CAJ | 학술논문

目的原核表达系统表达肠道病毒71型(Enterovirus 71,EV71) VP1蛋白,并探讨该基因工程蛋白阻断病毒感染及其制备抗体的中和活性.方法构建pET30a(+)-VP1表达载体、IPTG诱导表达,Western blot鉴定.His亲和纯化后,阻断试验分析该重组蛋白阻断病毒感染活性,免疫小鼠制备多抗分析多抗中和活性.结果构建的重组菌可有效表达VP1,用其免疫小鼠制备多抗ELISA效价达到1:3200,而中和活性只有1:16,并且该工程蛋白没有表现出阻断活性.结论本研究原核表达的VP1蛋白能刺激小鼠产生抗体,且抗体具有一定的中和活性,但是该蛋白没有阻断活性,表明该蛋白在原核表达系统中未能形成天然构象从而导致与受体结合的能力较差.
목적 원핵표체계통표체장도병독71형(Enterovirus 71,EV71) VP1단백,병탐토해기인공정단백조단병독감염급기제비항체적중화활성.방법 구건pET30a(+)-VP1표체재체、IPTG유도표체,Western blot감정.His친화순화후,조단시험분석해중조단백조단병독감염활성,면역소서제비다항분석다항중화활성.결과 구건적중조균가유효표체VP1,용기면역소서제비다항ELISA효개체도1:3200,이중화활성지유1:16,병차해공정단백몰유표현출조단활성.결론 본연구원핵표체적VP1단백능자격소서산생항체,차항체구유일정적중화활성,단시해단백몰유조단활성,표명해단백재원핵표체계통중미능형성천연구상종이도치여수체결합적능력교차.
Objective To express EV71 VP1 in prokaryotic expression system,initially evaluate the ability of blocking EV71 infection and the neutralizing activity of its polyclonal antibody.Methods Construct the prokaryotic expression plasmid pET30a (+)-VP1.Induced expression in Transetta (DE3) with IPTG and identified by Western blot.After purified with HisBind Protein Purification Kit,its ability of blocking EV71 infection and the neutralizing activity of its polyclonal antibody were analyzed.Results Plasmid PET-30a(+)-VPI was constructed successfully and the objective protein was expressed effectively.The ELISA titer of the polyclonal antibody was 1:3200 while neutralizing titer was 1:16 and the recombination protein lost the ability of blocking EV71 infection.Conclusion The recombination protein can stimulate mice to produce antibodies and the polyclonal antibody shew neutralizing activity but the recombination protein lost the binding activity to receptors probably due to the wrong advanced structure.