A kind of production method and application of recombinant small RNA

A production method and sequence technology, applied in the fields of molecular biology and medicine, can solve the problems of limited application scope, inability to express a variety of small RNAs, etc., achieve low toxicity and immunogenicity, improve complementary pairing conditions, functional Good results

Active Publication Date: 2022-03-29
西安荣清畅生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this biological expression method has been proven to have a limited scope of application, and many small RNAs cannot be expressed or the expression level is too low under this strategy

Method used

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  • A kind of production method and application of recombinant small RNA
  • A kind of production method and application of recombinant small RNA
  • A kind of production method and application of recombinant small RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: The hsa-mir-34a precursor was amplified by PCR technology, and the pBSKrnaSeph / hsa-mir-34a expression vector was constructed.

[0041] (1) According to the sequence of hsa-mir-34a (MI0000268) precursor in miRBase, primers were designed to amplify its precursor. At the same time, add 1-15nt homologous sequences on both sides of the vector insertion site at both ends of the primer. See Table 1 for the sequence (SEQ ID NO.4 and SEQ ID NO.5). The addition of homologous sequences can be assisted by the website http: / / bioinfo.clontech.com / infusion / convertPcrPrimersInit.do.

[0042] Table 1 hsa-mir-34a primer sequence

[0043]

[0044] (2) Synthesis of miR-34a precursor insert fragment

[0045] Human genomic DNA was used as a template, and the primers in Table 1 were used for PCR reaction. The reaction system was shown in Table 2:

[0046] Table 2 Polymerase in vitro amplification chain reaction system (50 μL)

[0047]

[0048]

[0049] After mixing ev...

Embodiment 2

[0066] Example 2: Expression of recombinant miR-27b with pBSKrnaSeph / hsa-mir-34a expression vector

[0067] (1) Design primers according to the mature hsa-mir-27b sequence (MIMAT0000419) in miRBase and the sequence on the pBSKrnaSeph / hsa-mir-34a expression vector.

[0068] (2) Synthesis of insert fragments

[0069] The two primers were used as templates to synthesize the insert by PCR. The reaction system was shown in Table 6, and the reaction conditions were the same as in Table 3.

[0070] Table 6 Polymerase in vitro amplification chain reaction system (50 μL)

[0071]

[0072] The rest of the build steps are the same as above.

[0073] figure 2 In the present invention, bacterial solution PCR is used to identify the recombinant miR-27b expression plasmid, and a band of ~500bp is amplified from the plasmid containing the insert fragment, as indicated by the arrow in the figure. The results showed that the recombinant miR-27b expression plasmid was constructed success...

Embodiment 3

[0074] Example 3: Expression of tRNA scaffold recombinant hsa-miR-27b

[0075] (1) A small amount of recombinant miR-27b expression plasmid was extracted

[0076] After 100 ng of recombinant hsa-miR-27b expression plasmid was transformed into HST08 competent bacteria, 5 mL of 2XYT medium was added and incubated overnight at 37°C with shaking at 200 rpm. After the bacterial solution was centrifuged at 10000 g for 2 min, the precipitate was collected. Add 180uL of 10mM magnesium acetate-Tris·HCl solution to the precipitate to resuspend, then add 200uL of saturated phenol, and shake at room temperature for 20-60min. After centrifuging at 10000 g for 10 min, the aqueous phase was collected, and 5M NaCl of 0.1 times the volume of the aqueous phase was added to precipitate macromolecular impurities. Add 2 times the volume of absolute ethanol to the supernatant, centrifuge at 10000 g for 10 min, and discard the supernatant. The residual ethanol was discarded with absorbent paper, ...

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Abstract

The invention discloses a production method of recombinant small RNA. In the method, tRNA is used as a scaffold, and a chimeric miRNA precursor is used to express target small RNA in Escherichia coli. In addition, the invention also discloses the application of the recombinant small RNA produced by the method. The recombinant small RNA designed and prepared by the present invention is obtained through bioengineering technology, and has the advantages of simple and convenient equipment, rapid production, high yield, low cost, and good functionality; tests by various technical means show that the method of the present invention can conveniently and quickly Prepare a variety of miRNA / siRNA / RNA aptamers, and produce recombinant small RNAs to meet the needs of scientific research and drug development.

Description

technical field [0001] The invention belongs to the field of molecular biology and medical technology, and specifically relates to a production method and application of recombinant small RNA. Background technique [0002] Noncoding RNA (noncoding RNA, ncRNA) has broad application prospects in the research of biological related fields and disease treatment. In the past two decades, small ncRNAs have been well established as key epigenetic factors of gene regulation. These small ncRNAs mainly participate in the formation of the silencing complex (RISC) through the RNAi pathway, thereby inducing gene silencing. The research on the function of ncRNA has given birth to a new treatment method, that is, the birth of RNA therapy. For example, reconstitution of tumor suppressor miRNA levels can significantly slow down tumor disease progression. [0003] However, the research on the function, pharmacological activity and therapeutics of ncRNA depends on the acquisition of relevant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/115C12N15/70C12N15/62
CPCC12N15/113C12N15/115C12N15/70C12N2310/16C12N2310/141C12N2310/14
Inventor 骞爱荣田野程群燕喻译锋罗晓庆裴佳伟王雪杨超飞
Owner 西安荣清畅生物科技有限公司
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