摘要
目的:在大肠杆菌中高效表达缺失型美洲商陆抗病毒蛋白PAP-C_(23),并制备其抗血清。方法:从成熟PAP中克隆了C端缺失23个氨基酸的基因PAP-C_(23),以PET101为表达载体构建了重组质粒,转化入大肠杆菌BL21(DE3),IPTG诱导表达。切胶回收表达的重组蛋白,冰浴研磨后用PBS溶解,再加入等体积福氏不完全佐剂,免疫家兔,制备抗PAP蛋白的多克隆抗体。结果:SDSPAGE分析表明,目的蛋白在BL21(DE3)中获得表达,表达产物以不溶的包涵体形式存在。通过对表达条件的优化,重组蛋白的表达量可占总包涵体蛋白的29.6%。间接ELISA法测定所制备抗血清效价为1:1000,Western blot分析结果显示,该抗血清与表达的重组蛋白和天然的PAP蛋白均发生特异性的免疫反应,结论:PAP-C_(23)基因在大肠杆菌中得到了正确表达,且重组蛋白的免疫原性较好,所制备的抗血清与天然PAP发生特异性免疫反应。
Pokeweed antiviral protein(PAP),a 29kDa protein isolated from Phytolacca americana,inhibits translation by catalyticaUy removing a specific adenine residue from die large rRNA of the 60s subunit of eukaryotic ribosomes.PAP-C23,a kind of deleted mutant of PAP gene,was amplified from mature pokeweed antiviral protein gene by PCR,then the amplified fragment was cloned into the expression vector PET101,and then the recombinant plasmid was transformed into Escherichia coli BL21(DE3) strain.Induced with IPTG,the recombinant gene was expressed.SDS-PAGE analysis showed that the recombinant protein existed in the form of inclusion body.The recombinant protein amount may accounted for 29.6%of the total inclusion proteins by optimizing the expression conditions.After recycled by cutting the gel which has been stained by KCL solution,the recombinant protein was grinded fully with a pesde in ice-bath and dissolved in phosphate buffered saline(PBS),and then added into same volume Freund's adjuvant incomplete.After emulsified completely,the mixed solution was used to inject rabbits,to prepare polyclonal antibody.Indirect enzyme linked immunosorbent assay(ELISA) showed that the titer of the antiserum was 1:1000.Western blot analysis showed that the antiserum could specifically react with expressed recombinant protein and nature PAP isolated from Phytolacca amercana,which also indicated that the recombinant PAP gene was expressed correcdy in Escherichia coli.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2008年第S1期61-64,共4页
China Biotechnology
基金
中国热带农业科学院中央级公益性科研院所基本科研业务费专项资金资助项目
