CAJ | 학술논문

[目的]构建敲除传染性法氏囊病病毒(IBDV)VP5蛋白跨膜区序列的原核表达载体,并进行目的蛋白的表达、分离和纯化.[方法]利用PCR技术分别扩增IBDV VP5基因的胞外片段和胞内片段,然后将2个片段及pET-28b(+)同时相接,即载体-胞内片段-胞外片段-载体,构建了敲除跨膜区基因片段的VP5重组表达质粒pET-VP5-FC及改进后的pET-VP5-SC.将表达质粒转化BL21(DE3),IPTG诱导后经Ni亲和层析及凝胶过滤层析纯化重组蛋白.[结果]得到可溶性表达的IBD VP5.[结论]为进一步研究VP5蛋白的结构与功能奠定了良好的基础,另外为本文的研究方法对其它跨膜蛋白的可溶性表达及进一步研究也有一定的借鉴意义.
[목적]구건고제전염성법씨낭병병독(IBDV)VP5단백과막구서렬적원핵표체재체,병진행목적단백적표체、분리화순화.[방법]이용PCR기술분별확증IBDV VP5기인적포외편단화포내편단,연후장2개편단급pET-28b(+)동시상접,즉재체-포내편단-포외편단-재체,구건료고제과막구기인편단적VP5중조표체질립pET-VP5-FC급개진후적pET-VP5-SC.장표체질립전화BL21(DE3),IPTG유도후경Ni친화층석급응효과려층석순화중조단백.[결과]득도가용성표체적IBD VP5.[결론]위진일보연구VP5단백적결구여공능전정료량호적기출,령외위본문적연구방법대기타과막단백적가용성표체급진일보연구야유일정적차감의의.
[Objective]The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out. Moreover, the expression, separation and purification of objective protein were carried out. [Method]PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then, the two fragments were simultaneously linked to pET-28b(+), and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed. Then, the expression plasmid was transformed into BL21(DE3).After IPTG induction, the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result]The soluble expressed VP5 of IBDV was obtained.[Conclusion]The research laid the foundation for further studying the structure and function of VP5 protein.