表达甲型H5N1禽流感病毒结构蛋白的重组痘苗病毒的构建与鉴定  

Construction and identification of recombinant vaccinia virus expressing structural proteins of H5N1 avian influenza A virus

作　　者：邱冉 张哲罡 闫璐瑶 李兴航 韩天 刘琛 年悬悬 张家友 赵巍 杨晓明 Qiu Ran;Zhang Zhegang;Yan Luyao;Li Xinghang;Han Tian;Liu Chen;Nian Xuanxuan;Zhang Jiayou;Zhao Wei;Yang Xiaoming(Laboratory 2 of Viral Vaccine Research,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,China;China National Biotec Group Co.,Ltd.,Beijing 100029,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究二室,430207 [2]中国生物技术股份有限公司,北京100029

出　　处：《国际生物制品学杂志》2020年第3期105-109,共5页International Journal of Biologicals

摘　　要：目的构建表达甲型H5N1禽流感病毒(H5N1 avian influenza A virus,H5N1 AIAV)NIBRG14株结构蛋白的重组痘苗病毒,为研制新型人用流感疫苗奠定基础。方法通过反转录PCR扩增H5N1 AIAV NIBRG14株的血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)编码基因,并将改造的HA、NA基因克隆至痘苗病毒穿梭质粒pSCCK。在重组质粒与痘苗病毒天坛株(vaccinia virus Tiantan,VTT)于Vero细胞中发生同源重组后,筛选同时表达HA和NA的重组痘苗病毒(rVTT-HA/NA),并对其进行鉴定。结果反转录PCR扩增的HA和NA基因大小约分别为1700和1400 bp,与预期相同。DNA测序证实,改造的HA和NA序列正确。对获得的rVTT-HA/NA进行PCR及测序表明,HA和NA基因正确插入VTT。蛋白质印迹显示,rVTT-HA/NA感染的Vero细胞表达的HA和NA能与相应的抗体发生反应。表达的HA具有血凝活性,血凝滴度为1∶32。结论重组痘苗病毒rVTT-HA/NA可稳定表达H5N1 AIAV NIBRG14株的HA和NA。Objective To construct a recombinant vaccinia virus expressing the structural proteins of H5N1 avian influenza A virus(AIAV)NIBRG14 strain,in order to lay the foundation for developing new influenza vaccine for human.Methods Full length genes encoding hemagglutinin(HA)and neuramindase(NA)of H5N1 AIAV NIBRG14 strain were amplified with reverse transcription PCR(RT-PCR),and modified HA and NA genes were cloned into shuttle plasmid pSCCK of vaccinia virus.After the recombinant plasmid homologously recombined with the vaccinia virus Tiantan(VTT)in Vero cells,the recombinant vaccinia virus(rVTT-HA/NA)co-expressing HA and NA of H5N1 AIAV NIBRG14 strain was selected and validated.Results The sizes of HA and NA genes amplified by RT-PCR were about 1700 and 1400 bp,respectively,and in line with expectations.The modified HA and NA sequences were correct according to DNA sequencing.The results of PCR and sequencing for the obtained rVTT-HA/NA showed that the HA and NA genes were inserted into VTT correctly.Western blotting confirmed that HA and NA expressed by rVTT-HA/NA infected Vero cells were able to react with corresponding antibodies.The expressed HA had hemagglutination activity,and its titer was 1∶32.Conclusion The recombinant vaccinia virus rVTT-HA/NA can stably express HA and NA of H5N1 AIAV NIBRG14 strain.

关 键 词：正黏病毒科 血凝素类 病毒 神经氨酸酶 痘苗病毒 疫苗 合成 

分 类 号：R392-33[医药卫生—免疫学]