E蛋白在Ⅱ型登革病毒感染过程中作用的研究
发布时间：2018-08-12 18:01
【摘要】：登革病毒（Dengue virus，DENV）是登革热(classical dengue fever，DF)和登革出血热/登革休克综合症(dengue hemorrhagic fever/dengue shock syndrome，DHF/DSS)的病原体[1]。近年来，随着旅游业的发展和地球温暖化，世界范围内DF和DHF/DSS的流行、暴发更加频繁，影响范围在不断扩大，发病率不断增加。据WHO最新统计，热带与亚热带地区每年有超过3.9亿人感染DENV。值得一提的是，2013年夏季，我国广东、云南有大范围的DF疫情，且有较多的重症病例。因而WHO再次发布报告，认为DF已成为全球蔓延最快的病种，亟待研制安全有效的疫苗、特效抗病毒药物进行防治。 DENV属于黄病毒科、黄病毒属的单股正链RNA病毒，有四种血清型(DENV1-4）[2]，DENV基因组大约为11kb，1个开放读码框编码三种结构蛋白包膜糖蛋白（E）、膜蛋白（M）和衣壳蛋白（C），以及七种非结构蛋白NS1、NS2a、NS2b、NS3、NS4a、NS4b和NS5[3，4]。病毒表面的包膜蛋白E蛋白是DENV病毒体上的包膜糖蛋白和最大的结构蛋白，在病毒吸附、与宿主细胞膜融合以及病毒组装过程中具有重要的作用，是DENV吸附于靶细胞与受体相互作用的病毒表面特异性吸附蛋白[5]。prM蛋白是未成熟病毒颗粒中的膜蛋白前体，prM蛋白的N端序列被蛋白酶切除后即生成M蛋白，只有含M蛋白的成熟病毒颗粒才能介导包膜糖蛋白E与细胞膜以酸性pH依赖型的方式发生融合[6]。DENV NS1蛋白的分子量约为42~50KDa，在DENV感染细胞内合成的NS1蛋白通过E蛋白C端末尾的疏水性信号序列进入内质网，随后信号肽酶作用于E/NS1的连接处最终产生成熟的NS1蛋白。成熟的NS1蛋白以细胞内、细胞膜和细胞外分泌的形式存在，NS1蛋白有助于免疫复合物的形成，诱发机体产生保护性抗体[7]。 DHF/DSS是DENV感染的重症，其特征性的临床表现之一是血管通透性增加引起的渗出和出血，但对于血管通透性增加的机制尚未完全阐明。已知整合素家族是重要的细胞外基质蛋白，其中的整合素β3和β1主要分布在血管内皮细胞表面，在维持血管壁完整性上发挥着重要作用。课题组前期研究发现DENV-2感染HMEC-1可以上调整合素β3的表达，且DENV E蛋白与整合素β3在HMEC-1内存在很明显的共定位现象，利用RNAi技术下调整合素β3的表达可明显抑制病毒的进入[8]，推测整合素β3可能是DENV入侵宿主细胞受体或共受体。但目前没有直接证据证明E蛋白与整合素β3之间的相互作用。因此，本研究应用Octet系统对重组E蛋白与可能受体整合素β3的亲和力进行分析，初步研究了E蛋白与整合素β3之间的相互作用。 GM-CSF在JEV prME DNA疫苗诱导的免疫应答中起抑制作用[9]，但其在DENV蛋白免疫中的作用尚不清楚。为此，本研究的第二部分工作是原核表达系统表达E蛋白，纯化DENV-2重组E蛋白后，对其免疫原性进行分析，同时设置GM-CSF佐剂组，研究GM-CSF在DENV E蛋白免疫中的作用。最后，我们利于实验室已经构建好的DENV prME、NS1+2a蛋白的真核表达质粒对动物的免疫保护作用进行了研究，希望能为DENV疫苗的研究奠定基础。具体实验结果如下： 一、DENV-2E蛋白的原核表达及免疫原性研究 1. DENV-2E蛋白的表达与纯化 以pReceiver-E重组质粒(本室保存)为模板，扩增目的基因E胞外区基因片段，构建重组质粒E/pGEX-6P-1，转化BL21菌，诱导表达，并摸索得到可溶性蛋白表达量相对较高的表达条件：诱导物IPTG浓度0.5mM，诱导温度20℃，诱导时间为15h；亲和层析纯化重组E蛋白，最后得到纯度较高的蛋白。 2.重组E蛋白免疫原性研究 免疫6周龄的BALB/c雌鼠，设置E蛋白组、E+pCAG-GM组、GST和生理盐水组。三次免疫2周后，断尾取血，ELISA法测定各组抗体效价；PRNT50检测血清的抗DENV-2中和抗体效价；ELISpot法检测脾细胞因子INF-γ、IL-2、IL-4、IL-10和IL-17水平。结果显示：重组E蛋白组抗体效价为1:3,200，中和抗体效价为1;320；而E+pCAG-GM组抗体效价为1:6,400，中和抗体效价为1:1,280，说明E蛋白可以诱导产生特异性抗体和中和抗体；E，E+pCAG-GM组IFN-γ、IL-2、IL-10和IL-17水平明显上升，与生理盐水组比较差异显著（p0.05）。由于IL-2和IFN-γ为Th1型细胞因子，IL-4和IL-10为Th2型细胞因子，IL-17为Th17细胞产生的细胞因子。细胞因子水平变化说明E蛋白组和E+pCAG-GM组诱导了Th1、Th2和Th17途径的免疫应答反应。 3.重组E蛋白对小鼠的保护作用 同上方法免疫6周龄的BALB/c雌鼠，设置E蛋白组、E+pCAG-GM组、GST和生理盐水组。三次免疫2周后，颅内注射1,000PFU的DENV-2，观察小鼠死亡情况。结果为E+pCAG-GM组、GST组和生理盐水组小鼠全部死亡，E蛋白组存活率为33.33%；结果说明，，重组E蛋白有一定的免疫保护作用，pCAG-GM有抑制E蛋白诱导的免疫应答的作用。 二、Octet系统分析DENV-2E蛋白与整合素β3的亲和力 运用基于光纤生物传感器的生物膜层光学干涉技术（BLI，BioLayerInterferometry）进行重组E蛋白与整合素β3的分子间的动力学分析，以确定重组E蛋白与整合素β3之间亲和力的强弱和特异性。Octet系统分析显示重组E蛋白与整合素β3平衡解离常数(KD)为3.14×10-9M，解离速率常数(Kdis)为5.11×10-5s-1。提示重组E蛋白与整合素β3的相互作用表现为易结合、难解离的特点，说明重组E蛋白与整合素β3有较强的特异性亲和力。 三、 prME及NS1+2a DNA的小鼠免疫原性研究 1. prME和NS1+2a DNA的免疫原性研究 6周龄BALB/c雌鼠分别免疫E蛋白、 E+pRe-prM-E、pRe-prM-E+pRe-NS1-NS2a，生理盐水组注射等量生理盐水。小鼠三次免疫2周后， ELISA的方法测定各组抗体效价；PRNT50检测血清的抗DENV-2中和抗体效价； ELISpot法检测脾细胞因子INF-γ、IL-2、IL-4、IL-10、IL-17水平。结果：E蛋白组抗体效价是1:3,200，中和抗体效价为1:320；E+pRe-prM-E组为1:800，中和抗体效价为1:160；pRe-prM-E+pRe-NS1-NS2a组，抗体效价最高，可达到1:12,800，中和抗体效价也是最高为1:2,560，并且pRe-prM-E+pRe-NS1-NS2a组INF-γ、IL-10和IL-17因子水平明显高于其他组（p0.05）；IL-4因子水平，各组间差异不显著。细胞因子水平说明pRe-prM-E和pRe-NS1-NS2a同时诱导了Th1和Th2途径，且Th17途径也参与了免疫应答过程；结果说明pRe-prM-E+pRe-NS1-NS2a组诱导的免疫应答水平明显优于其它组。 2. prME和NS1+2a DNA对小鼠保护作用的研究 将6周龄BALB/c雌鼠分别免疫E蛋白、 E+pRe-prM-E、pRe-prM-E+pRe-NS1-NS2a，生理盐水组注射等量生理盐水。三次免疫2周后，颅内注射1,000PFU的DENV-2，观察小鼠死亡情况。攻毒后第13d后，生理盐水组小鼠全部死亡；E蛋白组和E+pRe-prM-E组均在第12d出现死亡，最终存活率均为33.3%；而联合免疫质粒pRe-prM-E与pRe-NS1-NS2a组，整个实验过程小鼠均未见任何发病症状，存活率为100%。结果说明，pRe-prM-E和pRe-NS1-NS2a质粒联合免疫小鼠后，对小鼠的保护作用最好，可考虑作为候选DNA疫苗的分子靶点。 综上，本实验结果证明了E蛋白DENV在感染过程中的重要作用，动物实验表明同时免疫prME和NS1+2a DNA可以能够达到100%的免疫保护，可考虑作为候选疫苗的分子靶点。本研究为进一步研究DENV疫苗奠定了一定的基础。
[Abstract]:Dengue virus (DENV) is the pathogen of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)[1].In recent years, with the development of tourism and the warming of the earth, the prevalence of DF and DHF/DSS worldwide has become more frequent and influential. According to the latest statistics of WHO, more than 390 million people are infected with DENV every year in tropical and subtropical regions. It is worth mentioning that in the summer of 2013, Guangdong and Yunnan in China had a wide range of DF epidemics and a large number of severe cases. Therefore, the WHO issued a report again that DF has become the fastest spreading disease in the world. It is urgent to develop a safe and effective vaccine for the prevention and treatment of specific antiviral drugs.
DENV belongs to the family Flaviviridae. The flavivirus is a single stranded positive stranded RNA virus. It has four serotypes (DENV1-4) [2], a DENV genome of about 11 kb, an open reading frame encoding three structural protein envelope glycoproteins (E), membrane protein (M) and capsid protein (C), and seven non-structural proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 [3, 4]. Protein E is the envelope glycoprotein and the largest structural protein of DENV. It plays an important role in virus adsorption, fusion with host cell membrane and virus assembly. It is a virus surface-specific adsorbent protein that DENV adsorbs on target cells interacting with receptors [5]. The N-terminal sequence of prM protein was excised by proteinase to produce M protein. Only mature virus particles containing M protein could mediate the fusion of envelope glycoprotein E with cell membrane in an acidic pH-dependent manner [6]. The water-borne signal sequence enters the endoplasmic reticulum, and then the signal peptidase acts on the junction of E/NS1 to produce mature NS1 protein. The mature NS1 protein exists in the form of intracellular, cell membrane and extracellular secretion. NS1 protein contributes to the formation of immune complex and induces the body to produce protective antibodies [7].
DHF/DSS is a severe infection of DENV. One of the clinical manifestations of DHF/DSS is exudation and hemorrhage caused by increased vascular permeability. However, the mechanism of increased vascular permeability has not been fully elucidated. Previous studies have shown that DENV-2 infection with HMEC-1 can up-regulate the expression of integrin beta-3, and the co-localization of DENV-E protein and integrin beta-3 is obvious in HMEC-1. Adjusting the expression of integrin beta-3 by RNAi technology can significantly inhibit the entry of the virus [8], suggesting that integrin beta-3 may be able. However, there is no direct evidence to prove the interaction between E protein and integrin beta 3. Therefore, the affinity between recombinant E protein and integrin beta 3 was analyzed by Octet system, and the interaction between E protein and integrin beta 3 was preliminarily studied.
GM-CSF can inhibit the immune response induced by JEV prME DNA vaccine [9], but its role in DENV protein immunity is not clear. Therefore, the second part of this study is to express E protein in prokaryotic expression system, purify recombinant E protein of DENV-2, analyze its immunogenicity, and set up GM-CSF adjuvant group to study the role of GM-CSF in DENV protein immunity. Finally, we will benefit from the construction of DENV prME, NS1 + 2A protein eukaryotic expression plasmid to study the immune protection of animals, hoping to lay the foundation for the study of DENV vaccine.
Prokaryotic expression and immunogenicity of DENV-2E protein
1. expression and purification of DENV-2E protein
The recombinant plasmid E/pGEX-6P-1 was constructed and transformed into BL21 strain to induce the expression of the soluble protein. The expression conditions were as follows: the concentration of IPTG was 0.5mM, the induction temperature was 20 C, the induction time was 15 h; the affinity chromatography was used to purify the recombinant plasmid E/pGEX-6P-1. The recombinant E protein was obtained, and finally the protein with high purity was obtained.
2. immunogenicity of recombinant E protein
Immunized 6-week-old BALB/c female rats were divided into E protein group, E+pCAG-GM group, GST group and normal saline group. After 2 weeks of immunization, blood samples were taken from the tail and the antibody titers were determined by ELISA; PRNT50 was used to detect the antibody titers against DENV-2; ELISpot was used to detect the levels of spleen cytokines INF-gamma, IL-2, IL-4, IL-10 and IL-17. The titer of antibody was 1:3,200, and the titer of neutralizing antibody was 1:320; the titer of antibody was 1:6,400 in E+pCAG-GM group and 1:1,280 in E+pCAG-GM group, indicating that E protein could induce specific antibody and neutralizing antibody; the levels of IFN-gamma, IL-2, IL-10 and IL-17 in E, E+pCAG-GM group were significantly higher than those in normal saline group (p0.05). FN-gamma is a Th1 type cytokine, IL-4 and IL-10 are Th2 type cytokines, and IL-17 is a Th17 type cytokine. The changes of cytokine levels indicate that E protein group and E+pCAG-GM group induce the immune responses of Th1, Th2 and Th17 pathways.
3. protective effect of recombinant E protein on mice
After 2 weeks of immunization, 1,000 PFU DENV-2 was injected into the brain to observe the death of BALB/c mice. The results showed that all the mice in E+pCAG-GM group, GST group and normal saline group died and the survival rate of E protein group was 33.33%. PCAG-GM can inhibit the immune response induced by E protein.
Two, the affinity of DENV-2E protein to integrin beta 3 was analyzed by Octet system.
Bio-Layer Interferometry (BLI) based on fiber optic biosensor was used to analyze the molecular dynamics of recombinant E protein and integrin beta 3 in order to determine the affinity and specificity between recombinant E protein and integrin beta 3. Octet system analysis showed that the equilibrium dissociation of recombinant E protein and integrin beta 3 was normal. The number (KD) of recombinant E protein was 3.14 X 10-9M and the dissociation rate constant (Kdis) was 5.11 X 10-5s-1. It was suggested that the interaction between recombinant E protein and integrin beta 3 was easy to combine and difficult to dissociate, indicating that recombinant E protein had a strong specific affinity with integrin beta 3.
Immunogenicity of three, prME and NS1+2a DNA mice
Immunogenicity of 1. prME and NS1+2a DNA
Six-week-old BALB/c female mice were immunized with E protein, E+pRe-prM-E, pRe-prM-E+pRe-NS1-NS2a, and saline group was injected with the same amount of saline. Two weeks after the mice were immunized three times, the antibody titers of each group were determined by ELISA; PRNT50 was used to detect the antibody titers against DENV-2 in serum; and the spleen cytokines INF-gamma, IL-2, IL-4, IL-10, IL-17 water were detected by ELISpot. Results: The titer of antibody in E protein group was 1:3,200, the titer of neutralizing antibody was 1:320, the titer of neutralizing antibody in E + pRe-prM-E group was 1:800, the titer of neutralizing antibody was 1:160, the titer of antibody in pRe-prM-E + pRe-NS1-NS2a group was 1:12,800, and the titer of neutralizing antibody was 1:2,560, and the levels of INF-gamma, IL-10 and IL-17 in pRe-prM-E + pRe-NS1-NS2a group were clear. The levels of cytokines showed that pRe-prM-E and pRe-NS1-NS2a induced both Th1 and Th2 pathways, and Th17 pathway also participated in the immune response process; the results showed that pRe-prM-E+pRe-NS1-NS2a induced immune response level was significantly higher than other groups.
Protective effects of 2. prME and NS1+2a DNA on mice
The 6-week-old BALB/c female rats were immunized with E protein, E+pRe-prM-E, pRe-prM-E+pRe-NS1-NS2a, and the normal saline group was injected with the same amount of saline. Two weeks after the third immunization, 1,000 PFU DENV-2 was injected into the brain to observe the death of the mice. The survival rate was 100%. The results showed that the combined immunization of pRe-prM-E and pRe-NS1-NS2a plasmids had the best protective effect on mice and could be considered as the molecular target of candidate DNA vaccine.
In conclusion, the results of this study demonstrated that E protein DENV played an important role in the process of infection. Animal experiments showed that prME and NS1+2a DNA could achieve 100% immune protection and could be considered as molecular targets of candidate vaccines.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.8
[Abstract]:Dengue virus (DENV) is the pathogen of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)[1].In recent years, with the development of tourism and the warming of the earth, the prevalence of DF and DHF/DSS worldwide has become more frequent and influential. According to the latest statistics of WHO, more than 390 million people are infected with DENV every year in tropical and subtropical regions. It is worth mentioning that in the summer of 2013, Guangdong and Yunnan in China had a wide range of DF epidemics and a large number of severe cases. Therefore, the WHO issued a report again that DF has become the fastest spreading disease in the world. It is urgent to develop a safe and effective vaccine for the prevention and treatment of specific antiviral drugs.
DENV belongs to the family Flaviviridae. The flavivirus is a single stranded positive stranded RNA virus. It has four serotypes (DENV1-4) [2], a DENV genome of about 11 kb, an open reading frame encoding three structural protein envelope glycoproteins (E), membrane protein (M) and capsid protein (C), and seven non-structural proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 [3, 4]. Protein E is the envelope glycoprotein and the largest structural protein of DENV. It plays an important role in virus adsorption, fusion with host cell membrane and virus assembly. It is a virus surface-specific adsorbent protein that DENV adsorbs on target cells interacting with receptors [5]. The N-terminal sequence of prM protein was excised by proteinase to produce M protein. Only mature virus particles containing M protein could mediate the fusion of envelope glycoprotein E with cell membrane in an acidic pH-dependent manner [6]. The water-borne signal sequence enters the endoplasmic reticulum, and then the signal peptidase acts on the junction of E/NS1 to produce mature NS1 protein. The mature NS1 protein exists in the form of intracellular, cell membrane and extracellular secretion. NS1 protein contributes to the formation of immune complex and induces the body to produce protective antibodies [7].
DHF/DSS is a severe infection of DENV. One of the clinical manifestations of DHF/DSS is exudation and hemorrhage caused by increased vascular permeability. However, the mechanism of increased vascular permeability has not been fully elucidated. Previous studies have shown that DENV-2 infection with HMEC-1 can up-regulate the expression of integrin beta-3, and the co-localization of DENV-E protein and integrin beta-3 is obvious in HMEC-1. Adjusting the expression of integrin beta-3 by RNAi technology can significantly inhibit the entry of the virus [8], suggesting that integrin beta-3 may be able. However, there is no direct evidence to prove the interaction between E protein and integrin beta 3. Therefore, the affinity between recombinant E protein and integrin beta 3 was analyzed by Octet system, and the interaction between E protein and integrin beta 3 was preliminarily studied.
GM-CSF can inhibit the immune response induced by JEV prME DNA vaccine [9], but its role in DENV protein immunity is not clear. Therefore, the second part of this study is to express E protein in prokaryotic expression system, purify recombinant E protein of DENV-2, analyze its immunogenicity, and set up GM-CSF adjuvant group to study the role of GM-CSF in DENV protein immunity. Finally, we will benefit from the construction of DENV prME, NS1 + 2A protein eukaryotic expression plasmid to study the immune protection of animals, hoping to lay the foundation for the study of DENV vaccine.
Prokaryotic expression and immunogenicity of DENV-2E protein
1. expression and purification of DENV-2E protein
The recombinant plasmid E/pGEX-6P-1 was constructed and transformed into BL21 strain to induce the expression of the soluble protein. The expression conditions were as follows: the concentration of IPTG was 0.5mM, the induction temperature was 20 C, the induction time was 15 h; the affinity chromatography was used to purify the recombinant plasmid E/pGEX-6P-1. The recombinant E protein was obtained, and finally the protein with high purity was obtained.
2. immunogenicity of recombinant E protein
Immunized 6-week-old BALB/c female rats were divided into E protein group, E+pCAG-GM group, GST group and normal saline group. After 2 weeks of immunization, blood samples were taken from the tail and the antibody titers were determined by ELISA; PRNT50 was used to detect the antibody titers against DENV-2; ELISpot was used to detect the levels of spleen cytokines INF-gamma, IL-2, IL-4, IL-10 and IL-17. The titer of antibody was 1:3,200, and the titer of neutralizing antibody was 1:320; the titer of antibody was 1:6,400 in E+pCAG-GM group and 1:1,280 in E+pCAG-GM group, indicating that E protein could induce specific antibody and neutralizing antibody; the levels of IFN-gamma, IL-2, IL-10 and IL-17 in E, E+pCAG-GM group were significantly higher than those in normal saline group (p0.05). FN-gamma is a Th1 type cytokine, IL-4 and IL-10 are Th2 type cytokines, and IL-17 is a Th17 type cytokine. The changes of cytokine levels indicate that E protein group and E+pCAG-GM group induce the immune responses of Th1, Th2 and Th17 pathways.
3. protective effect of recombinant E protein on mice
After 2 weeks of immunization, 1,000 PFU DENV-2 was injected into the brain to observe the death of BALB/c mice. The results showed that all the mice in E+pCAG-GM group, GST group and normal saline group died and the survival rate of E protein group was 33.33%. PCAG-GM can inhibit the immune response induced by E protein.
Two, the affinity of DENV-2E protein to integrin beta 3 was analyzed by Octet system.
Bio-Layer Interferometry (BLI) based on fiber optic biosensor was used to analyze the molecular dynamics of recombinant E protein and integrin beta 3 in order to determine the affinity and specificity between recombinant E protein and integrin beta 3. Octet system analysis showed that the equilibrium dissociation of recombinant E protein and integrin beta 3 was normal. The number (KD) of recombinant E protein was 3.14 X 10-9M and the dissociation rate constant (Kdis) was 5.11 X 10-5s-1. It was suggested that the interaction between recombinant E protein and integrin beta 3 was easy to combine and difficult to dissociate, indicating that recombinant E protein had a strong specific affinity with integrin beta 3.
Immunogenicity of three, prME and NS1+2a DNA mice
Immunogenicity of 1. prME and NS1+2a DNA
Six-week-old BALB/c female mice were immunized with E protein, E+pRe-prM-E, pRe-prM-E+pRe-NS1-NS2a, and saline group was injected with the same amount of saline. Two weeks after the mice were immunized three times, the antibody titers of each group were determined by ELISA; PRNT50 was used to detect the antibody titers against DENV-2 in serum; and the spleen cytokines INF-gamma, IL-2, IL-4, IL-10, IL-17 water were detected by ELISpot. Results: The titer of antibody in E protein group was 1:3,200, the titer of neutralizing antibody was 1:320, the titer of neutralizing antibody in E + pRe-prM-E group was 1:800, the titer of neutralizing antibody was 1:160, the titer of antibody in pRe-prM-E + pRe-NS1-NS2a group was 1:12,800, and the titer of neutralizing antibody was 1:2,560, and the levels of INF-gamma, IL-10 and IL-17 in pRe-prM-E + pRe-NS1-NS2a group were clear. The levels of cytokines showed that pRe-prM-E and pRe-NS1-NS2a induced both Th1 and Th2 pathways, and Th17 pathway also participated in the immune response process; the results showed that pRe-prM-E+pRe-NS1-NS2a induced immune response level was significantly higher than other groups.
Protective effects of 2. prME and NS1+2a DNA on mice
The 6-week-old BALB/c female rats were immunized with E protein, E+pRe-prM-E, pRe-prM-E+pRe-NS1-NS2a, and the normal saline group was injected with the same amount of saline. Two weeks after the third immunization, 1,000 PFU DENV-2 was injected into the brain to observe the death of the mice. The survival rate was 100%. The results showed that the combined immunization of pRe-prM-E and pRe-NS1-NS2a plasmids had the best protective effect on mice and could be considered as the molecular target of candidate DNA vaccine.
In conclusion, the results of this study demonstrated that E protein DENV played an important role in the process of infection. Animal experiments showed that prME and NS1+2a DNA could achieve 100% immune protection and could be considered as molecular targets of candidate vaccines.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.8
【共引文献】
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