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Method for constructing small interfering RNA plasmid of chicken TBK1 gene, and use thereof

A construction method and small interference technology, which can be used in DNA/RNA fragments, genetic engineering, recombinant DNA technology, etc., and can solve the problems of transient, difficult and persistent interference effects, and low interference efficiency.

Active Publication Date: 2018-08-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is convenient to operate, the interference efficiency is not high, and the interference effect is instantaneous and difficult to last

Method used

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  • Method for constructing small interfering RNA plasmid of chicken TBK1 gene, and use thereof
  • Method for constructing small interfering RNA plasmid of chicken TBK1 gene, and use thereof
  • Method for constructing small interfering RNA plasmid of chicken TBK1 gene, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The construction of the eukaryotic expression vector of the siRNA of embodiment 1 targeting chicken TBK1 gene

[0062] The chicken TBK1 nucleotide sequence is shown in SEQ ID NO.7. According to the online website analysis, I designed the small interfering RNA sequence targeting TBK1 by myself, and after BLAST analysis confirmed that it had no homology with other known genes, I designed and synthesized 2 small interfering RNA sequences according to the instructions and requirements of the pGPU6-Neo vector. the corresponding DNA sequence. figure 1 For the design of the small interfering RNA sequence for chicken TBK1 in this embodiment, figure 2 Plasmid map for pGPU6-Neo. The sequences were dissolved in an appropriate amount of deionized water, and annealed according to the program (90°C for 4 min, 70°C for 10 min, 50°C for 15 min, 37°C for 20 min, 10°C for 10 min) to form double-stranded DNA fragments with sticky ends. The pGPU6-Neo vector was digested with BamHI an...

Embodiment 2

[0063] Example 2 Cell Culture and Transfection

[0064] DF-1 cells were cultured in DMEM containing 10% fetal bovine serum, 100 U / m L penicillin, 100 U / m L streptomycin at 37°C, 5% CO 2 Routine cultivation in an incubator. Cells were inoculated in 25T cell flasks 24 hours before transfection, and the inoculation density was such that the cells grew to 70%-90% abundance during transfection. Mix 3.8 μg of plasmids pGPU6-shTBK1-78 and pGPU6-shTBK1-983 with 500 μL of DMEM, then mix 12.5 μL of liposomes with 500 μL of DMEM, mix the two gently, let stand at room temperature for 20 minutes, and add to the cells , 37°C, 5% CO 2 Routine cultivation in an incubator.

Embodiment 3

[0065] Example 3 Western blot detection

[0066] Collect samples from DF-1 cells transfected with pGPU6-shTBK1-78 and pGPU6-shTBK1-983 in Example 2, respectively, with protein lysate, add SDS loading buffer, boil water bath for 15min, centrifuge at 4°C and take the supernatant Perform SDS-PAGE, transfer to PVDF membrane with semi-dry transfer after electrophoresis, incubate with 5% skimmed milk powder at room temperature for 2h, then add 1:3000 diluted Myc tag antibody and incubate overnight at 4°C. Wash the membrane 5 times with TBST, 10min each time, add diluted horseradish peroxidase-conjugated mouse secondary antibody, incubate at room temperature for 2h, wash the membrane 5 times with TBST, 10min each time, and then Substrate chemical reagents are added dropwise to the membrane and developed immediately.

[0067] image 3 To detect the protein expression map of TBK1 in DF-1 cells by western blotting, M in the figure represents the protein Marker; shNC represents the s...

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Abstract

The invention provides a method for constructing a small interfering RNA plasmid of chicken TBK1 gene. The construction method comprises the following steps: S1, designing two interference target sequences for chicken TBK1; S2, respectively synthesizing two complementary single-stranded DNA sequences used for constructing an shRNA fragment according to the two interference target sequences designed in step S1, and respectively introducing corresponding restriction enzyme cutting sites to the two complementary single-stranded DNA sequences; and S3, respectively performing denaturation annealingon the two complementary single-stranded DNA sequences to respectively form double-stranded DNA fragments having sticky ends; and respectively connecting the double-stranded DNA fragments with a vector to obtain the small interfering RNA of the chicken TBK1 gene. Cell level experiments confirm that the constructed small interfering RNA plasmid targeting the chicken TBK1 gene can efficiently and specifically inhibit the expression of the chicken TBK1 gene, and provides a platform for further study of the effect of TBK1 in chicken antiviral innate immunity.

Description

technical field [0001] The invention relates to a method for constructing a small interfering RNA plasmid targeting chicken-derived TANK binding kinase 1 (TANK binding kinase-1, TBK1) gene. Specifically, it relates to a construction method and application of a chicken TBK1 gene small interfering RNA plasmid. Background technique [0002] Under ideal circumstances, the body's natural immunity can successfully block the invasion of viruses, but viruses can also successfully escape the body's innate immune mechanism through various methods such as viral gene mutations, and there is always a constant confrontation between the virus and the body , co-evolution. According to research reports, many viral proteins can escape the innate antiviral immune response by acting on TBK1. For example, murine hepatitis virus A59), vaccinia virus, dry sloth virus, herpes virus and the like. In recent years, with the development of the poultry industry, the abuse of veterinary drugs has incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N5/10
CPCC12N5/0656C12N15/1137C12N15/85C12N2310/14C12N2510/00C12N2800/106C12N2310/531
Inventor 孙建和程玉强朱雯娴严亚贤
Owner SHANGHAI JIAO TONG UNIV
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