重组黄热病毒E蛋白结构域Ⅲ作为亚单位疫苗的抗原性和免疫原性

目的：表达纯化黄热病毒（YFV）囊膜蛋白（E蛋白）结构域Ⅲ,研究其作为亚单位疫苗预防YFV、日本脑炎病毒（JEV）感染的可能。方法：扩增YFVE蛋白结构域Ⅲ（YFDⅢ）的cDNA片段333bp,将其连接到原核表达载体pET-32a（＋）中,构建原核表达载体pET-YFDⅢ,转化感受态大肠杆菌Rosetta（DE3）,IPTG诱导表达重组YFDⅢ;用纯化的YFDⅢ免疫新西兰兔和BALB/c鼠,检测相关抗体滴度。结果：在大肠杆菌中可溶性表达了YFDⅢ融合蛋白,表达量约占菌体蛋白的50%;Western印迹及ELISA分析表明,纯化的YFDⅢ具有良好的抗原性和免疫原性;利用纯化的YFDⅢ免疫新西兰兔,获得了高达1∶4×105滴度的抗YFV抗体和1∶2×104滴度的抗JEV抗体;利用纯化的YFDⅢ免疫BALB/c鼠,获得了1∶7×104滴度的抗YFV抗体和1∶2×103滴度的抗JEV抗体。结论：重组YFDⅢ有较好的免疫原性,具有开发成亚单位疫苗的潜能。     

乙型脑炎病毒SA14-14-2株E蛋白主要抗原片段的高效表达

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,将其亚克隆入酵母表达载体pPICZα-A,以电穿孔法转化酵母X-33,用Zeocin平板筛选重组子,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物.由于糖基化不同,所表达产物有两种,其相对分子质量分别为44kDa和50kDa,表达量较高,约为290mg/L,经Western印迹验证,有较好的抗原性.在ELISA试验中,我们直接以PBS透析后的酵母上清包被,能够很明显地区分出乙脑阴阳性血清,与RT-PCR检测的相符率达95%,为制备JEV的诊断抗原和基因工程疫苗提供了依据.     

流行性乙型脑炎病毒减毒株SA14-14-2E基因稳定性研究

为研究乙脑病毒减毒株SA14-14-2 E蛋白基因稳定性,将乙脑病毒减毒株SA14-14-2在原代地鼠肾细胞(PHK)上传至18代,应用RT-PCR分别扩增PHK6代、PHK7代、PHK8代、PHK13代、PHK18代E蛋白基因并测序后,与Genebank中乙脑病毒减毒株SA14-14-2(D90195)进行比较分析。PHK6、PHK7、PHK8代病毒与D90195 E蛋白核苷酸和氨基酸序列完全相同。PHK13、PHK18代病毒与D90195E蛋白核苷酸序列同源性分别为99.8%、99.7%,与D90195E蛋白氨基酸序列同源性分别为99.6%、99.4%。各代次病毒E蛋白与减毒相关氨基酸未发生改变,同时所有突变的氨基酸均非SA14原有的,故不是恢复性突变。结果表明乙脑病毒减毒株SA14-14-2的遗传学特性稳定,从分子水平证明乙脑病毒减毒株SA14-14-2及其生产的疫苗具有安全性。     

日本脑炎病毒E蛋白在酵母双杂交系统中的表达及其对报告基因激活作用的检测

用日本脑炎病毒(JEV)E蛋白基因片段构建酵母双杂交诱饵载体,并检测其表达产物对酵母细胞有无毒性作用及对报告基因有无激活作用。用RT—PCR从JEV感染的鼠脑中扩增出JEV E蛋白基因片段,克隆入pUCl9质粒,经测序正确后,再亚克隆入酵母双杂交诱饵载体pGBKT7中。将重组质粒导入酵母菌AHl09,检测其表达产物在酵母细胞中对报告基因有无激活作用。成功获得JEV E蛋白基因片段,表达的E蛋白对酵母菌AHl09无毒性,对报告基因亦无激活作用。为利用酵母双杂交GAL4系统3进行JEV细胞受体蛋白的研究奠定了基础。     

蜱传脑炎病毒东北株E蛋白的核酸和氨基酸序列

从我国东北林区死者脑组织中,分离到蜱传脑炎病毒ＨＬＪ－１株,测定了它的Ｅ蛋白基因序列和推导的氨基酸序列,以及Ｅ蛋白抗原位点反应图谱。将该株与远东亚型Ｓｏｆｙｎ株和中欧亚型Ｎｅｕｄｅｒｆｌ株做了同源性比较,ＨＬＪ－１株与Ｓｏｆｙｎ株Ｅ蛋白基因的核苷酸及氨基酸的同源性分别为９４．３％和９８．８％;与Ｎｅｕｄｏｅｒｆｌ的核苷酸和氨基酸的同源性分别为８３．７％和９６％。在Ｅ蛋白的核苷酸序列中,ＨＬＪ－１和     

流行性乙型脑炎病毒E蛋白结构域Ⅲ的抗原表位鉴定与功能研究

流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)是一种严重危害人畜健康的虫媒病毒.表面囊膜蛋白(E蛋白)是该病毒的主要结构蛋白.E蛋白在介导病毒与宿主细胞的吸附、融合,决定病毒的血凝活性、细胞嗜性以及决定病毒毒力和诱导宿主产生保护性免疫反应中起重要作用.E蛋白结构域Ⅲ(EⅢ)是诱导中和抗体的重要区域.为确定乙型脑炎EⅢ的抗原表位,实验首先克隆了JEV疫苗株SA14-14-2的EⅢ区域,并用pGEX-6P-1载体进行融合表达,免疫印迹分析表明,该融合蛋白能被抗JEV血清识别.为了进一步对该结构域进行抗原表位作图,设计了14个覆盖该区域且部分重叠的短肽.将各短肽与GST进行融合表达与纯化.短肽融合蛋白经JEV阳性血清免疫印迹和EUSA免疫反应性扫描分析,结果鉴定出,E39(306TEKFSFAKNPVDTGHG320)、EA5-l(355VTNPFVATSSA366)、FA8-1(377FGDSYIV384)和E49(385VGRGDKQINHHWHKAG400)4个线性抗原表位.分别将4个抗原表位融合蛋白免疫小鼠,制备各抗原表位单因子血清,结果经体外病毒中和试验表明,E39为具有病毒中和活性的抗原表位.试验结果为进一步分析JEVE蛋白结构与功能以及诊断试剂和表位疫苗的研究提供了重要工作基础.     

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.     

Inhibition of japanese encephalitis virus infection by flavivirus recombinant e protein domain III

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.     

A bio‐nanocapsule containing envelope protein domain III of Japanese encephalitis virus protects mice against lethal Japanese encephalitis virus infection

An engineered bio‐nanocapsule (BNC) comprising modified hepatitis B surface antigen L protein was used as a physical scaffold for envelope protein domain III (D3) of Japanese encephalitis virus (JEV). At the N terminus, the BNC contained a two‐tandem repeat of the Z domain (ZZ) derived from Staphylococcus aureus protein A (ZZ‐BNC). The Lys‐rich ZZ moiety exposed on the surface of ZZ‐BNC was used for chemical conjugation with the JEV D3 antigen, which had been expressed and purified from Escherichia coli. Immunization of mice with D3 loaded on the surface of ZZ‐BNC (ZZ‐BNC:D3) augmented serum IgG response against JEV and increased protection against lethal JEV infection. The present study suggests that innocuous recombinant antigens, when loaded on the surface of ZZ‐BNC, can be transformed to immunogenic antigens.     

Investigation of the genotype III to genotype I shift in Japanese encephalitis virus and the impact on human cases

Japanese encephalitis is a mosquito borne disease and is the leading cause of viral encephalitis in the Asia-Pacific area. The causative agent, Japanese encephalitis virus(JEV) can be phylogenetically classified into five genotypes based on nucleotide sequence. In recent years, genotype I(GI) has displaced genotype III(GIII) as the dominant lineage, but the mechanisms behind this displacement event requires elucidation. In an earlier study, we compared host variation over time between the two genotypes and observed that GI appears to have evolved to achieve more efficient infection in hosts in the replication cycle, with the tradeoff of reduced infectivity in secondary hosts such as humans. To further investigate this phenomenon, we collected JEV surveillance data on human cases and, together with sequence data, and generated genotype/case profiles from seven Asia-Pacific countries and regions to characterize the GI/GIII displacement event. We found that, when comprehensive and consistent vaccination and surveillance data was available, and the GIII to GI shift occurred within a well-defined time period, there was a statistically significant drop in JEV human cases. Our findings provide further support for the argument that GI is less effective in infecting humans, who represent a dead end host. However, experimental investigation is necessary to confirm this hypothesis. The study highlights the value of alternative approaches to investigation of epidemics, as well as the importance of effective data collection for disease surveillance and control.     

DC-SIGN promotes Japanese encephalitis virus transmission from dendritic cells to T cells via virological synapses

Skin-resident dendritic cells (DCs) likely encounter incoming viruses in the first place, and their migration to lymph nodes following virus capture may promote viral replication. However, the molecular mechanisms underlying these processes remain unclear. In the present study, we found that compared to cell-free viruses, DC-bound viruses showed enhanced capture of JEV by T cells. Additionally, JEV infection was increased by co-culturing DCs and T cells. Blocking the C-type lectin receptor DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) with neutralizing antibodies or antagonists blocked JEV transmission to T cells. Live-cell imaging revealed that DCs captured and transferred JEV viral particles to T cells via virological synapses formed at DC-T cell junctions. These findings indicate that DC-SIGN plays an important role in JEV transmission from DCs to T cells and provide insight into how JEV exploits the migratory and antigen-presenting capabilities of DCs to gain access to lymph nodes for dissemination and persistence in the host.

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Immunogenicity of a recombinant MVA and a DNA vaccine for Japanese encephalitis virus in swine

We previously reported that mice immunized with recombinant modified vaccinia virus Ankara (MVA) encoding Japanese encephalitis virus (JEV) prM and E genes were completely protected against JEV challenge (Nam, J.H., Wyatt, L.S., Chae, S.L., Cho, H.W., Park, Y.K., Moss, B. Vaccine 1999,17: 261-268). In this study, we examined the immunogenicity in swine of this recombinant MVA (vJH9) or a DNA vaccine (pcJH-1) expressing the same JEV genes. Although the booster effect in mice with a combination of vJH9, pcJH-1 and inactivated JEV commercial vaccine was not apparent by measuring JEV antibodies, the recombinant MVA vaccine (vJH9) and the DNA vaccine (pcJH-l) efficiently produced neutralizing antibodies in swine and 2 doses of each showed a booster effect in mice and swine. Therefore, both vJH9 and pcJH-1 are good candidates for a second generation JEV vaccine.     

An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus

Japanese encephalitis virus(JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus–host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope(E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.     

流行性乙型脑炎病毒E基因的克隆、表达及初步应用

参照GenBank中的日本乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14株序列设计了一对特异性引物,用PCR方法从SA14-14扩增E基因全长,然后克隆到pMD18-T载体中,转化宿主菌DH5a,提取阳性克隆质粒进行双酶切鉴定,将目的片段定向克隆到pET32a( )中,转化入BL21(DE3),经IPTG诱导可表达分子量约73ka的蛋白,Western blotting试验呈阳性,表明E基因得到表达。以纯化的表达产物为核心抗原,猪抗JEV血清为一抗,HRP标记羊抗猪IgG抗体为二抗建立间接ELISA方法,并初步检测了一些血清样品,结果提示表达的蛋白具有很好的应用开发价值。     

Development of antiviral carbon quantum dots that target the Japanese encephalitis virus envelope protein

Japanese encephalitis is a mosquito-borne disease caused by the Japanese encephalitis virus (JEV) that is prevalent in Asia and the Western Pacific. Currently, there is no effective treatment for Japanese encephalitis. Curcumin (Cur) is a compound extracted from the roots of Curcuma longa, and many studies have reported its antiviral and anti-inflammatory activities. However, the high cytotoxicity and very low solubility of Cur limit its biomedical applications. In this study, Cur carbon quantum dots (Cur-CQDs) were synthesized by mild pyrolysis-induced polymerization and carbonization, leading to higher water solubility and lower cytotoxicity, as well as superior antiviral activity against JEV infection. We found that Cur-CQDs effectively bound to the E protein of JEV, preventing viral entry into the host cells. In addition, after continued treatment of JEV with Cur-CQDs, a mutant strain of JEV was evolved that did not support binding of Cur-CQDs to the JEV envelope. Using transmission electron microscopy, biolayer interferometry, and molecular docking analysis, we revealed that the S123R and K312R mutations in the E protein play a key role in binding Cur-CQDs. The S123 and K312 residues are located in structural domains II and III of the E protein, respectively, and are responsible for binding to receptors on and fusing with the cell membrane. Taken together, our results suggest that the E protein of flaviviruses represents a potential target for the development of CQD-based inhibitors to prevent or treat viral infections.