摘要
本试验旨在表达并纯化鞭毛蛋白,为研究并获得高效蛋白佐剂奠定基础。用PCR扩增鼠伤寒沙门氏菌鞭毛蛋白基因fljB、fljB’和fliC,将扩增产物克隆至pMD19-T Simple Vector上,构建了克隆质粒pMD19-fljB、pMD19-fljB’和pMD19-fliC。克隆质粒经双酶切后将片段克隆至pET-30a中,构建原核表达质粒pET30a-fljB、pET30a-fljB’fliC和pET30a-fliC。经PCR、酶切及测序鉴定后将阳性表达质粒转化至大肠杆菌BL21(DE3)中,诱导表达后的菌体经超声处理取上清,用Ni柱进行纯化。Western blotting证实了表达的蛋白能与豚鼠抗鞭毛蛋白血清发生特异性反应。通过优化表达条件,鞭毛蛋白fljB、fliC和fljB’fliC均以可溶性表达,纯化得到的鞭毛蛋白为后续评价其佐剂效应奠定基础。
This study was aimed to express and gain high efficient adjuvants. The fljB, fljB＇ purify the flagellins, and contribute to research and and fliC genes were amplified from Salmonella Typhimurium genomic DNA by PCR,and the amplified PCR products were cloned into pMD19-T Simple Vector to construct cloned plasmid pMD19-fljB, pMD19-fljB;and pMD19-fljC. The cloned plasmids were digested by double enzymes, and purified genes were subcloned into pET-30a vector to construct prokaryotie expression vector pET30a-fljB, pET30a-fljB; fliC and pET30a-fliC. Transformed colonies were screened by PCR, restriction enzyme analysis and sequencing. Then the positive expression vectors were transformed into E. coli BL21（DE3） strain. After induced by IPTG, the expressed proteins were extracted from E. coli BL21 （DE3） strain using Ultrusonic Cell Disrupter System. The fljB,fljB; fliC and fliC were purified by Ni-NTA Columns. The recombinant proteins were further confirmed by Western blotting analysis using anti-flagellin serum from Guinea pig. Through optimizing the expression conditions, flagellins were expressed in dissoluble form. The purified flagellins would lay the foundation for researching their adjuvant properties later.
出处
《中国畜牧兽医》
CAS
北大核心
2015年第4期871-876,共6页
China Animal Husbandry & Veterinary Medicine
基金
兰州市生物医药专项
关键词
鞭毛蛋白
原核表达
纯化
flagellin
prokaryotic expression
purification
