Prokaryotic expression of GII.6 norovirus P protein and preparation of polyclonal antibody
10.3760/cma.j.cn112866-20190908-00142
- VernacularTitle:GII.6型诺如病毒P蛋白原核表达及多克隆抗体制备
- Author:
Tao KANG
1
;
Wei CHEN
;
Siqi XIN
;
Yuyang ZHANG
;
Rongliang YUAN
;
Congwen SHAO
;
Shenrong JING
Author Information
1. 昆明理工大学医学院 650500
- Keywords:
GII.6;
P protein;
Oligosaccharide binding assay;
Prokaryotic expression;
Polyclonal antibody
- From:
Chinese Journal of Experimental and Clinical Virology
2020;34(2):191-196
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To get norovirus (NoV) GII.6 P protein through prokaryotic expression and prepare the polyclonal antibody.Methods:NoV GII.6 P region gene was amplified and cloned into prokaryotic expression vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein GII.6 P was expressed by induced Isopropyl β-D-Thiogalactoside (IPTG) and then purified with Ni-NTA Affinity Column. The binding ability of recombinant GII.6 P was determined by oligosaccharide binding assay and the polyclonal antibody serum was prepared by immunizing BALB/c mice. The titer of GII.6 P polyclonal antibody was determined by ELISA, and the specificity of the antibody was detected by western blot (WB). The effectiveness of GII.6 P polyclonal antibody was assessed. Results:The recombinant GII.6P-pET28a plasmid was constructed successfully and the recombinant GII.6 P protein was expressed with relative molecular mass of 40 ×10 3. The purity of GII.6P protein was more than 90% after purification. The oligosaccharide binding showed that the GII.6 P protein binds to B, le b and H2, but does not bind to A, H1, and le a type; the titer of GII.6 P polyclonal antibody was 1∶160 000. WB indicated that the antibody had high specificity and the cross experiments did not show affinity to GII.4. Conclusions:The GII.6P protein has been expressed successfully and the GII.6 P polyclonal antibody with high titer was prepared, which provides an effective tool for detection and vaccine development for NoV GII.6.
