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PPA-linker-Thanatin fusion protein and preparation method thereof

A technology of fusion protein and protein, applied in the direction of hybrid peptide, recombinant DNA technology, chemical instruments and methods, etc., can solve the problem of low expression activity of fusion protein, and achieve the effect of development and application prospects of major new drug markets

Inactive Publication Date: 2011-11-16
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the use of E. coli to express the fusion protein of Pinellia palmata lectin and death protein, some problems have also been overcome: in the three-step PCR process, that is, in the construction of the fusion gene, the deletion and mutation of the base will cause the expression of the fusion protein, so it must be carried out. The annealing temperature optimization of each step of PCR and the PCR results should be cloned into T vector for sequencing analysis; in the process of using IPTG to induce expression at 37°C, the expression activity of the fusion protein is low, try to use low temperature to induce expression, and the induction time and IPTG concentration After appropriate heightening, the activity of the fusion protein has been significantly improved

Method used

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  • PPA-linker-Thanatin fusion protein and preparation method thereof
  • PPA-linker-Thanatin fusion protein and preparation method thereof
  • PPA-linker-Thanatin fusion protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Obtaining of the cDNA gene of Pinellia palmata lectin

[0039] Total RNA was extracted from the tuber of Pinellia pedatisecta Schott. According to the TransScript First-Strand cDNA Synthesis SuperMix instruction manual, the total RNA of Pinellia pedatisecta Schott was reverse-transcribed into single-stranded cDNA.

[0040] The synthesis system of First-Strand cDNA is as follows:

[0041]

[0042] The temperature of the PCR instrument was controlled at 50°C for 30 minutes; at 85°C for 5 minutes, the RT Enzyme was inactivated. cDNA after electrophoresis ( figure 1 ) shows that the cDNA is a diffuse band below 1 kb, which is in line with the expected result. Using cDNA as a template, two pairs of primers PPAL1 and PPAL2 were designed according to the complete sequence of Pinellia pedatisecta agglutinin (PPA) mRNA reported on NCBI (GeneBank: HM593586.1), respectively:

[0043]

[0044] Among them, PPAL1 and PPAL2 were respectively introduced into restric...

Embodiment 2

[0051] The cloning of embodiment 2, ppa-linker-thanatin recombinant gene

[0052] The gene ppa encoding Pinellia palmata lectin was amplified by three-step PCR with primers P1, P2, P3, and P4, and the recombinant gene ppa-linker-thanatin was amplified. The overlapping genes are connected to the 5' end of the ppa ORF through P1, P2, and P3, respectively. The PCR products of each step were purified by tapping and cloned into the T vector, and after identification by plasmid PCR and enzyme digestion, Sangon was commissioned for sequencing.

[0053]

[0054] The PCR system is as follows:

[0055]

[0056]

[0057] P1, P4 product (ppal1, primers P1, P4, annealing temperature 55 degrees), P2, P4 product (ppal2, primers P2, P4, annealing temperature 65 degrees), P3, P4 product (ppal3, primers P3, P4, annealing temperature 56 degrees) were respectively connected to the pEasy T1 carrier, and the connection system was the same as in Example 1, identified by PCR and enzyme dig...

Embodiment 3

[0060] Embodiment 3, the construction of ppa-linker-thanatin prokaryotic expression vector

[0061] ppa-linker-thanatin (pt) gene and pET28a gene were digested by SalI and XhoI, and the target fragment was recovered. Under the action of T4 DNA ligase, the expression vector pET28a-pt was constructed, and the vector was transformed into T1 competent cells, at 37°C Plasmids were extracted after culturing for 12 hours, identified by PCR and enzyme digestion ( Figure 9 ) shows that the vector was constructed successfully.

[0062] The amino acid sequence corresponding to the expected recombinant gene is shown in SEQ ID NO: 2 (ie, the second item in the sequence listing).

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Abstract

The invention discloses a PPA-linker-thanatin fusion protein of which the amino acid sequence is as shown in SEQ ID NO: 2. The invention also discloses a preparation method of the PPA-linker-thanatin fusion protein, comprising the following steps of: acquiring target genes of recombinant pinellia pedatisecta schott agglutinin and thanatin by a two-step PCR (Polymerase Chain Reaction) method, cloning the target genes onto an E.coli expression vector pET-28a-c(+), and acquiring the expressed protein by IPTG (isopropyl thiogalactoside) induction, wherein the expressed protein is the PPA-linker-thanatin fusion protein, namely the expression product of the pinellia pedatisecta schott agglutinin-thanatin. The expression product of the pinellia pedatisecta schott agglutinin-thanatin has biological activity and has inhibiting effect on the growth of tumor cells.

Description

technical field [0001] The invention relates to the preparation of genetic engineering for expressing recombinant protein with Escherichia coli and its value in clinical application. Background technique [0002] In recent years, top international journals such as Cell, Nature, and Science have published reviews, monographs, and monographs to review the research progress and significance of the relationship between inflammation and tumors, which has aroused the interest and attention of scholars from all over the world. Studies have shown that inflammation is closely related to tumors, and chronic inflammation is related to more than 1 / 4 of cancers. Cytokines, free radicals, prostaglandins, growth factors and other inflammatory response mediators in the inflammatory microenvironment can induce genetic and epigenetic changes such as DNA methylation, tumor suppressor gene point mutations, and post-translational modifications, resulting in the maintenance of a normal intracellu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70
Inventor 徐涛吕正兵刘学锋田辉
Owner ZHEJIANG SCI-TECH UNIV
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