摘要
目的人博卡病毒(HBoV)是近几年发现的一种导致人支气管炎的病原体,本研究旨在表达纯化其VP1衣壳蛋白独有区(VP1-U)抗原,为免疫学诊断治疗奠定基础。方法优化HBoV VP1-U DNA密码子,以适应大肠杆菌原核表达密码子亲嗜性。将密码子优化改造的DNA序列克隆到pET30a(+)原核表达载体,C末端融合表达组氨酸亲和纯化标签。LB培养基增菌,IPTG诱导表达。采用自行设计的纯化缓冲液体系,镍株亲和纯化,超滤浓缩。结果VP1-U蛋白表达效率约为50%,纯化活性蛋白浓度为3.3mg/ml。结论经过密码子优化,合理设计工艺流程,原核表达系统可以高效表达纯化VP1-U活性蛋白抗原。
Objective To express and purificate of HBoV VP1-U protein antigen.Methods HBoV VP1-U codons was Optimized to accommodate E.coil codon tropism,and The DNA sequence which fused to an C-terminal（His）6-tagged that allowed single-step isolation by Ni2＋affinity chromatography,was cloned into pET30a（＋）vector.The recombinant plasmid was transformed into E.coli strain BL21（DE3）induced expression by IPTG.Extracts protein from cell was loaded onto Ni2＋affinity column and（His）6-tagged proteins are selectively eluted with self-designed buffers system.Results The VP1-U expression efficiency was approximately 50%,the concentration of the purified active protein was about 3.3mg/mL.Conclusion By codons optimization and rational process,VP1-U active protein antigen can be efficiently expressed and purified in E.coil prokaryotic expression systems.
出处
《中国实验诊断学》
2014年第8期1226-1229,共4页
Chinese Journal of Laboratory Diagnosis
