Preparation and purification of recombinant proteins of elastin-like polypeptide for displaying two epitopes of NT-proBNP
10.13481/j.1671-587x.20200305
- Author:
Xin FU
1
Author Information
1. Medical Research Center, Dalian University
- Publication Type:Journal Article
- Keywords:
Amino-terminal pro-brain natriuretic peptide;
Elastic-like polypeptide;
Epitope;
Escherichia colit heart failure
- From:
Journal of Jilin University(Medicine Edition)
2020;46(3):458-463
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To use Escherichia coli ( E. coli) to prepare the recombinant proteins of elastin-like polypeptide (ELP) for displaying the two epitopes of N-terminal pro-brain natriuretic peptide (NT-proBNP)» and to provide the basis for the low-cost and high-efficiency preparation of NT-proBNP detection calibrator. Methods: The epitopes of 13 20 and 63 71 amino acid residues of NT-proBNP were designed and fused with ELP by flexible chain; three kinds of fusion proteins were obtained. The genes encoding the three fusion proteins mentioned above were synthesized by genetic engineering technique and cloned into the pET-28a ( +) vector; the recombinant proteins were induced and expressed automatically in F. coli BL21 (DE3), the recombinant proteins were purified with inverse transition cycling (ITC), and the abilities of their antibodies specificly binding epitopes were detected by Western blotting and ELISA methods. Results: Three kinds of vectors of ELP for displaying NT-proBNP epitopes were successfully constructed, and the corresponding recombinant proteins were expressed in the E. coli. The western blotting and direct ELISA results showed that the specific epitopes displayed by ELP had the better binding ability with the relative antibodies. The results of Sandwich ELISA showed that the protein concentrations of recombinant proteins of ELP for displaying two epitopes of NT-proBNP had a double logarithmic linear dose-dependent relationship with the absorbance (A) value at 450 nm ( r=0.919 1-P<0.01). Conclusion: The ELF is successfully used to display the two epitopes of NT-proBNP. and lay a foundation for the low-cost and high-efficiency preparation of the NT-proBNP detection calibrator.
