Novel epothilone biosynthetic gene P3 promoter as well as preparation method and application thereof

A promoter and gene expression technology, applied in the fields of biochemistry and molecular biology, can solve the problem of few promoters of epothilone biosynthesis genes, and achieve the effect of promoting wide application, promoting improvement and promoting yield

Active Publication Date: 2019-04-26
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few reports on the promoter ...

Method used

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  • Novel epothilone biosynthetic gene P3 promoter as well as preparation method and application thereof
  • Novel epothilone biosynthetic gene P3 promoter as well as preparation method and application thereof
  • Novel epothilone biosynthetic gene P3 promoter as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Cloning and analysis of promoter P3:

[0016] 1. Cloning of the promoter sequence

[0017] two,

[0018] 1. Design primers:

[0019] P3 upstream primer: 5'-TGGCGTCGGGCGCGGGG-3'

[0020] P3 downstream primer: 5'-TTGGGGATTGGAGAC-3'

[0021] 2. Extract the genomes of three bacterial strains of S. cellulosus So ce M1, So ce M4 and So ce M6 with the bacterial genome extraction kit, according to the above p3 primer (comprising the P3 upstream primer and the P3 downstream primer) PCR, with Epothilium The P3 and P5 fragments ( figure 1 ), but the non-epothilone-producing S. cellulosus So ce M1 and So ceM6 could not amplify any fragments, indicating that the P3 and P5 fragments are specific fragments of epothilones producing bacteria.

[0022] The PCR reaction system is as follows:

[0023]

[0024] The PCR amplification procedure is as follows:

[0025]

[0026] After recovery, the obtained PCR products were connected to pMD18-T vector, connected over...

Embodiment 2

[0027] Example 2: P3 promoter functional verification

[0028] The P3 promoter was amplified by PCR, and XhoI and HindIII restriction sites were introduced at the 5' end and 3' end, respectively, and the gel was cut and recovered, digested with XhoI and HindIII at 37°C for 4 hours, and the product was recovered, and simultaneously used XhoI and HindIII Digest pGL3-Basic at 37°C for 2 hours to recover large fragments. Ligate the digested P3 promoter sequence and pGL3-Basic vector at 22°C for 3.0 h, transform into Escherichia coli DH5α, pick clones for expansion and culture, use P3 upstream primers and P3 downstream primers to carry out bacterial liquid PCR amplification respectively, and the obtained Positive clones were sequenced and verified, thus obtaining the PGL3-P3 recombinant vector (double enzyme digestion verification such as figure 2 shown), which is to insert the P3 promoter into the pGL3-Basic vector. The promoter pgpd was also inserted into the pGL3-Basic vector...

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Abstract

The invention discloses a novel epothilone biosynthetic gene P3 promoter as well as a preparation method and application thereof. A nucleotide sequence of the P3 promoter is shown as SEQ ID NO. 1. Thepromoter of a So ce M4 epothilone biosynthetic gene is amplified by adopting a degenerate primer designing method for the first time; the promoter is subjected to cloning and promoter element analysis; and a luciferase reporter gene vector is adopted to verify the functions of the obtained epothilone biosynthetic gene promoter. The obtained novel epothilone biosynthetic gene cluster promoter canlay a molecular biological foundation for transcriptional regulation of sprangium cellulosum epothilone biosynthesis and heterologous expression of an epothilone biosynthetic gene cluster; the improvement of the yield of epothilone is promoted; and wide application of the epothilone in the aspect of biomedicines is promoted.

Description

Technical field: [0001] The invention belongs to the fields of biochemistry and molecular biology, and in particular relates to a novel epothilone biosynthetic gene P3 promoter and its preparation method and application. Background technique: [0002] Sonocystis cellulosus is a myxobacteria that can produce abundant biologically active secondary metabolites. Epothilone is a sixteen-membered macrolide with broad-spectrum and high-efficiency anti-tumor activity produced by Sonocystis cellulosus class of compounds. Due to its better water solubility, lower side effects, broader antitumor activity and good antitumor activity against drug-resistant tumor cells, epothilone has become the most promising compound to replace paclitaxel. Epothilone derivatives have been approved for the clinical treatment of advanced breast cancer, but the low yield and high price of epothilone limit its wide application. The biosynthetic gene clusters of epothilones have been identified, and there ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/70
CPCC12N15/10C12N15/70C07K14/37
Inventor 叶伟章卫民刘桃妹李浩华李赛妮黄自磊朱牧孜许丽琼
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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