CAJ | 학술논문

目的：构建发热伴血小板减少综合征布尼亚病毒（ SFTSV）核蛋白（ NP）基因的原核表达载体，获得纯化并具有抗原性的重组表达蛋白。方法：提取病毒RNA，逆转录为cDNA后 PCR 扩增得到目的片段；通过连接pMD18-T构建克隆载体，测序正确后连接至原核表达载体pMAL-c2x中，转化大肠杆菌JM 109，IPTG诱导表达、纯化MBP-NP融合蛋白。采用SDS-PAGE、Western blot对融合蛋白进行分析；建立间接ELISA方法对SFTSV患者血清NP抗体进行检测。结果：成功构建了NP基因的原核表达载体pMAL-c2x-NP，通过优化表达条件获得了纯化的带MBP标签的融合蛋白，经免疫印迹杂交证实重组蛋白具有抗原性。用建立的ELISA方法检测96例SFTSV患者血清，阳性率为40．6％；实时荧光定量RT-PCR 方法阳性率为55．2％，两者符合率为77．1％（ Kappa＝0．550， P＜0.001）。结论：成功构建了SFTSV NP基因的原核表达系统，诱导表达了具有抗原性的MBP-NP融合蛋白，为进一步研发SFTSV的ELISA诊断试剂盒奠定了基础。
목적：구건발열반혈소판감소종합정포니아병독（ SFTSV）핵단백（ NP）기인적원핵표체재체，획득순화병구유항원성적중조표체단백。방법：제취병독RNA，역전록위cDNA후 PCR 확증득도목적편단；통과련접pMD18-T구건극륭재체，측서정학후련접지원핵표체재체pMAL-c2x중，전화대장간균JM 109，IPTG유도표체、순화MBP-NP융합단백。채용SDS-PAGE、Western blot대융합단백진행분석；건립간접ELISA방법대SFTSV환자혈청NP항체진행검측。결과：성공구건료NP기인적원핵표체재체pMAL-c2x-NP，통과우화표체조건획득료순화적대MBP표첨적융합단백，경면역인적잡교증실중조단백구유항원성。용건립적ELISA방법검측96례SFTSV환자혈청，양성솔위40．6％；실시형광정량RT-PCR 방법양성솔위55．2％，량자부합솔위77．1％（ Kappa＝0．550， P＜0.001）。결론：성공구건료SFTSV NP기인적원핵표체계통，유도표체료구유항원성적MBP-NP융합단백，위진일보연발SFTSV적ELISA진단시제합전정료기출。
Aim:To construct a MBP-tag prokaryotic expression plasmid containing the nucleocapsid protein ( NP) gene of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV)and to obtain the purified fusion protein with antigenic activity.Methods: Total RNA from the virus was extracted to synthesize cDNA .The NP gene was cloned into a pMD 18-T cloning vector.After sequencing was confirmed ,the NP gene digested from the pMD 18-T-NP was inserted into the pMAL-c2x vector.The E.coli JM109 strain was transformed with the recombinant vector and induced to express MBP-NP fusion protein through IPTG.The production was analyzed with SDS-PAGE and Western blot.An indirect ELISA method was estab-lished to detect the NP antibody from SFTSV patients serum .Results:The recombinant expression vector pMAL-c2x-NP was constructed successfully .Moreover,the purified recombinant protein with MBP-tag was acquired with optimized induc-tion and Amylose resin .A special SFTSV antigenicity of this fusion protein was identified by western blot .The established ELISA method showed a positive rate of 406.% for 96 clinical samples .Compared with the real-time RT-PCR,the coinci-dence rate of indirect ELISA was 77.1%(Kappa=0.550,P<0.001).Conclus ion:A prokaryotic expression system con-taining the NP gene of SFTSV was constructed and the fusion protein possessing antigenicity was efficiently expressed after induction.The established indirect ELISA provided a basis for the development of ELISA kit .