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Single-subunit RNA polymerase and its purification method and application in RNA synthesis

A technology of RNA polymerase and purification method, applied to single-subunit RNA polymerase, its purification method and application field in RNA synthesis, can solve problems such as uneven transcription products

Active Publication Date: 2018-05-11
RNASYN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Syn5 RNA polymerase has shown obvious advantages in many aspects of performance, especially in several weak links of T7 system, but for the in vitro transcription of RNA rich in higher order structures, Syn5 RNA polymerase and T7 RNA polymerase have the same transcripts. The disadvantage of uniformity, which undoubtedly brings demands and challenges to the research and development of more novel RNA polymerases

Method used

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  • Single-subunit RNA polymerase and its purification method and application in RNA synthesis
  • Single-subunit RNA polymerase and its purification method and application in RNA synthesis
  • Single-subunit RNA polymerase and its purification method and application in RNA synthesis

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Embodiment 1

[0048] Embodiment 1 KP34 RNA polymerase gene amplification and protein expression purification

[0049] (1) KP34 RNA polymerase gene amplification and protein expression

[0050] The gene of KP34 RNA polymerase was amplified by PCR, cloned into the prokaryotic expression vector pET-28b between the restriction sites NdeI and NotI, and the 5' end of the gene was fused with a 48-base DNA fragment, The DNA fragment encodes the histidine tag shown in the sequence table SEQ ID NO.2 and the flexible connecting peptide between the tag and KP34 RNA polymerase. The connecting peptide sequence is Ser-Ser-Gly-Leu- Val-Pro-Arg-Gly-Ser-His, the resulting recombinant vector was transformed into E.coli BL21(DE3), and then the bacteria were placed in LB medium containing 50ug / ml kana at 37°C and cultured on a shaker until OD 600 The value is close to 1.2, and then adding a final concentration of 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) to induce expression at 30°C for 3h.

[0051] (2...

Embodiment 2

[0059] Embodiment 2 utilizes KP34 RNA polymerase to carry out in vitro transcription

[0060] (1) Acquisition and purification of transcription reaction templates

[0061] Amplify the pUC19 vector sequence by PCR method, so that the front primer has the promoter sequence of KP34 RNA polymerase. The total volume of the PCR reaction system is 20ul: 2X PrimeSTAR Max 10ul, pUC19 plasmid 1ng, double distilled water to make up to 20ul. Amplification conditions: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 45 s, 35 cycles; final extension at 72°C for 5 min, storage at 16°C for 2 min. The PCR product was detected by agarose gel electrophoresis to see if a single band was amplified. Use PCR product purification kit DNA Clean&Concentrator TM -5 (purchased from ZYMO RESEARCH Biotechnology Company) to purify the PCR product, the specific purification steps are as follows:

[0062] a. Add DNA Binding Buffer with 5 t...

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Abstract

The invention discloses a single-subunit RNA polymerase and its purification method and application in RNA synthesis. The single-subunit RNA polymerase is a single-subunit RNA polymerase derived froma phi KMV phage or an other phage single-subunit RNA polymerase with a protein sequence which is 25% or more homologous to the single-subunit RNA polymerase derived from the phi KMV phage, contains acharacteristic amino acid sequence shown in the formula of SEQ ID NO. 1 in the sequence table and has the total amino acid sequence number of 800 and 830. The research result shows that the RNA polymerase has a long-distance relationship with the existing RNA tool enzyme and high transcription efficiency. Compared with the existing T7 RNA polymerase and Syn5 RNA polymerase, the single-subunit RNApolymerase solves the problem that the synthesis of RNA rich in a stable and high-level structure from the T7 RNA polymerase and Syn5 RNA polymerase produces complex products. The single-subunit RNA polymerase produces same transcription products.

Description

technical field [0001] The invention belongs to the research field of microbial nucleic acid metabolizing enzymes, in particular to single-subunit RNA polymerase, its purification method and its application in RNA synthesis. Background technique [0002] RNA (ribonucleic acid) is the most important class of macromolecules for the transmission of genetic information in all organisms, and it also plays a variety of regulatory roles in organisms. RNA research has become one of the hottest fields in biological research in recent years. RNA mainly includes mRNA, tRNA, rRNA and other categories. With the rapid development of biological technology in recent years, scientists have discovered some novel RNAs such as microRNA (miRNA) (Cheng et al., 2005), long non-coding RNA (lncRNA) (Shi et al., 2016), nanostructure RNA (Geary et al., 2014), etc. also play a vital role in organisms. The designed RNA sequence can encode and be translated into any protein, so it can theoretically rep...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12P19/34
CPCC12N9/1247C12P19/34C12Y207/07006
Inventor 朱斌吴慧陆雪玲夏恒黄锋涛
Owner RNASYN BIOTECH CO LTD
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