欧亚类禽型H1N1猪流感病毒HA蛋白的表达及免疫原性评估

doi:10.3864/j.issn.0578-1752.2019.05.014

摘要/Abstract

摘要：

【目的】表达欧亚类禽型（eurasian avian-like,EA）H1N1猪流感病毒（SIV）的血凝素（HA）蛋白,并对其免疫原性进行测定。【方法】利用RT-PCR技术扩增病毒A/swine/Zhejiang/245/2013（H1N1）(ZJ245)的HA基因,将其克隆至真核表达载体pCAGGS中,获得重组质粒pCAGGS-HA(ZJ245),将其转染293T细胞,采用间接免疫荧光（IFA）检测和Western blot检测HA蛋白在体外的表达;将重组质粒pCAGGS-HA(ZJ245)经肌肉注射途径、以100 μg/只剂量免疫16只6周龄BALB/c小鼠（I组和II组）,间隔3周后进行加强免疫（二免）;同等数量的小鼠以相同方式注射100 μL无菌PBS作为非免疫对照组（III组和IV组）。首免和二免每周采血,分别采用血凝抑制（HI）试验和病毒中和（VN）试验两种方法测定不同类型的血清抗体效价;二免2周后,其中两组小鼠（I组和III组）用50 μL（10 ^6.0EID₅₀）的ZJ245病毒经滴鼻感染途径进行攻毒,另外两组（II组和IV组）用A/swine/Heilongjiang/44/2009（H1N1）（HLJ44）病毒进行攻毒。攻毒后14 d内每天观察小鼠的临床症状、统计发病与死亡情况,且每天称量小鼠体重,在体重下降比率超过25%时判定为小鼠死亡。攻毒后第3 天,每组随机剖杀3只小鼠,分别采集脑、鼻甲、肺、脾和肾等脏器,匀浆处理后通过接种10日龄的非免疫鸡胚测定脏器的病毒含量。通过小鼠体重变化以及脏器滴定的病毒含量评估重组质粒pCAGGS-HA(ZJ245)的免疫保护效果。【结果】经酶切鉴定和测序验证表明ZJ245的HA基因的已正确克隆至真核表达质粒pCAGGS中获得重组质粒pCAGGS-HA(ZJ245),经体外转染293T细胞后,IFA和Western blot证实了病毒的HA蛋白能够正确表达,并具有良好的生物学活性;小鼠的免疫与攻毒试验结果表明,重组质粒pCAGGS-HA(ZJ245)首免后一周可检测到针对同源病毒ZJ245的低水平HI和VN抗体,加强免疫后抗体水平明显升高,HI抗体达到76.88、VN抗体达到152.5;同时针对异源病毒HLJ44也产生了较低水平的HI和VN抗体。10 ^6.0EID₅₀的同源病毒ZJ245攻毒时,与非免疫组小鼠相对比,重组质粒pCAGGS-HA(ZJ245)的免疫完全阻止了因病毒攻击而导致的小鼠体重下降以及肺脏和鼻甲内病毒的复制;10 ^6.0EID₅₀的异源病毒HLJ44攻毒时,相比非免疫组小鼠,质粒免疫组小鼠体重的下降比率、以及肺脏和鼻甲内的病毒滴度均显著降低（P<0.0001、P <0.001、P <0.05）。【结论】重组质粒pCAGGS-HA(ZJ245)能够有效表达病毒HA蛋白,免疫后可使小鼠获得完全抵抗ZJ245感染及部分抵御HLJ44感染的免疫保护力,表明重组质粒 pCAGGS-HA(ZJ245)具有良好的免疫原性。

关键词: 欧亚类禽型H1N1猪流感病毒, HA蛋白, 重组质粒, 免疫原性, DNA疫苗

Abstract:

【Objective】This study aimed to construct the recombinant plasmid expressing HA gene of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) and then evaluate its immunogenicity in mice. 【Method】HA gene of A/swine/Zhejiang/ 245/2013(H1N1) (ZJ245) was amplified by RT-PCR, and inserted into an eukaryotic expression vector pCAGGS. The recombinant plasmid, designated as pCAGGS-HA(ZJ245), was transfected into 293T cells, and the expressed HA protein was identified by indirect fluorescence assay (IFA) and Western blot. In order to evaluate the immunogenicity of the recombinant plasmid pCAGGS- HA(ZJ245), sixteen six-week-old female BALB/c mice were immunized with 100 μg of the recombinant plasmid by intramuscular injection, and then were boosted once with a 3-week interval. Another group of sixteen mice received 100 μL of phosphate-buffered saline (PBS) were used as unvaccinated control. Serum samples were collected every week after prime and boost immunization in order to detect the hemagglutinin inhibition (HI) antibodies, virus neutralization (VN) antibodies, respectively. Two weeks after the boost immunization, pCAGGS-HA(ZJ245)-immunized and PBS-inoculated mice were intranasally challenged with 50 μL(10 ^6.0EID₅₀) of the homologous ZJ245 and heterologous A/swine/Heilongjiang/44/2009(H1N1) (HLJ44), respectively. All mice per group were monitored daily for clinical signs of infection and body weight changes for two weeks. The mice that lost more than 25% of their initial body weight were euthanized on humane ground. On day 3 post-challenge three mice per group were euthanized and their organs including brain, nasal turbinate, lung, spleen and kidney were collected for virus titration in eggs. Immune efficacy of the recombinant plasmid pCAGGS-HA(ZJ245) was evaluated by body weight loss and virus replication titer in mice, respectively. 【Result】The recombinant plasmid pCAGGS-HA(ZJ245) was constructed by inserting HA gene of ZJ245 virus and verified by restriction endonuclease analysis and plasmid sequencing. IFA and Western blot analysis confirmed that the HA protein could be correctly expressed by the recombinant plasmid pCAGGS-HA(ZJ245) and had a good biological activity in vitro. Immunization and challenge trial indicated that low levels of HI and VN antibody against the homologous ZJ245 virus were initially detected one week after the first immunization, and significantly increased after the second immunization, with the HI titer of 76.88 and the VN titer of 152.5, respectively. Meanwhile, low levels of HI and VN antibodies against the heterologous HLJ44 virus were also detected. Compared with PBS-inoculated mice, the weight loss and viral replication rate of the pCAGGS-HA(ZJ245)-vaccinated mice when challenged with 10 ^6.0EID₅₀of the homologous ZJ245 virus were completely inhibited. When challenged with 10 ^6.0EID₅₀of the heterologous HLJ44 virus, the extent of weight loss and viral titer of the challenge virus detected in the pCAGGS-HA(ZJ245)- vaccinated mice were significantly lower than those in the PBS-inoculated mice (P<0.0001, P<0.001, P<0.05).【Conclusion】The recombinant plasmid pCAGGS-HA(ZJ245) could efficiently express HA protein, and provided complete protection for the immunized mice against the homologous ZJ245 virus infection and partial cross-protection against the heterologous HLJ44 virus infection, which indicated that the recombinant plasmid pCAGGS-HA(ZJ245) had good immunogenicity.

Key words: Eurasian avian-like H1N1 swine influenza virus, hemagglutinin protein, recombinant plasmid, immunogenicity, DNA vaccine

贾云慧,许程志,隋金钰,吴运谱,许榜丰,陈艳,杨焕良,乔传玲,陈化兰. 欧亚类禽型H1N1猪流感病毒HA蛋白的表达及免疫原性评估[J]. 中国农业科学, 2019, 52(5): 930-938.

JIA YunHui,XU ChengZhi,SUI JinYu,WU YunPu,XU BangFeng,CHEN Yan,YANG HuanLiang,QIAO ChuanLing,CHEN HuaLan. Immunogenicity Evaluation of Eukaryotic Expressing Plasmids Encoding HA Protein of Eurasian Avian-Like H1N1 Swine Influenza Virus[J]. Scientia Agricultura Sinica, 2019, 52(5): 930-938.

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