Heterologous expression and purification method for human RANTES protein having chemotaxis

A technology of heterologous expression and purification method, applied in the field of bioengineering pharmacy, can solve the problems of low protein activity, imperfect post-translational processing mechanism of protein expression, unfavorable research work, etc. The effect of easy purification

Inactive Publication Date: 2009-04-15
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of RANTES protein is mainly limited to the expression system of Escherichia coli. The post-translational processing mechanism of this system is not perfect for protein expression, and the activity of the protein produced is low, which is not conducive to the later research work

Method used

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  • Heterologous expression and purification method for human RANTES protein having chemotaxis
  • Heterologous expression and purification method for human RANTES protein having chemotaxis
  • Heterologous expression and purification method for human RANTES protein having chemotaxis

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Experimental program
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Effect test

Embodiment 1

[0074] Embodiment 1: Cloning of human RANTES gene

[0075] According to the reported human RANTES gene sequence (NCBI accession number: NM 002985), two primers F1 (SEQ ID NO: 3) and R1 (SEQ ID NO: 4) were designed, RNA was extracted from human blood, and human RANTES was amplified by RT-PCR Gene fragment. The primer sequences are as follows:

[0076] Upstream primer F1: 5'----ATGAAGGTCTCCGCGGCAGCCC----3'

[0077] Downstream primer R1: 5'----CTAGCTCATCTCCAAAGAGTTGATG----3'

[0078] The reverse transcription system is 20ul, which contains 2ul of 10×RT reaction buffer, 25mM MgCl 2 4ul, 10mMdNTP 2ul, 200U / ul RNase Inhibitor 0.5ul, AMV Reverse Transcriptase 1ul, downstream primer R1 (20μM) 1ul, total RNA (2μg / μl) 2ul. The reaction conditions were: incubate at 42°C for 60 minutes, heat at 80°C for 5 minutes, and cool on ice for 5 minutes. Then, PCR amplification was carried out using the above-mentioned reverse transcription product cDNA as a template. The PCR reaction system...

Embodiment 2

[0079] Embodiment 2: Construction of human RANTES fusion protein expression vector

[0080] In order to facilitate the separation and purification of human RANTES protein, a 6×His purification tag was fused to the N-terminus of the target protein, and an enterokinase (EK) cleavage site was added between the His-tag and RANTES protein to construct a human RANTES fusion protein. For this purpose, a pair of primers F2 and R2 were designed, and the primer sequences are as follows:

[0081] F2: 5'----GCGAATTC CATCATCATCATCATCAT GATGACGATGACAAG ATGAAGGTC----3'

[0082] R2: 5'----GC GCGGCCGC CTA GCT CAT CTC CAA AGA GT----3'

[0083] At the 5' end of the upstream primer F2, an EcoR I restriction site, a DNA sequence of 6 histidines and an enterokinase recognition site (Asp-Asp-Asp-Asp-Lys) (SEQ ID NO : 6), a NotI restriction site (SEQ ID NO: 7) was added to the 5' end of the downstream primer R2, and then the human RANTES fusion protein gene fragment (SEQ ID NO: 7) was amplified by ...

Embodiment 3

[0084] Embodiment 3: Construction of human RANTES fusion protein engineered yeast

[0085] pPIC9K is an integrative vector that can integrate foreign genes into the chromosomes of Pichia pastoris cells through homologous recombination, and can achieve multi-site integration to obtain multiple copies. Generally speaking, the expression level and copy number of foreign proteins Positive correlation. Since pPIC9K contains the Kanamycin (Kan) gene that can resist G418, the Kan gene and the foreign gene will be integrated into the chromosome together during transformation, so that the transformant has G418 resistance, and the resistance level is positively correlated with the number of Kan genes. Therefore, we obtained high-expression recombinant transformants through G418 resistance screening of transformants. The screened transformants also need to identify the phenotype, that is, to determine whether they are Mut+ or Muts. Transformants can be spotted on both MD and MM plates ...

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Abstract

The invention relates to heterogeny expression and purification method of a human RANTES protein with chemotaxis. A Pichia pastoris expression system with the advantages of high expression, high stability, high exudation and low cost is adopted and the protocols in molecular biology and the protein expressing and purifying technology which include cloning a human RANTES gene, building a human RANTES fusion protein expression vector and the methanol induction expressing and purifying authentication of the human RANTES fusion protein through PT-PCR, and the like are adopted for realizing the exudation expression of the human RANTES protein. In order to be convenient for the separation and purification of the human RANTES protein, a 6 multiplied by His purifying label is fused at the N end of a target protein, and moreover, an enterokinase(EK) restriction enzyme locus is added between His-tag and the RANTES protein for building the RANTES fusion protein; then the human RANTES protein with higher purity and larger yield is obtained through affinity chromatography and EK restriction enzyme locus; and the protein can be used for preparing the medicaments for curing multiple sclerosis arthritis, rheumatoid arthritis, organ transplantation rejection, cardiovascular and cerebrovascular disease, knub and HIV.

Description

technical field [0001] The invention belongs to the field of bioengineering and pharmacy, and in particular relates to a heterologous expression and purification method of human RANTES protein with chemotaxis. Background technique [0002] Multiple sclerosis, rheumatoid arthritis, organ transplant rejection, cardiovascular and cerebrovascular diseases, tumors, and HIV / AIDS are many important diseases that endanger human health. These diseases have brought great suffering to human beings. People take away health and life due to these diseases, especially HIV, and there is no effective treatment so far. Over the years, researchers have devoted a great deal of time and money to finding ways to treat these diseases. [0003] Chemokine (CK) is a group of cytokines with chemotactic effect, which can attract immune cells to the local immune response and participate in immune regulation and immunopathological reactions. Most of them are small molecular proteins with less than 100 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/19C12N15/81C07K14/52A61K38/19A61P19/02A61P29/00A61P37/06A61P9/00A61P35/00A61P31/18
Inventor 陈国平胡宗利邓晓洁刘志昭顾峰
Owner CHONGQING UNIV
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