甲型流感病毒各亞型假病毒系統(tǒng)的構(gòu)建及H1亞型特異性保守表位的鑒定
發(fā)布時(shí)間：2019-06-20 06:06
【摘要】：甲型流感病毒可引起年度季節(jié)性流感和反復(fù)的流感大流行,嚴(yán)重危害人民健康。近年來(lái)甲型H1N1流感的流行和人感染H5N1禽流感病毒病例不斷增加,進(jìn)一步提示加強(qiáng)甲型流感病毒的研究和防控的重要性。血凝素(HA)和神經(jīng)氨酸酶(NA)是甲型流感病毒編碼的表面糖蛋白。根據(jù)HA和NA的抗原性,甲型流感病毒可分為16個(gè)HA亞型(H1～H16)和9個(gè)NA亞型(N1-N9)。HA和NA的重組和重配產(chǎn)生新的病毒是造成流感大流行的重要原因。理論上16個(gè)HA和9個(gè)NA亞型可自由組合形成144種亞型流感病毒,多數(shù)亞型流感病毒已在自然界被發(fā)現(xiàn),但引發(fā)大流行的流感病毒的亞型種類(lèi)仍無(wú)法預(yù)測(cè)。因此,加強(qiáng)亞型特異性快速診斷、加強(qiáng)監(jiān)測(cè)能力以及時(shí)發(fā)現(xiàn)流行的流感亞型的變化是應(yīng)對(duì)流感大流行重要基礎(chǔ)。為此,本研究構(gòu)建了涵蓋各亞型流感假病毒系統(tǒng),為流感病毒中和抗體的快速檢測(cè)鑒定以及深入研究HA和NA的功能提供材料和手段；另一方面,通過(guò)建立基于表位的甲型流感病毒亞型特異性檢測(cè)技術(shù)為發(fā)展新型的流感病毒亞型檢測(cè)、監(jiān)測(cè)技術(shù)提供基礎(chǔ)和依據(jù)。 一、甲型流感病毒各亞型假病毒系統(tǒng)的構(gòu)建 構(gòu)建了16個(gè)HA亞型(H1～H16)的真核表達(dá)質(zhì)粒,用相應(yīng)的亞型特異性抗體對(duì)HA的表達(dá)進(jìn)行了免疫印跡驗(yàn)證,結(jié)果表明所有16個(gè)亞型HA均獲得表達(dá)。對(duì)除H5和H7外的14個(gè)亞型HA的切割位點(diǎn)進(jìn)行了改造,將切割位點(diǎn)由單堿性氨基酸突變?yōu)槎鄩A性氨基酸,免疫印跡結(jié)果表明切割位點(diǎn)改造不影響HA的表達(dá),其中H1、H6、H9、H10、H11、H12、H13、H14、H15和H16HA經(jīng)改造后發(fā)生切割,H2、H3、H4和H8HA改造后仍未切割。同時(shí),構(gòu)建了9個(gè)亞型NA (N1～N9)的真核表達(dá)質(zhì)粒,用亞型特異性抗體(N3～N9)進(jìn)行免疫印跡檢測(cè)驗(yàn)證了相應(yīng)NA的表達(dá),并用商品化的神經(jīng)氨酸酶檢測(cè)試劑盒對(duì)細(xì)胞裂解液的酶活性進(jìn)行了檢測(cè),結(jié)果表明9個(gè)NA的裂解液中具有很強(qiáng)的神經(jīng)氨酸酶活性,間接證明了9個(gè)NA亞型的正確表達(dá)。 將16個(gè)HA亞型和9個(gè)NA亞型組合包裝假病毒,144種假病毒感染293T細(xì)胞的結(jié)果根據(jù)HA的切割情況及與不同NA組合的感染能力可分為四組。H5、H6、H7和H94個(gè)HA亞型均切割表達(dá)且與9個(gè)NA形成的假病毒均能感染細(xì)胞；H1、H10～H168個(gè)HA亞型能切割表達(dá)但只有與部分NA形成的假病毒具有感染性；H2和H8兩個(gè)HA亞型不切割但與部分NA形成的假病毒具有感染性；H3和H4兩個(gè)HA亞型不切割且與所有NA形成的假病毒都不能感染細(xì)胞。這些結(jié)果提示不同亞型HA在切割性和假病毒感染性上存在很大差異,HA和NA在假病毒水平上存在相互作用的可能,其具體機(jī)制還需要進(jìn)一步的研究。胰酶處理增強(qiáng)了原先不能感染的H3和H4假病毒的感染性。我們對(duì)于構(gòu)建的假病毒通過(guò)以下三種方法進(jìn)行了驗(yàn)證,電鏡下能觀(guān)察到完整的病毒粒子形態(tài),免疫印跡結(jié)果表明HA均摻入相應(yīng)的假病毒,血凝試驗(yàn)表明摻入假病毒的HA具有生物學(xué)活性。最后,用H1、H3、H5和H7假病毒與相應(yīng)抗體進(jìn)行了假病毒中和試驗(yàn),結(jié)果表明H1、H3、H5、H7(?)病毒可被相應(yīng)抗體中和,初步說(shuō)明我們所構(gòu)建的假病毒可用于中和抗體的檢測(cè)和評(píng)價(jià)。 二、甲型流感病毒H1亞型特異性保守表位的鑒定和應(yīng)用 用H1N1甲型流感(H1N1pdm)病毒HA蛋白胞外區(qū)的合成肽庫(kù)和H1N1甲流病人恢復(fù)期血清進(jìn)行肽掃描分析,成功鑒定了五條免疫優(yōu)勢(shì)肽,分別是P3(38-52aa)、 P5(58-72aa)、P15(158-172aa)、P16(168-182aa)和P31(318-332aa)。除P15外,其余四條合成肽偶聯(lián)物在小鼠中誘導(dǎo)出較強(qiáng)的抗體滴度,免疫印跡和ELISA試驗(yàn)表明P3、P5和P31三條肽含有HlNlpdm病毒的優(yōu)勢(shì)表位。進(jìn)一步的免疫印跡分析表明P5和P31只與H1亞型HA蛋白反應(yīng),且與多個(gè)不同年代來(lái)源的H1亞型HA均反應(yīng)，，，說(shuō)明P5和P31為H1亞型保守的特異性表位。。氨基酸序列比對(duì)分析和In silico保守性分析表明P5表位在H1亞型HA中高度保守,但在H2～H16HA中兒乎不存在。P5表位是一個(gè)從未被報(bào)道的線(xiàn)性表位,它位于傳統(tǒng)的五個(gè)抗原位點(diǎn)區(qū)之外。以此表位作為抗原建立的合成肽ELISA方法與傳統(tǒng)的血凝抑制試驗(yàn)相比具有很高的相關(guān)性(X2=58.01,P0.01)。兩種方法的一致性為87%,以血凝抑制試驗(yàn)作為標(biāo)準(zhǔn)方法,合成肽ELISA的靈敏度和特異性分別為96.5%和74.4%。 綜上所述,本研究首次建立了可涵蓋甲型流感病毒已知的16個(gè)HA亞型及9個(gè)NA亞型的假病毒系統(tǒng),并在假病毒水平上提示了HA和NA存在相互作用的可能；利用合成肽掃描技術(shù)首次發(fā)現(xiàn)一個(gè)H1亞型特異且高度保守的線(xiàn)性表位,利用該表位建立了H1亞型抗體檢測(cè)技術(shù),這些結(jié)果為發(fā)展流感病毒的有效監(jiān)測(cè)和檢測(cè)提供了基礎(chǔ)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R373
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R373
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