RTA和RTA-KEEL/KDEL基因的表达及细胞毒性  

Expression of Ricin A Chain and RTA-KEEL/KDEL Gene and Cytotoxicity of Expressed Product

作　　者：廖鹏[1] 马青原[2] 刘文森[2] 万家余[2] 邢丽杰[3] 李吉平[2] 高宏伟[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130062 [3]石河子大学食品学院,石河子832000

出　　处：《中国生物制品学杂志》2008年第6期475-478,共4页Chinese Journal of Biologicals

摘　　要：目的构建蓖麻毒素A链(RTA)和RTA-KEEL/KDEL基因表达载体,并检测重组蛋白的细胞毒性作用。方法用PCR法从蓖麻毒素基因组中扩增出RTA及RTA-KEEL/KDEL基因,连接T载体,测序正确后,定向插入质粒pET-28a(+)中,构建重组表达质粒pET-28a-RTA和pET-28a-RTA-KEEL/KDEL,转化感受态E.coliBL21(DE3),IPTG诱导表达,并对表达产物进行鉴定。结果所表达的RTA及RTA-KEEL/KDEL非融合蛋白经SDS-PAGE分析,相对分子质量约为31000,Westernblot分析具有反应原性,MTS法检测,RTA-KEEL/KDEL对CHO细胞具有比RTA更强的杀伤作用。结论已成功构建RTA和RTA-KEEL/KDEL表达载体,表达的重组蛋白具有生物学活性。Objective To construct an expression vector for ricin A chain（RTA） and RTA-KEEL/KDEL gene and determine the cytotoxicity of expressed recombinant protein. Methods Amplify RTA and RTA-KEEL / KDEL genes from the genome of ricin by PCR and subclone to vector pMD18-T respectively. Transform the constructed recombinant plasmids to E.coli DH5α, screen positive recombinant strains and extract plasmids for restriction analysis and sequencing. Digest recombinant plasmids pMD18-T-RTA, pMD18-T-RTA-KEEL and pMD18-T-RTA-KDEL, identified by sequencing, with Hind Ⅲ and Nco Ⅰ, and insert the obtained RTA, RTA-KEEL and RTA-KDEL gene fragments into vector pET-28a（ ＋ ） respectively. Transform the constructed recombinant plasmids pET-28a-RTA, pET-28a-RTA-KEEL and pET-28a-RTA-KDEL to E.coli BL21 （DE3） for expression under induction of IPTG. The expressed product was identified by Western blot and determined for cytotoxicity by MTS assay. Results All the relative molecular masses of expressed RTA, RTA-KEEL and RTA-KDEL non-fusion proteins were about 31 000. The expressed proteins showed reacto- genicity as proved by Western blot. MTS assay proved that the killing rates of RTA-KEEL and RTA-KDEL to CHO cells were higher than that of RTA. Conclusion The expression vectors for RTA and RTA-KEEL/KDEL were successfully constructed, and the expressed proteins showed cytotoxicity.

关 键 词：蓖麻毒素A链 非融合蛋白 原核表达 细胞毒性 

分 类 号：Q782[生物学—分子生物学]