摘要
采用RT-PCR方法从接种了猪瘟疫苗的家兔脾组织中扩增出了P7基因,将其克隆到表达载体pSMK中,测序验证后转化入表达菌BL21中进行诱导表达。结果显示,重组菌可以表达出分子质量约为19ku的重组融合蛋白,经终浓度为1mmol/L的IPTG诱导2h的表达效果较好。表达产物经Ni柱纯化,可得到纯度较好的目的蛋白。纯化的蛋白置于4℃2d后进行Western-blot检测,发现有多聚体形成;戊二醛交联结果表明,P7蛋白可形成同源寡聚体。此研究结果为进一步研究猪瘟病毒P7基因表达蛋白的性质和功能奠定了基础。
P7 gene of classical swine fever virus（CSFV） was amplified from the vaccinated rabbit＇s spleen by RT-PCR. The gene was cloned into the expression vector pSMK. After sequencing, the right re- combinant plasmid was transformed into BL21. The transformed bacteria were induced by IPTG and ex- pressed. The expressed recombinant protein was about 19 ku in molecular mass. In result,1 mmol/L IPTG and 2 h for induction were the best conditions for the expression of P7 protein. The protein was purified using Ni column. The expressed protein was rested for 2 days at 4 ℃ and the homo-oligomers were found by Western-blot. At last the protein was homologous oligomerization by glutaraldehyde. The result laid the founda- tion for further study on the nature and function of the expressed CSFV P7 protein.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2014年第3期235-239,共5页
Chinese Veterinary Science
基金
国家自然科学基金青年科学基金项目(31101838,31100688)
甘肃省科技专项(1102NKD033,1102NKD034,1104WCGA185)
教育部留学归国人员科研启动基金项目
中国农业科学院基本科研业务费预算增量项目(2013ZL035)
关键词
猪瘟病毒
P7蛋白
原核表达
同源寡聚
classical swine fever virus
P7 protein
prokaryotic expression
homologous oligomerization
