棉铃虫效应蛋白 HARP1 体外表达与纯化体系的建立.

[Objectives]This article aimed to establish the expression and purification system of the effector protein HARP1 from cotton bollworm in vitro to express and purify the HARP1 protein in vitro and lay the foundation for analyzing the molecular mechanism of the interaction between HARP1 protein and JAZ proteins in plant jasmonate signal pathway. [Methods]The codon optimization of harp1 gene sequence was carried out. Protein prediction software was used to predict signal peptide and mature peptide in HARP1. Based on the above information, the expression plasmid of the target protein was constructed and induced to express, and the HARP1 protein was purified by various methods including affinity chromatography, protease digestion, dialysis, ion exchange chromatography and gel filtration chromatography. SDS-PAGE and Western blot were used to analyze and detect the expression and purity of protein. [Results]Through optimization, the codon adaptation index (CAI) of harp1 gene increased from 0.39 to 0.95. The protein prediction software predicted that the signal peptide of HARP1 was composed of the 21 amino acids on the N-terminal. The cleavage site of the signal peptide and the mature peptide was between Alanine21 and Leucine22,and the mature peptide was from Leucine22 to Arginine122. The expression plasmid pETDuet1-His6-MBP-3 C-HARP1（22-122）was constructed, and IPTG could induce the expression of the target protein. The fusion protein His6-MBP-3 C-HARP1（22-122）was purified by Ni-NTA Column. 3 C protease could remove the His6-MBP tag, and the HARP1（22-122）protein was successfully separated from His6-MBP tag using anion exchange column. Superdex 200 increase Gel Filtration Column was used for further purification to obtain monomer HARP1（22-122）protein. [Conclusions]The in vitro expression and purification system of HARP1 was successfully established. [ABSTRACT FROM AUTHOR]