Establishment of prokar yotic expression system of autoantigen human bactericidal/permeability-incre-asing fold-containing B1
10.3760/cma.j.issn.1007-7480.2019.07.007
- VernacularTitle:结缔组织病肺间质病相关自身抗原人杀菌/渗透增强蛋白B1蛋白原核表达体系的建立
- Author:
Linhong GUO
1
;
Zhuoli ZHANG
;
Wei ZHOU
Author Information
1. 北京大学第一医院风湿免疫科 100034
- Keywords:
Autoantigens;
Bactericidal/permeability-increasing fold-containing B1;
Prokaryotic ex-pression;
Protein purification;
Lung diseases;
interstitial
- From:
Chinese Journal of Rheumatology
2019;23(7):465-471
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a prokaryotic expression system of interstitial lung disease associated autoantigen human bactericidal/permeability-increasing fold-containing B1 (BPIFB1), providing tools for the study on its function in immune responese. Methods The coding region of BPIFB1 gene was amplified with specific primers from recombinant pGEM-C20ORF114 plasmid and cloned into the pET28a-MBP-His and pGEX-5X-1 vectors. The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were transfected into Top10 cells. The positive clones were selected and sequenced. The correct clones of pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST were transfected into prokaryotic expression strain Rosetta (DE3) and induced by Isopropyl β-D-Thiogalactoside (IPTG). The expression of recombinant BPIFB1 fusion protein was analyzed by SDS-PAGE and Western blotting, and purified by urea modified and renaturation and affinity chromatography of nickel NTA-resin. Results The polymerase chain reaction (PCR) produced specific product with the molecular weight equivalent to that of BPIFB1. The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were cloned by double restriction enzyme digestion and ligation and confirmed by sequencing. The SDS-PAGE result showed that both BPIFB1-MBP and BPIFB1-GST fusion proteins were mainly expressed in the form of inclusion bodies. The Western blotting result revealed that the recombinant BPIFB1-MBP-His protein could be recognized by Anti-6 ×His antibody. The purified soluble BPIFB1-MBP fusion protein was obtained by urea denaturation, affinity chromatography of nickel NTA-resin and then renaturation after purification. Conclusion The BPIFB1 prokaryotic expression system is established by construct recombinant plasmid pET-BPIFB1-MBP-His, and an approach of renaturation after nickel resin affinity purification in denatured condition.
