Cloning and expression of rotavirus nonstructural protein 4 and its interference effect on intrancellu-lar calcium homeostasis
10.3760/cma.j.issn.0254-5101.2013.08.002
- VernacularTitle:轮状病毒非结构蛋白4的克隆、表达及其对胞内钙离子的干扰
- Author:
Ling LIN
;
Pan ZHANG
;
Xiangfang LIN
;
Caixia LIU
;
Jinhong YANG
;
Xiangyang LI
- Publication Type:Journal Article
- Keywords:
Rotavirus;
Nonstructural protein 4;
86-175 amino acid peptide;
Cloning and expres-sion;
Intracellular free calcium
- From:Chinese Journal of Microbiology and Immunology
2013;(8):568-573
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct prokaryotic expression systems of genes of intact rotavirus non -structural protein 4 (NSP4) and 86 to 175 amino acid peptide of NSP4 (NSP486-175) and to identify the bio-activities of the recombinant expression products .Methods Colloidal gold assay was used to screen group A rotavirus antibody-positive stools , from which the viral RNA was extracted and transcribed into cDNA .Then NSP4 gene and NSP486-175 gene were amplified by PCR and inserted into pET-28a(+) vector to construct the prokaryotic expression plasmids of pET-28a(+)-NSP4 and pET-28a(+)-NSP486-175 , which were transformed into E.coli BL21( DE3) and inducted by IPTG .The expressed NSP486-175 protein was purified by Ni-NTA af-finity chromatography and examined by Western blot .Fluo-3 AM ester was used to label intracellular free Ca2+( [ Ca2+] i) in Caco-2 cells and laser scanning confocal microscopy was performed to detect the effect of NSP486-175 protein on [Ca2+]i.Results DNA sequencing indicated that NSP4 gene and NSP486-175 gene were consistent with the corresponding sequences in NCBI .The intact NSP4 had difficulty in expressing in E.coli. NSP486-175 protein mainly existed in soluble form with a relative molecular weight of 10×103 .Fluorescence in-tensity and distribution were changed by exogenous addition of NSP 486-175 to cultured Caco-2 cells.Conclu-sion The expression vectors of pET-28a(+)-NSP4 and pET-28a(+)-NSP486-175 were successfully construc-ted and the target protein could induce [Ca2+]i imbalance.This phenomenon preliminarily indicates that the prokaryotic expressed NSP486-175 protein has biological activity and can be further used to elucidate rotavirus pathogenic mechanisms and perform vaccine research .
