摘要
目的原核表达、纯化人星状病毒(human astrovirus,HAstV)非结构蛋白nsP1a/3,并制备多克隆抗体。方法酶切质粒nsP1a/3-pCDNA3.1,获得nsP1a/3基因,克隆至原核表达载体pET-28a,构建重组原核表达质粒nsP1a/3-pET-28a,转化大肠埃希菌BL2(DE3),IPTG诱导表达,并优化表达条件。表达的重组蛋白经Ni2+亲和层析纯化后,经腹腔免疫SD大鼠,共4次,最后1次免疫7 d后,经心脏采血,分离血清,ELISA法检测多抗效价,Western blot鉴定多抗的特异性。结果重组表达质粒nsP1a/3-pET-28a经双酶切和测序鉴定构建正确;表达的重组蛋白相对分子质量约为22500,以包涵体形式表达;纯化的重组蛋白纯度为86.6%,浓度为1.26mg/ml;制备的多克隆抗体效价较高,可达1∶30 000,且特异性较好。结论成功地原核表达、纯化了HAstV非结构蛋白nsP1a/3,并制备了特异性良好的鼠多克隆抗体,为进一步研究nsP1a/3蛋白在病毒侵染靶细胞时的作用机制奠定了基础。
Objective To express human astrovirus（HAst V） nonstructural protein ns P1 a / 3 in prokaryotic cells,purify the expressed protein and prepare its polyclonal antibody.Methods Plasmid ns P1 a / 3-p ET-28 a was transformed to E.coli BL21（DE3） and induced with IPTG,and the condition for expression was optimized.The expressed recombinant protein was purified by nickel ion affinity chromatography.SD rats were immunized i.p.with the purified protein for 4times,of which the heart blood was collected 7 d after the last immunization,and the sera were separated.The titer of polyclonal antibody was determined by ELISA,while the specificity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid ns Pla / 3-p ET-28 a was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 22 500,existed in a form of inclusion body.The purity and concentration of purified recombinant protein were 86.6% and 1.26 mg / ml respectively.The prepared polyclonal antibody reached a titer of 1 ∶ 30 000 and showed high specificity.Conclusion The ns Pla / 3 of HAst V was successfully expressed in prokaryotic cells and purified,which laid a foundation of further study on the mechanism of the protein in invasion of target cells with virus.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第11期1127-1131,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(31360619
31160193)
关键词
人星状病毒
nsP1a/3
原核细胞
基因表达
纯化
多克隆抗体
Human astrovirus（HAst V）
nsP1a/3 protein
Prokaryotic cells
Gene expression
Purification
Polyclonal antibody
作者简介
通讯作者：井申荣，E-mail：jingshenrong@163．com
