction pathway JAK / STAT (Janus kinase / signal transduction and activators of transcription). The SOCS family includes eight members (CIS, SOCS1-7), among which SOCS1 is related to the signal transduction of various cytokines. SOCS1 is a negative regulator of various cytokines. DCs activated by TLR signals produce a large number of cytokines, including IL-12, TNF-α, IL-6, IFN-α / β, IFN-γ, etc., most of which Regulated by SOCS1. Studies have shown that SOCS1 is involved in the occurrence, maturation and activation of DC, and plays an important role in the negative feedback regu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00C12N15/864C12N15/66
Inventor 吕成伟
Owner 吕成伟
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