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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: 2013-03-06
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 the cDNA gene of Pinellia ternata lectin

[0039] The total RNA was extracted from the tubers of Pinellia pedatisecta Schott. According to the instructions for TransScript First-Strand cDNA Synthesis SuperMix, 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 is controlled at 50°C for 30min; the RT Enzyme is inactivated at 85°C for 5min. After cDNA electrophoresis ( figure 1 ) Shows that the cDNA is a diffuse band below 1kb, which is in line with the expected result. Using cDNA as template, according to the complete sequence of Pinellia pedatisecta agglutinin (PPA) mRNA (GeneBank: HM593586.1) reported on NCBI, two pairs of primers PPAL1 and PPAL2 were designed, respectively:

[0043]

[0044] Among them, PPAL1 and PPAL2 were introduced into restriction sites Nco I and Xho I, respectively. R...

Embodiment 2

[0051] Example 2. Cloning of ppa-linker-thanatin recombinant gene

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

[0053]

[0054] The PCR system is as follows:

[0055]

[0056]

[0057] P1, P4 products (ppal1, primers P1, P4, annealing temperature 55 degrees), P2, P4 products (ppal2, primers P2, P4, annealing temperature 65 degrees), P3, P4 products (ppal3, primers P3, P4, annealing temperature 56 degrees) were respectively connected to pEasy T1 vector, the connection system was the same as in Example 1, and was identified by PCR and restriction digestion ( Image 6 , ...

Embodiment 3

[0060] Example 3. Construction of ppa-linker-thanatin prokaryotic expression vector

[0061] The ppa-linker-thanatin(pt) gene and pET28a gene were digested with SalI and XhoI to recover the target fragments. Under the action of T4DNA ligase, the expression vector pET28a-pt was constructed, and the vector was transformed into T1 competent cells at 37℃ After culturing for 12 hours, the plasmid was extracted, PCR identification and restriction enzyme digestion identification ( Picture 9 ) Shows that the vector was successfully constructed.

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

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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 genetic engineering preparation of recombinant protein expressed by Escherichia coli and its value in clinical application. Background technique [0002] In recent years, top international journals such as Cell, Nature, Science, etc. have published reviews, monographs, and treatises on the research progress and significance of the relationship between inflammation and tumors, which has attracted 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 the occurrence of more than a quarter 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, and cause the maintenance of normal cellular...

Claims

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Application Information

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Patent Type & Authority Patents(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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