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Expressing and purifying method of recombinant human-derived LECT2 protein in Pichia pastoris

A-LECT2, Pichia pastoris technology, applied in the field of bioengineering

Active Publication Date: 2012-07-11
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports at home and abroad on the preparation of recombinant human LECT2 protein expressed by Pichia pastoris and further purified

Method used

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  • Expressing and purifying method of recombinant human-derived LECT2 protein in Pichia pastoris
  • Expressing and purifying method of recombinant human-derived LECT2 protein in Pichia pastoris
  • Expressing and purifying method of recombinant human-derived LECT2 protein in Pichia pastoris

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Experimental program
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Effect test

Embodiment 1

[0032] Expression and purification method of recombinant human LECT2 protein in Pichia pastoris of the present invention ， The artificially synthesized human LECT2 gene was inserted into the extracellular expression vector pPICZαA; after enzyme digestion and linearization, electroporation was introduced into Pichia pastoris X33 to obtain recombinant engineering strains; after shake flask fermentation and methanol induction, the secretion of recombinant human LECT2 protein was realized Expression; and then purified by column chromatography to obtain a recombinant human LECT2 protein with a purity of more than 95%, the steps are as follows:

[0033] (1) Construction of recombinant expression plasmids

[0034]Extract RNA from human peripheral blood cells, reverse transcribe and synthesize cDNA to obtain the human LECT2 protein gene sequence, the accession number is NM002302, use this as a template to design primers for PCR amplification of human LECT2 protein, and the PCR amplifi...

Embodiment 2

[0044] The method for expressing and purifying the recombinant human LECT2 protein in Pichia pastoris of the present invention specifically comprises the following steps:

[0045] 1. Construction of human LECT2 gene Pichia pastoris expression vector

[0046] Total RNA was extracted from human peripheral blood cells, reverse-transcribed to synthesize cDNA, and primers were designed according to the published human source LECT2 gene sequence (NM_002302). The forward primer was 5'-GGAATTCGGGCCATGGGCTAATATATG-3', and the reverse primer was 5'-GGGTACCCAGGTATGCAGTAGGGTCAC-3'. The PCR reaction conditions were carried out according to the conventional settings. LECT2 was amplified by PCR, and the PCR product containing LECT2 was recovered with a gel-cutting recovery kit (OMEGA), and the recovered product was recovered with a restriction endonuclease Eco RI and Kpn I (Takara) double enzyme digestion, the digested PCR product was ligated with the same digested plasmid pPICZαA with T4...

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Abstract

The invention discloses an expressing and purifying method of a recombinant human-derived LECT2 protein in Pichia pastoris, and the method is characterized by comprising the following steps of: inserting an artificially synthesized human-derived LECT2 gene into an extracellular expression vector pPICZalphaA to construct a recombinant expression plasmid; carrying out enzyme digestion and linearization, then carrying out electric shock, and introducing the recombinant expression plasmid to Pichia pastoris X33 to obtain a recombinant engineering gene; carrying out shake flask fermentation and methanol induction to realize secretory expression of the recombinant human-derived LECT2 protein; and then, purifying by utilizing column chromatography to prepare the recombinant human-derived LECT2 protein with the purity of over 95%. The expressing and purifying method of the recombinant human-derived LECT2 protein in Pichia pastoris has the advantages that the secretory expressing and purifying method of the recombinant human-derived LECT2 protein in Pichia pastoris is firstly proposed, a great number of recombinant human-derived LECT2 proteins with high activity are expressed by using the Pichia pastoris, and the expressing and purifying method has the advantages of stability, high yield, high activity and the like and can be used for pharmacy and diagnosis detection.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for secreting, expressing and purifying recombinant human LECT2 protein in Pichia pastoris. Background technique [0002] LECT2 protein, leukocyte cell-derived chemotaxin 2 (leukocyte cell-derived chemotaxin 2) is a multifunctional protein with a molecular weight of about 16 kDa and three intramolecular disulfide bonds. LECT2 protein was initially identified as a chemokine, but more and more literatures show that LECT2 protein is more similar to cytokine and is a multifunctional protein. Reports have shown that in the mouse model of alkaline liver injury in canavalin, the expression of LECT2 protein is transiently reduced; in the development of rheumatoid arthritis, the level of blood LECT2 protein is negatively correlated with the severity of the disease; it can trigger the process of liver regeneration Early events, manifested as concomitant inhibition of hepatocyte prol...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/19C12N1/19C07K14/52C07K1/18C07K1/16C12R1/84
Inventor 陈炯史雨红章瑞程陆新江李长红李明云
Owner NINGBO UNIV
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