CAJ | 학술논문

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3．3 mg/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。
목적：인박잡병독（HBoV）시근궤년발현적일충도치인지기관염적병원체，본연구지재표체순화기 VP1의각단백독유구（VP1-U）항원，위면역학진단치료전정기출。방법우화 HBoV VP1-U DNA 밀마자，이괄응대장간균원핵표체밀마자친기성。장밀마자우화개조적 DNA 서렬극륭도 pET30a（＋）원핵표체재체，C 말단융합표체조안산친화순화표첨。LB 배양기증균，IPTG 유도표체。채용자행설계적순화완충액체계，얼주친화순화，초려농축。결과VP1-U 단백표체효솔약위50％，순화활성단백농도위3．3 mg/ml。결론경과밀마자우화，합리설계공예류정，원핵표체계통가이고효표체순화 VP1-U 활성단백항원。
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 re-combinant 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 pro-tein was about 3.3 mg/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.