甲型流感病毒各亚型假病毒系统的构建及H1亚型特异性保守表位的鉴定

发布时间：2019-06-20 06:06

【摘要】：甲型流感病毒可引起年度季节性流感和反复的流感大流行,严重危害人民健康。近年来甲型H1N1流感的流行和人感染H5N1禽流感病毒病例不断增加,进一步提示加强甲型流感病毒的研究和防控的重要性。血凝素(HA)和神经氨酸酶(NA)是甲型流感病毒编码的表面糖蛋白。根据HA和NA的抗原性,甲型流感病毒可分为16个HA亚型(H1～H16)和9个NA亚型(N1-N9)。HA和NA的重组和重配产生新的病毒是造成流感大流行的重要原因。理论上16个HA和9个NA亚型可自由组合形成144种亚型流感病毒,多数亚型流感病毒已在自然界被发现,但引发大流行的流感病毒的亚型种类仍无法预测。因此,加强亚型特异性快速诊断、加强监测能力以及时发现流行的流感亚型的变化是应对流感大流行重要基础。为此,本研究构建了涵盖各亚型流感假病毒系统,为流感病毒中和抗体的快速检测鉴定以及深入研究HA和NA的功能提供材料和手段；另一方面,通过建立基于表位的甲型流感病毒亚型特异性检测技术为发展新型的流感病毒亚型检测、监测技术提供基础和依据。一、甲型流感病毒各亚型假病毒系统的构建构建了16个HA亚型(H1～H16)的真核表达质粒,用相应的亚型特异性抗体对HA的表达进行了免疫印迹验证,结果表明所有16个亚型HA均获得表达。对除H5和H7外的14个亚型HA的切割位点进行了改造,将切割位点由单碱性氨基酸突变为多碱性氨基酸,免疫印迹结果表明切割位点改造不影响HA的表达,其中H1、H6、H9、H10、H11、H12、H13、H14、H15和H16HA经改造后发生切割,H2、H3、H4和H8HA改造后仍未切割。同时,构建了9个亚型NA (N1～N9)的真核表达质粒,用亚型特异性抗体(N3～N9)进行免疫印迹检测验证了相应NA的表达,并用商品化的神经氨酸酶检测试剂盒对细胞裂解液的酶活性进行了检测,结果表明9个NA的裂解液中具有很强的神经氨酸酶活性,间接证明了9个NA亚型的正确表达。将16个HA亚型和9个NA亚型组合包装假病毒,144种假病毒感染293T细胞的结果根据HA的切割情况及与不同NA组合的感染能力可分为四组。H5、H6、H7和H94个HA亚型均切割表达且与9个NA形成的假病毒均能感染细胞；H1、H10～H168个HA亚型能切割表达但只有与部分NA形成的假病毒具有感染性；H2和H8两个HA亚型不切割但与部分NA形成的假病毒具有感染性；H3和H4两个HA亚型不切割且与所有NA形成的假病毒都不能感染细胞。这些结果提示不同亚型HA在切割性和假病毒感染性上存在很大差异,HA和NA在假病毒水平上存在相互作用的可能,其具体机制还需要进一步的研究。胰酶处理增强了原先不能感染的H3和H4假病毒的感染性。我们对于构建的假病毒通过以下三种方法进行了验证,电镜下能观察到完整的病毒粒子形态,免疫印迹结果表明HA均掺入相应的假病毒,血凝试验表明掺入假病毒的HA具有生物学活性。最后,用H1、H3、H5和H7假病毒与相应抗体进行了假病毒中和试验,结果表明H1、H3、H5、H7(?)病毒可被相应抗体中和,初步说明我们所构建的假病毒可用于中和抗体的检测和评价。二、甲型流感病毒H1亚型特异性保守表位的鉴定和应用用H1N1甲型流感(H1N1pdm)病毒HA蛋白胞外区的合成肽库和H1N1甲流病人恢复期血清进行肽扫描分析,成功鉴定了五条免疫优势肽,分别是P3(38-52aa)、 P5(58-72aa)、P15(158-172aa)、P16(168-182aa)和P31(318-332aa)。除P15外,其余四条合成肽偶联物在小鼠中诱导出较强的抗体滴度,免疫印迹和ELISA试验表明P3、P5和P31三条肽含有HlNlpdm病毒的优势表位。进一步的免疫印迹分析表明P5和P31只与H1亚型HA蛋白反应,且与多个不同年代来源的H1亚型HA均反应，，，说明P5和P31为H1亚型保守的特异性表位。。氨基酸序列比对分析和In silico保守性分析表明P5表位在H1亚型HA中高度保守,但在H2～H16HA中儿乎不存在。P5表位是一个从未被报道的线性表位,它位于传统的五个抗原位点区之外。以此表位作为抗原建立的合成肽ELISA方法与传统的血凝抑制试验相比具有很高的相关性(X2=58.01,P0.01)。两种方法的一致性为87%,以血凝抑制试验作为标准方法,合成肽ELISA的灵敏度和特异性分别为96.5%和74.4%。综上所述,本研究首次建立了可涵盖甲型流感病毒已知的16个HA亚型及9个NA亚型的假病毒系统,并在假病毒水平上提示了HA和NA存在相互作用的可能；利用合成肽扫描技术首次发现一个H1亚型特异且高度保守的线性表位,利用该表位建立了H1亚型抗体检测技术,这些结果为发展流感病毒的有效监测和检测提供了基础和手段。
[Abstract]:Influenza A virus can cause annual seasonal influenza and repeated influenza pandemic, seriously endangering the health of the people. In recent years, the prevalence of influenza A (H1N1) and the number of human-infected H5N1 avian influenza virus have been increasing, and the importance of strengthening the research and prevention and control of influenza A virus is further indicated. Hemagglutinin (HA) and neuraminidase (NA) are the surface glycoproteins encoded by the influenza A virus. According to the antigenicity of HA and NA, the influenza A virus can be divided into 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1-N9). The recombination and reformulation of HA and NA results in a new virus which is an important cause of the pandemic. In theory,16 HA and 9 NA subtypes can be freely combined to form 144 subtypes of influenza virus. Most of the influenza viruses have been found in nature, but the types of influenza viruses that trigger the pandemic still cannot be predicted. As a result, enhanced subtype-specific rapid diagnosis, enhanced monitoring capability, and the discovery of a change in the prevalence of influenza subtypes are an important basis for responding to the pandemic. To this end, the study constructed a system of pseudoviral influenza viruses, which provide materials and means for rapid detection and identification of influenza viruses and antibodies, as well as in-depth study of the function of HA and NA; on the other hand, The invention provides the basis and the basis for developing novel influenza virus subtype detection and monitoring technology by establishing an epitope-based influenza A virus subtype specific detection technology. I. Influenza A virus subtype pseudovirus system The eukaryotic expression plasmid of 16 HA subtypes (H1-H16) was constructed, and the expression of HA was verified by the corresponding subtype-specific antibody. The results showed that all the 16 subtypes of HA were obtained. It is expressed that the cleavage site of 14 subtypes of HA except H5 and H7 is modified, the cleavage site is mutated from a single basic amino acid to a polybasic amino acid, and the immunoblotting results show that the transformation of the cleavage site does not affect the expression of the HA, wherein H1, H6, H9, H10, H11, H12, H13, After the transformation of H14, H15 and H16HA, the cut, H2, H3, H4 and H8HA were modified and still Eukaryotic expression plasmid of 9 subtypes of NA (N1 to N9) was constructed, and the expression of the corresponding NA was verified by immunoblotting with the subtype specific antibody (N3-N9), and the enzymatic activity of the cell lysate was carried out by the commercial neuraminidase detection kit. The results showed that 9 NA had strong neuraminidase activity in the lysis solution, and it was proved that the 9 NA subtypes were positive. Exact expression. The results of the combination of 16 HA subtypes and 9 NA subtypes on the pseudovirus,144 pseudoviral infection 293T cells were based on the cleavage of the HA and the infection capacity with the different NA combinations It can be divided into four groups. The H5, H6, H7 and H94 HA subtypes are all cut and expressed and the pseudoviruses formed with 9 NA can infect the cells; H1, H10-H168 HA subtypes can cut the expression, but only the pseudoviruses formed with the part NA has infectious; two HA subtypes of H2 and H8 do not cut but are infectious with the pseudovirus formed by part NA; both HA subtypes of H3 and H4 do not cut and the pseudoviruses formed with all NA do not These results suggest that HA and NA of different subtypes have a great difference in the infectivity of the pseudovirus, and HA and NA may be involved in the level of the pseudovirus, and the specific mechanism of HA and NA may also need to be One-step study. The pancreatic enzyme treatment enhanced the previously uninfected H3 and H4 pseudodiseases. The infection of the virus was confirmed by the following three methods. The complete virus particle morphology was observed under the electron microscope. The results of the immunoblotting showed that the HA was incorporated into the corresponding pseudovirus. The results showed that H1, H3, H and H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, H _ 3, 5 The H7 (?) virus can be neutralized by the corresponding antibody, and a preliminary description of the pseudovirus that we construct can be used for neutralizing antibodies Detection and evaluation of influenza A virus H1 subtype-specific conservative The identification and application of the epitopes were carried out by peptide scanning analysis of the synthetic peptide library of the HA protein extracellular region of the H1N1 influenza A (NN1pdm) virus HA protein and the convalescent serum of the H1N1 influenza A patient. The five immunodominant peptides were successfully identified, P3 (38-52aa), P5 (58-72aa), P15 (1 58-172aa), P16 (168-182aa) and P31 (3 18-332aa). In addition to P15, the four synthetic peptide conjugates induced strong antibody titres in mice, and the immunoblotting and ELISA tests showed that the three peptides of P3, P5 and P31 contained HlNlp. Further immunoblotting analysis indicated that P5 and P31 were only reactive with the H1 subtype of HA protein, and were all reactive with the H1 subtype HA from multiple different age sources, indicating that P5 and P31 were H1 subtypes. The conservative analysis of amino acid sequence and the conservative analysis of In silico showed that the P5 epitope was highly conserved in the H1 subtype HA, but in H2-H1 It is no longer in the 6HA. The P5 epitope is a linear table that has never been reported, which is in the traditional In addition to the five antigenic site regions, the synthetic peptide ELISA method established as an antigen with this epitope has a high correlation (X2 = 58) compared to the conventional hemagglutination inhibition assay. 01, P0.01). The consistency of the two methods was 87%. The sensitivity and specificity of the synthetic peptide ELISA were 96% and 96%, respectively. In the light of the above, the first time in this study was the establishment of a pseudoviral system that could cover the known 16 HA subtypes and 9 NA subtypes of the influenza A virus and presented HA and HA at the level of the pseudovirus. An H1 subtype of specific and highly conserved linear epitope is first discovered by using synthetic peptide scanning technique, and the H1 subtype of antibody detection technology is established by using the epitope. These results are effective monitoring of the development of influenza virus.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373

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