CAJ | 학술논문

目的探究利用大肠埃希菌原核表达系统表达的肠道病毒71型(EV71)衣壳蛋白VP1的抗原性和免疫原性.方法将EV71 H3-TY株的VP1基因通过PCR扩增,并引入Nde Ⅰ和Xho Ⅰ酶切位点,定向克隆至pET44a-EV71 VP1表达载体,转化大肠埃希菌BL21(DE3),并用IPTG诱导VP1的表达.然后将表达的重组VP1 (rVP1)经超声、离心和Ni-NTA亲和层析柱纯化并透析复性,用Western免疫印迹法和先前建立的夹心ELISA等方法检测其抗原活性,同时用rVP1免疫兔子并通过测定其诱导的特异性抗体水平来确定其免疫原性.结果 rVP1在大肠埃希菌中主要表达于包涵体中,但能被顺利且有效地纯化和复性.SDS-PAGE显示表达的rVP1相对分子质量约为34 000,与预期大小相符.纯化的rVP1可分别与兔抗EV71多克隆抗体、大鼠抗EV71 VP1单克隆抗体(单抗)和小鼠抗EV71 VP1单抗进行特异性免疫应答.rVP1免疫兔诱导产生的特异性抗体效价高达729 000,高于本底效价,且差异有统计学意义(t=2.646,P＜0.01).结论 EV71 VP1在大肠埃希菌原核表达系统中得以成功表达,纯化的rVP1具有良好的抗原活性和免疫原性.
목적 탐구이용대장애희균원핵표체계통표체적장도병독71형(EV71)의각단백VP1적항원성화면역원성.방법 장EV71 H3-TY주적VP1기인통과PCR확증,병인입Nde Ⅰ화Xho Ⅰ매절위점,정향극륭지pET44a-EV71 VP1표체재체,전화대장애희균BL21(DE3),병용IPTG유도VP1적표체.연후장표체적중조VP1 (rVP1)경초성、리심화Ni-NTA친화층석주순화병투석복성,용Western면역인적법화선전건립적협심ELISA등방법검측기항원활성,동시용rVP1면역토자병통과측정기유도적특이성항체수평래학정기면역원성.결과 rVP1재대장애희균중주요표체우포함체중,단능피순리차유효지순화화복성.SDS-PAGE현시표체적rVP1상대분자질량약위34 000,여예기대소상부.순화적rVP1가분별여토항EV71다극륭항체、대서항EV71 VP1단극륭항체(단항)화소서항EV71 VP1단항진행특이성면역응답.rVP1면역토유도산생적특이성항체효개고체729 000,고우본저효개,차차이유통계학의의(t=2.646,P＜0.01).결론 EV71 VP1재대장애희균원핵표체계통중득이성공표체,순화적rVP1구유량호적항원활성화면역원성.
Objective To express enterovirus 71 capsid protein VP1 by E.coli prokaryotic expression system and explore its antigencity and immunogenicity.Methods The coding sequence of EV71 strain H3-TY VP1 was amplified by PCR and cloned into the pET44a vector at Nde Ⅰ and Xho Ⅰ sites.The resultant plasmid pET44a-EV71 VP1 was transformed into E.coli BL21 (DE3) and its expression was induced by IPTG.The re-combinant VP1 protein (rVP1) was purified through ultrasonification,centrifugation,Ni-NTA chromatography and reconstituted by dialysis.The antigencity of rVP1 was examined by Western blotting and the sandwich ELISA method established previously,and its immunogenicity was checked by measuring the titer of specific antibody in rabbit serum induced by inoculated rVP1.Results The rVP1 was purified and reconstituted efficiently though it expressed mainly in inclusion bodies.As expected,SDS-PAGE revealed the molecular weight of rVP1 was 34 000.In Western blotting,the purified rVP1 gave a positive signal towards rabbit anti-EV71 pAb,rat anti-EV71 VP1 mAb and mouse anti-EV71 VP1 mAb,respectively.The titer of rVP1-induced specific antibody in rabbit serum was up to 729 000,apparently much higher than the blank titer (t=2.646,P＜0.01).Conclusions EV71 VP1 is successfully expressed in E.coli prokaryotic expression system and the purified rVP1 shows good antigencity and immunogenicity.