2015-2016年度中国甲型H1N1亚型流感病毒疫苗候选株的制备及鉴定  

Preparation and identification of influenza H1N1 subtype vaccine candidate strain in China

作　　者：唐静[1] 辛丽[1] 李晓丹[1] 陈永坤[1] 郭俊峰[1] 黄维娟[1] 成艳辉[1] 王大燕[1] 舒跃龙[1] Tang Jing Xin Li Li Xiaodan Chen Yongkun Guo Junfeng Huang Weijuan Cheng Yanhui Wang Dayan Shu Yuelong(National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Key Laboratory for Medical Virology, Ministry of Health, Beijing 102206, China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室,北京102206

出　　处：《中华实验和临床病毒学杂志》2017年第4期348-352,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的 为获得2015-2016年度中国流行的甲型H1N1亚型流感病毒疫苗候选株,制备流感病毒重配株并对其进行鉴定.方法 采用经典重配的方法,将H1N1亚型流感病毒流行株与H3N2亚型的鸡胚高产重配母本株（X-157株）在鸡胚上进行混合培养.用H3亚型的HA蛋白抗血清和X-157株全病毒抗血清对混合培养病毒进行阴性筛选.阴性筛选后HA滴度较高的病毒用Real-Time PCR法对表面蛋白基因型进行鉴定.对表面蛋白基因型正确的毒株用限制性内切酶酶切鉴定法鉴定其内部基因组成.进一步对HA和NA基因进行Sanger法测序,并用表面基因无氨基酸位点突变的毒株免疫雪貂,进行双向血凝抑制（Two-way hemagglutination inhibition test,HI）试验.结果 Real-Time PCR筛选出5株表面蛋白基因型正确的毒株.经内部基因鉴定其中4株为6＋2组成,1株为5＋3组成.5株重配株的HA和NA基因均未发生氨基酸位点突变.5株重配株HA滴度均维持在1 024以上.最终选取的12号重配株免疫原性良好,HI滴度达5 120,双向HI试验均通过.重配后疫苗株在鸡胚上的产量是重配前野毒株的64倍.结论 成功制备了2015-2016年度中国流行的甲型H1N1亚型流感病毒疫苗株,为疫苗贮备和疾病防控奠定了基础.Objective Influenza H1N1 subtype vaccine candidate strains from a 2015-2016 year epidemic strain in China were prepared and identified by themethod of classical reassortment.Methods The influenza H1N1 epidemic strain and H3N2 high-yield reassortant parental strain （X-157） were mixed and inoculatcd into embryonated chicken eggs by the classical reassortmentmethod.The negative selection of mixed culture virus was carried out with the antiserum of H3 protein and the antiserum of X-157 strain.Realtime PCRmethod was used to test the HA and NA genes.Restriction enzyme digestionmethod was used to identify the internal genes.HA and NA genes of selected strains were sequenced.The strain which HA and NA genes possessed the same amino acid constitution with the wild type virus was selected and immunized to ferret.Two-way test was carried out.Results Five strains with expected HA and NA genes were selected by real-time PCR.Internal genes were identified,with 4 strains had 6 ＋ 2 constitution,1 strain had 5 ＋ 3 constitution.Comparing with the wild type virus,HA and NA genes of the 5 strains had no mutation.HA titer of reassortant strains was above 1 024.HI titer of the selected NO.12 reassortment strain reached 5 120,and two-way test was passed.The yield of reassortant strain was 64 times that of the wild type strain.Conclusions A circulating influenza A （H1N1） strain of influenza A （2015-2016） was successfully prepared in China and laid the foundation for vaccine storage and disease prevention and control.

关 键 词：流感病毒A型 经典重配 H1N1亚型 流行株 

分 类 号：R392-33[医药卫生—免疫学]