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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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, ...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com