巴泰病毒N_(5aa-90aa)重组肽段的原核表达纯化及多克隆抗体的制备

Prokaryotic Expression,Purifi cation and Preparation of Polyclonal Antibodies of Peptide N_(5aa-90aa) of Batai virus

巴泰病毒(BATV)是一种虫媒病毒,属于布尼亚病毒科成员,其核衣壳N蛋白具有高度保守性,可诱导机体产生较好的抗体反应,常用做诊断抗原。本研究通过对N蛋白强抗原性肽段N_(5aa-90aa)进行密码子优化,将其插入到pET-32a(+)载体,并转化到大肠杆菌BL21(DE3)感受态细胞中表达,通过不同IPTG浓度和诱导时间确定最佳蛋白表达条件。利用镍柱亲和层析方法对rHis-N_(5aa-90aa)肽段进行纯化。通过Western blot对rHis-N5aa-90aa肽段进行验证,将rHis-N_(5aa-90aa)肽段免疫新西兰白兔制备多克隆抗体,间接ELISA测定其抗体效价。结果显示:本研究成功构建了pET-32a-N_(5aa-90aa)重组质粒,在IPTG终浓度为0.1 mmol/L、37℃诱导5 h可获得大量重组蛋白;SDS-PAGE电泳结果显示重组rHis-N_(5aa-90aa)肽段分子质量约28.6 kDa,蛋白浓度为0.256 mg/mL;经Western blot分析纯化的rHis-N_(5aa-90aa)肽段与His-tag抗体呈阳性反应,证明rHis-N_(5aa-90aa)成功表达,间接ELISA测定制备的兔抗N_(5aa-90aa)肽段多克隆抗体的效价为1∶25600,并且Western blot和间接免疫荧光分析其具有良好的特异性。本研究表达并纯化了BATV的rHis-N_(5aa-90aa)肽段,为BATV的ELISA诊断方法的建立和基因疫苗的研制奠定基础。

Batai virus(BATV)is an arbovirus belonging to the Bunyaviridae family and its nucleocapsid protein is highly conserved and used as the diagnostic antigen.In this study,the strong antigenic peptide N5aa-90aa of N protein was inserted into pET-32a(+)vector and transformed into E.coli BL21(DE3).Optimum expression conditions were optimized with IPTG concentration and induction time.The rHis-N_(5aa-90aa) peptide was purified with nickel affinity chromatography and its activity was examined in ELISA,Western blot and IFA.The polyclonal antibodies were prepared by immunization of rabbits with the rHis-N_(5aa-90aa) and titrated in in indirect ELISA.The results showed that the recombinant plasmid pET-32a-N_(5aa-90aa) was successfully constructed and a large number of recombinant proteins were obtained with induction of IPTG at 0.1 mmol/L at 37℃for 5 h.SDS-PAGE showed that the molecular weight of the recombinant rHis-N_(5aa-90aa) peptide was about 28.6 kDa and the protein concentration was 0.256 mg/mL.The results showed that the purified protein had good immunoreactivity and specificity.The prepared polyclonal antibody titer against N_(5aa-90aa) peptide was 1∶25600 as titrated in ELISA,and also showed good specificity in Western blot and indirect immunofluorescence analysis.In this study,rHis-N_(5aa-90aa) peptide of BATV was expressed and purified,which laid a foundation for the development of an ELISA diagnostic method and vaccine research for BATV.