H1N2亚型猪流感病毒进化分析及抗HA蛋白单克隆抗体的制备
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摘要
猪流感病毒(Swine Influenza Virus, SIV)是猪呼吸道疾病的主要病原之一,目前已发现的猪流感病毒有H1N1、H1N2、H1N7、H2N3、H3N1、H3N2、H3N6、H4N6、H5N1及H9N2亚型。我国从2004年分离到第一株H1N2猪流感病毒以来,连续又在不同省份分离到H1N2亚型重配猪流感病毒,这类病毒能否引起区域性的或者全国性的猪流感暴发,值得更深一步研究。
本研究根据H1N2毒株的参考序列设计RT-PCR及测序引物,对本实验室分离、鉴定的的两株H1N2亚型猪流感病毒(SW/HeB/10/06和SW/TJ/1/07)进行了全基因组序列测定,并利用生物信息学软件对其8个基因片段进行进化分析。结果SW/HeB/10/06和SW/TJ/1/07两个分离毒株的亲缘关系较远,8个基因片段的同源性:PB2为81.4%,PB1为83.2%,PA为83.0%,HA为90.1%,NP为81.6%,NA为91.7%,M为84.1%,NS为80.4%。毒株SW/TJ/1/07的HA及NA基因与我国的第一株H1N2分离株(SW/ZJ/1/04)同源性最高,其6个内部基因分别与欧洲分离的两株H1N1亚型猪流感病毒具有最高的同源性;毒株SW/HeB/10/06的HA、NA及NS基因片段与禽流感病毒A/Turkey/MO/24093/99(H1N2)同源性最高;其它5个内部基因分别与H1N1人流感病毒和H3N2及H1N2猪流感病毒的相应基因片段具有最高同源性,SW/HeB/10/06为我国分离到的第一株人-禽-猪流感基因三重配病毒。以上结果表明我国所出现的H1N2毒株遗传背景较为复杂。
通过血凝抑制(HI)交互试验,从实验室分离的4株H1亚型猪流感病毒中筛选出一株具有抗原代表性的毒株SW/HeN/11/05 (H1N1)作为免疫原,制备抗H1亚型猪流感病毒的血凝素蛋白的单克隆抗体。将SW/HeN/11/05毒株接种9～11日龄鸡胚进行增殖,收取尿囊液,浓缩、纯化,与佐剂混匀,免疫6周龄BALB/c雌性小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合。采用HI和酶联免疫吸附试验(ELISA)方法筛选阳性细胞克隆,经连续多次克隆培养后获得2株能稳定分泌抗H1亚型HA蛋白特异性单克隆抗体的细胞株4E6B2和3E11C4。两株单克隆抗体能够与H1亚型猪流感病毒毒株发生特异性的反应,而与H3、H5、H9亚型猪流感病毒及其它常见猪病病原无交叉反应。
总之,本研究分析了两株H1N2亚型猪流感病毒的遗传进化关系,为两株亲缘关系较远的重配猪流感病毒,这两个H1N2亚型毒株的分离为猪作为混合器产生新型重配流感病毒提供了更进一步的证据;同时,制备了两株抗H1亚型猪流感病毒HA蛋白的单克隆抗体细胞株,为下一步建立H1亚型猪流感病毒诊断方法提供了物质基础和条件。
Swine influenza virus (SIV) is one of important pathogens that cause respiratory disease in pigs. At present, the subtypes of SIV includes H1N1, H1N2, H1N7, H2N3, H3N1, H3N2, H3N6, H4N6, H5N1 and H9N2. Several reassortant H1N2 SIV strains were isolated from some provinces in recent years, since the first H1N2 SIV strain was isolated in China in 2004. Whether or not an epidemic swine influenza disease induced by these viruses would break out in pigs should be further studied.
In the present study, the specific primers for RT-PCR and sequencing were designed based on the preference sequences of H1N2 viruses. All the eight gene fragments of SW/HeB/10/06 and SW/TJ/1/07 are sequenced and genetically analysised with bioinformatic softwares. Results of genetic analysis showed that SW/HeB/10/06 and SW/TJ/1/07 are far in relatives. Homology of eight gene fragments of the two strains is: PB2 81.4%,PB1 83.2%,PA 83.0%,HA 90.1%,NP 81.6%,NA 91.7%,M 84.1%,NS 80.4%. HA and NA gene fragments of SW/TJ/1/07 shows highest homology with corresponding genes of SW/ZJ/1/04 (H1N2), and other internal gene fragments with two H1N1 SIV strains in europe. HA, NA and NS gene fragments of SW/HeB/10/06 shows highest homology with A/Turkey/MO/24093/99,and other internal gene fragments with H1N1 human influenza virus, H3N2 and H1N2 swine influenza strains. SW/HeB/10/06 is proved to be a new reassorted virus different from other H1N2 SIV, which is the first H1N2 SIV including human,avian and swine influenza genes in mainland.
We proceed HI test among 4 H1 influenza viruses and serum. According to the results, SW/HeN/11/05 was selected to be propagated in embryonated eggs. Allantoic fluids were harvested and puried. Mixed with Freunds adjuvant, the viruses were injected into BALB/c female mice. Two monoclonal antibody(McAb)hybridoma cell strains were developed by fusing SP2/0 cells with spleen cells of mouse that had been immunized with SIV SW/HeN/11/05 (H1N1).Cloned several times, positive hybridoma cells strains 4E6B2,3E11C4 were determined by HI and ELISA . In HI test, The two antibodies show specific activity in HI, no activity with H3, H5 and H9 swine influenza virus and other pathogens of swine.
In summary, genetic relationship of these two SIV stains were analysed in the study. The results showed that the two new reassorted SIV strains were far in relatives. The strains SW/TJ/1/07 and SW/HeB/10/06 provides evidence for producing new reassorted influenza viruses with pig as a vessel. Meanwhile, two monoclonal antibody(McAb)hybridoma cell strains against HA protein were developed, providing materials and conditions for detecting H1 SIV.
本研究根据H1N2毒株的参考序列设计RT-PCR及测序引物,对本实验室分离、鉴定的的两株H1N2亚型猪流感病毒(SW/HeB/10/06和SW/TJ/1/07)进行了全基因组序列测定,并利用生物信息学软件对其8个基因片段进行进化分析。结果SW/HeB/10/06和SW/TJ/1/07两个分离毒株的亲缘关系较远,8个基因片段的同源性:PB2为81.4%,PB1为83.2%,PA为83.0%,HA为90.1%,NP为81.6%,NA为91.7%,M为84.1%,NS为80.4%。毒株SW/TJ/1/07的HA及NA基因与我国的第一株H1N2分离株(SW/ZJ/1/04)同源性最高,其6个内部基因分别与欧洲分离的两株H1N1亚型猪流感病毒具有最高的同源性;毒株SW/HeB/10/06的HA、NA及NS基因片段与禽流感病毒A/Turkey/MO/24093/99(H1N2)同源性最高;其它5个内部基因分别与H1N1人流感病毒和H3N2及H1N2猪流感病毒的相应基因片段具有最高同源性,SW/HeB/10/06为我国分离到的第一株人-禽-猪流感基因三重配病毒。以上结果表明我国所出现的H1N2毒株遗传背景较为复杂。
通过血凝抑制(HI)交互试验,从实验室分离的4株H1亚型猪流感病毒中筛选出一株具有抗原代表性的毒株SW/HeN/11/05 (H1N1)作为免疫原,制备抗H1亚型猪流感病毒的血凝素蛋白的单克隆抗体。将SW/HeN/11/05毒株接种9～11日龄鸡胚进行增殖,收取尿囊液,浓缩、纯化,与佐剂混匀,免疫6周龄BALB/c雌性小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合。采用HI和酶联免疫吸附试验(ELISA)方法筛选阳性细胞克隆,经连续多次克隆培养后获得2株能稳定分泌抗H1亚型HA蛋白特异性单克隆抗体的细胞株4E6B2和3E11C4。两株单克隆抗体能够与H1亚型猪流感病毒毒株发生特异性的反应,而与H3、H5、H9亚型猪流感病毒及其它常见猪病病原无交叉反应。
总之,本研究分析了两株H1N2亚型猪流感病毒的遗传进化关系,为两株亲缘关系较远的重配猪流感病毒,这两个H1N2亚型毒株的分离为猪作为混合器产生新型重配流感病毒提供了更进一步的证据;同时,制备了两株抗H1亚型猪流感病毒HA蛋白的单克隆抗体细胞株,为下一步建立H1亚型猪流感病毒诊断方法提供了物质基础和条件。
Swine influenza virus (SIV) is one of important pathogens that cause respiratory disease in pigs. At present, the subtypes of SIV includes H1N1, H1N2, H1N7, H2N3, H3N1, H3N2, H3N6, H4N6, H5N1 and H9N2. Several reassortant H1N2 SIV strains were isolated from some provinces in recent years, since the first H1N2 SIV strain was isolated in China in 2004. Whether or not an epidemic swine influenza disease induced by these viruses would break out in pigs should be further studied.
In the present study, the specific primers for RT-PCR and sequencing were designed based on the preference sequences of H1N2 viruses. All the eight gene fragments of SW/HeB/10/06 and SW/TJ/1/07 are sequenced and genetically analysised with bioinformatic softwares. Results of genetic analysis showed that SW/HeB/10/06 and SW/TJ/1/07 are far in relatives. Homology of eight gene fragments of the two strains is: PB2 81.4%,PB1 83.2%,PA 83.0%,HA 90.1%,NP 81.6%,NA 91.7%,M 84.1%,NS 80.4%. HA and NA gene fragments of SW/TJ/1/07 shows highest homology with corresponding genes of SW/ZJ/1/04 (H1N2), and other internal gene fragments with two H1N1 SIV strains in europe. HA, NA and NS gene fragments of SW/HeB/10/06 shows highest homology with A/Turkey/MO/24093/99,and other internal gene fragments with H1N1 human influenza virus, H3N2 and H1N2 swine influenza strains. SW/HeB/10/06 is proved to be a new reassorted virus different from other H1N2 SIV, which is the first H1N2 SIV including human,avian and swine influenza genes in mainland.
We proceed HI test among 4 H1 influenza viruses and serum. According to the results, SW/HeN/11/05 was selected to be propagated in embryonated eggs. Allantoic fluids were harvested and puried. Mixed with Freunds adjuvant, the viruses were injected into BALB/c female mice. Two monoclonal antibody(McAb)hybridoma cell strains were developed by fusing SP2/0 cells with spleen cells of mouse that had been immunized with SIV SW/HeN/11/05 (H1N1).Cloned several times, positive hybridoma cells strains 4E6B2,3E11C4 were determined by HI and ELISA . In HI test, The two antibodies show specific activity in HI, no activity with H3, H5 and H9 swine influenza virus and other pathogens of swine.
In summary, genetic relationship of these two SIV stains were analysed in the study. The results showed that the two new reassorted SIV strains were far in relatives. The strains SW/TJ/1/07 and SW/HeB/10/06 provides evidence for producing new reassorted influenza viruses with pig as a vessel. Meanwhile, two monoclonal antibody(McAb)hybridoma cell strains against HA protein were developed, providing materials and conditions for detecting H1 SIV.
引文
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