摘要
目的:探讨自裂解肽T2A对猴B病毒g D和g B蛋白共表达的可行性。方法:利用一点褐翅蛾病毒(Ta V)的2A元件(T2A)连接猴B病毒膜蛋白g D和g B基因,构建多顺反子表达质粒,制备CHO-S工程细胞,采用Western印迹和ELISA检测蛋白表达及重组蛋白的免疫学特性。结果:g D和g B蛋白实现了共表达且保持了独立和完整的分子结构,但g B的表达水平明显低于g D;ELISA结果显示,单独表达的g D和g B以及共表达产物都能与B病毒阳性血清反应,但共表达产物的反应强度高于单独表达的g D和g B;重组蛋白经变性处理后,g B蛋白的吸光度值降幅最大。结论:自裂解肽T2A能够实现不同分子共表达且保持独立和完整的分子结构;共表达猴B病毒g D和g B分子有利于提高抗体检测的敏感性。
Objective： To explore the feasibility of co-expressing monkey B virus（BV） gD and gB by self-cleav-age peptide T2 A. Methods： A polycistronic expression vector contains T2 A from insect viruses Ta V linked mon-key B virus gD and gB gene was constructed and transfected CHO-S cells. Cell culture supernatants from stablyexpressing gD, gB and gD-gB were detested by Western blot and ELISA to expound the expression level and im-munological characteristics of recombinant proteins. Results： Recombinant proteins BV-gD or BV-gB were detect-ed in cell culture supernatant of only gD or gB gene expressing cells, and simultaneously existed in gD and gB co-expressing cells, with the independence and integrity of the molecular structure. However, the expression levelof gB was significantly lower than gD. ELISA results showed that expression products of gD, gB and gD-gB react-ed with positive sera of BV. But the intensity of the reaction was higher in gD-gB than gD and gB individual. Af-ter denaturation treatment, OD value of ELISA declined in three expression products and the largest decline ap-peared in gB. Conclusion： T2 A has self-cleaving activity in CHO-S cells and could be a potential tool for co-ex-pression of proteins. Co-expression of BV gD and gB would improve the sensitivity of antibody detection.
出处
《生物技术通讯》
CAS
2016年第2期163-167,共5页
Letters in Biotechnology
基金
国家自然科学基金(31172276
31272514)
作者简介
徐刚（1967-），男，副教授
通信作者：法云智，（E-mail）fycz2004@163．com
通信作者：叶华虎，（E-mail）huahuy512@126．com
