摘要
为获得含有口蹄疫病毒(foot-and-mouth disease virus,FMDV)3D蛋白C端多个Th表位的重组表达蛋白,根据口蹄疫3DC端160个氨基酸序列,设计优化并人工合成相应的核酸序列,将其克隆至pET28a,构建原核表达质粒pET28a-C160,并将重组质粒转化BL21(DE3),0.5mmol/L IPTG诱导后,收集菌液进行SDS-PAGE和Western-blot鉴定。结果显示,在25ku处有一条明显的蛋白表达条带,且重组蛋白能与豚鼠抗O型口蹄疫病毒阳性血清发生特异性反应。对表达产物进行可溶性分析,结果显示,表达蛋白全部以包涵体形式存在,经变复性后获得较高质量浓度的纯化蛋白。可见:用纯化的3DC160蛋白作为Th佐剂与口蹄疫主抗原混合制成疫苗,免疫后能刺激猪体产生较高水平的口蹄疫抗体。
To obtain a recombinant protein containing multiple T helpers （Th） epitopes of the 3D pro- tein of foot and mouth disease virus （FMDV）,the nucleotides encoding these epitopes were optimized to be expressed in Escherichia coli （E. coli） BL21 （DE3）. Following the induction of 0.5 mmol/L iso- propyl-beta-D-thiogalactopyranoside （IPTG）, recombinant cells exhibited an apparent 25 ku band in sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. Western blot revealed that the recombinant protein could react specifically with Guinea pig anti O type foot-and-mouth disease vi- rus serum. The high purity and activity protein obtained after purified by Ni-NTA resins. The results showed that the recombinant protein was expressed efficiently in E. coli as inclusion bodies and could be renatured and purified. The purified 3D C160 protein as adjuvant mixed with the antigens of foot- and-mouth disease virus to make vaccine, after immunization it can stimulate pig to produce higher lev- el of antibodies against foot-and-mouth disease.
出处
《西北农业学报》
CAS
CSCD
北大核心
2013年第4期11-16,共6页
Acta Agriculturae Boreali-occidentalis Sinica
基金
河南省级重点实验室建设专项基金项目(122300413217)
NSFC-河南人才培养联合基金(U1204327)
关键词
口蹄疫病毒
3D蛋白
TH表位
原核表达
蛋白纯化
Foot-and-mouth disease virus
3D polymerase
Helper T lymphocyte epitope
Prokaryoticexpression
Purification of protein
