流感病毒血凝素茎干部通用疫苗的构建及免疫原性研究

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摘要为了构建基于血凝素(hemagglutinin , HA)茎干部的流感通用疫苗并研究其免疫原性，本研究以A/Puerto Rico/8/1934(H1N1)流感病毒为模板，扩增HA茎干部区域主要蛋白HA2(HA的344~566 aa)和完整茎干部HA-stem(缺失53 aa~276 aa的HA)，与真核表达载体pCAGGS连接获得重组质粒。将重组质粒瞬时转染至HEK-293T细胞，利用RT-PCR及间接免疫荧光实验检测其表达情况。将6~8周龄健康雌性BALB/c小鼠(Mus musculus)分为4组，即2个重组质粒组及pCAGGS质粒对照组和磷酸盐缓冲液(phosphate buffer saline, PBS)对照组，分别于0和30 d免疫2次，在免疫当天(第0天)及免疫后第60天采集小鼠眼眶血，测定其HI抗体、细胞中和抗体和IFN-γ水平。结果表明，成功构建了重组质粒pCAGGS-HA2和pCAGGS-HA-stem，且重组质粒转染HEK-293T细胞后均能检测到目的蛋白及其mRNA。免疫后第60天pCAGGS-HA2和pCAGGS-HA-stem免疫组对A/Puerto Rico/8/1934(H1N1)病毒的HI抗体效价均为5 log2，中和抗体效价分别为1∶27和1∶24。对异源病毒A/Chicken/Hebei/4/2008(H9N2)病毒的HI抗体效价分别为3 log2和6 log2，中和抗体效价分别为1∶24和1∶22。pCAGGS-HA2和pCAGGS-HA-stem组INF-γ含量分别为(2362.40±98.24) ng/L和(2497.06±120.44) ng/L。pCAGGS-HA2及pCAGGS-HA-stem诱导产生的抗体及INF-γ水平与免疫前及对照组相比差异显著(P＜0.05)。结果表明，pCAGGS-HA2和pCAGGS-HA-stem能够诱导对异源病毒的交叉反应，有望作为候选的流感通用疫苗。

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	马礼照
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关键词 ：流感病毒, 血凝素蛋白, 茎干部, 通用型疫苗

Abstract：In order to construct a Hemagglutinin-stem based universal vaccine and investigate its immunogenicity, the hemagglutinin (HA) gene of A/Puerto Rico/8/1934(H1N1) virus was used as template in this study to amplify the stem region, namely HA2(main protein of HA stem region) and HA-stem (whole stem region). The amplicons were inserted into pCAGGS vector, and the constructed recombinant plasmids were transiently transfected into HEK-293T cells. Then, the proteins expression was identified by RT-PCR and indirect immunofluorescence (IFA) assay. 6~8 weeks female BALB/c mice (Mus musculus) were divided into 2 recombinant plasmids group, pCAGGS group, and PBS group. The mice were intramuscularly inoculated twice at 0 and 30 d . Orbital blood was sampled at 0 and 60 d post immunization, and subjected to HI antibody titration, neutralizing antibody titration and INF-γ detection. The results showed that recombinant plasmids pCAGGS-HA2 and pCAGGS-HA-stem were successfully constructed, and could normally express target proteins in HEK-293T cells. pCAGGS-HA2 and pCAGGS-HA-stem induced antibody against A/Puerto Rico/8/1934(H1N1)in mice with HI titre 5 log2, and neutralizing antibody 1∶27 and 1∶24, respectively. While HI titre and neutralizing antibody against A/Chicken/Hebei/4/2008(H9N2) was 3 log2 and 6 log2, 1∶24 and 1∶22. The INF-γ level in pCAGGS-HA2 and pCAGGS-HA-stem group was (2362.40±98.24)ng/Land (2497.06±120.44) ng/L.The antibody titre and INF-γ level induced by pCAGGS-HA2 and pCAGGS-HA-stem was significantly higher than those induced by control group(P＜0.05). It indicates that pCAGGS-HA2 and pCAGGS-HA-stem can induce cross reaction with heterologous influenza virus, and may be a hopeful candidate universal influenza virus.

Key words： Influenza virus Hemagglutinin (HA) Stem region Universal vaccine

收稿日期: 2016-05-04 出版日期: 2016-10-01

基金资助:青岛市科技计划基础研究项目;财政部中国亚太经合组织合作基金项目

通讯作者: 黄娟 E-mail: happyhj888@163.com;juanhuang@qau.edu.cn

引用本文:

马礼照刘焕李桂梅黄娟单虎. 流感病毒血凝素茎干部通用疫苗的构建及免疫原性研究[J]. , 2016, 24(11): 1764-1770.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I11/1764

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