抗登革病毒2型E蛋白单克隆抗体的制备和生物学特性的鉴定

为了制备可用于胶体金快速检测试纸条的抗登革病毒2型（DEN2）E蛋白单克隆抗体（mAb）,通过基因克隆获得E基因与质粒pET32a（＋）的重组质粒,将重组质粒转化入大肠杆菌BL21,IPGT诱导表达重组蛋白。以DEN2重组E蛋白免疫BALB／c小鼠,采用杂交瘤技术制备抗DEN2E蛋白的mAb,以间接ELISA法和Western blot进行mAb特异性鉴定;同时采用间接ELISA法鉴定mAb的Ig亚类。结果表明获得1株可分泌特异性mAb的杂交瘤细胞（7C7）,其抗体亚类为IgG1。Western blot显示该株mAb能特异识别重组pET32a-DEN2E蛋白。因此,成功制备出抗DEN2E的1株mAb,为建立快速特异橙测登革病毒感染的实验方法提供了有力的工具。     

登革病毒非结构蛋白NS3研究进展

登革病毒非结构蛋白NS3是一种多功能蛋白,N端具有Ser蛋白酶活性,C端具有RNA解旋酶及NTP磷酸酶、5'RNA-Z磷酸酶等活性,参与病毒前体的加工和病毒RNA的复制及基因组RNA的5’端加帽。NS3具有良好的免疫原性,存在多个登革病毒特异性CD4^ ,CD8^ T细胞表位,且多具有型间交叉免疫特性。登革病毒非结构蛋白NS3已成为有吸引力的抗病毒靶标。     

衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

GM-CSF作为Ⅱ型登革病毒E蛋白免疫佐剂的免疫效果观察

目的观察粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)作为II型登革病毒E蛋白免疫佐剂的保护效果,评估GM-CSF作为蛋白疫苗免疫佐剂的可行性。方法提取GM-CSF质粒(pCAG-GM)、将表达II型登革病毒E蛋白的重组质粒(E/pGEX-6P-1)进行诱导,表达产物用亲和层析纯化。将BALB/c小鼠随机分为E蛋白组、佐剂组(E蛋白+GM-CSF)和对照组进行免疫,采用酶联免疫吸附试验(ELISA)检测血清抗体终点效价;采用蚀斑减少中和试验(PRNT)检测II型登革病毒中和抗体水平;酶联免疫斑点试验(ELISPOT)检测小鼠免疫后细胞因子的水平;用攻毒试验观察各组小鼠保护率。结果 E蛋白组和佐剂组小鼠血清中抗体终点效价、中和抗体水平均在免疫后有一定的升高,差异无统计学意义(P0.05);佐剂组细胞因子(IFN-γ和IL-10)水平较E蛋白组均有明显上升,差异具有统计学意义(P0.05);但攻毒试验显示,佐剂组小鼠全部死亡,生存率为0,而E蛋白组的小鼠保护率为33%。结论 GM-CSF免疫佐剂可抑制II型登革病毒E蛋白的免疫保护作用,其作为蛋白疫苗免疫佐剂仍需慎重。     

重组Ⅱ型登革病毒NS1的表达及其免疫原性的研究

目的:表达制备重组Ⅱ型登革病毒非结构蛋白NS1,检测该重组表达蛋白的免疫原性,为检测试剂盒的研制提供基础。方法:根据Ⅱ型登革病毒NS1蛋白基因序列(GenBank登录号:NC_001474),对基因序列进行编码密码子优化后进行全基因合成,随后,经双酶切将目的片段克隆到表达载体pET28a上,通过IPTG诱导表达;表达产物经Western blot鉴定后,进一步进行蛋白纯化和透析复性,制备获得重组Ⅱ型登革病毒NS1蛋白。收获的NS1蛋白免疫小鼠,制备多克隆抗体,通过间接酶联免疫学方法测定其效价,检测其免疫原性。结果:成功构建了Ⅱ型登革病毒NS1蛋白的表达载体。Western blot显示表达的重组蛋白能够被NS1单抗特异性的结合,纯化复性后的重组蛋白在小鼠体内表现出良好的免疫原性。结论:重组表达的NS1蛋白有良好的免疫原性,为以后的NS1单克隆抗体的制备和登革病毒检测试剂盒的研制提供了良好的基础。     

抗登革病毒NS1单抗的制备及检测NS1方法的建立

制备抗登革病毒NS1蛋白单克隆抗体,建立检测NS1的ELISA方法。表达1~4型登革病毒NS1蛋白,将1型NS1蛋白纯化后免疫BALB/c小鼠,通过杂交瘤技术制备单克隆抗体。经ELISA、Western blotting、间接免疫荧光筛选和鉴定单克隆抗体,进行纯化和HRP标记。通过鉴定每两株单抗之间是否存在竞争作用,选择非竞争单抗组合并建立NS1捕获法ELISA。结果获得7株高滴度抗NS1单抗,捕获法ELISA可以检出10ng/mL NS1。原核表达登革病毒NS1蛋白制备的单抗可以和天然病毒抗原反应,NS1捕获法ELISA可以用于登革病毒感染检测。     

登革病毒血清型特异单克隆抗体的制备和鉴定

登革病毒 (Dengue virus,DENV) 是全球传播最为广泛的虫媒病毒,由于缺乏快速鉴别感染病毒血清型的诊断技术,导致异型交叉感染引起重症登革出血热病例居高不下。为实现免疫学方法快速鉴别诊断不同血清型DENV感染,本研究采用哺乳动物细胞293T表达并纯化了4种DENV血清型NS1蛋白,免疫小鼠后通过杂交瘤技术制备了针对NS1蛋白的单克隆抗体。利用酶联免疫吸附方法 (Enzyme-linked immunosorbent assay,ELISA)、间接免疫荧光法 (Indirect immunofluorescence assay,IFA)、免疫斑点杂交试验 (Dot blotting) 以及蛋白质免疫印迹试验 (Western blotting) 确认所制备的单克隆抗体能够有效识别天然病毒NS1以及重组NS1蛋白。获得的单克隆抗体包含2株可识别1–4型DENV NS1蛋白的通用型抗体及3株分别针对DENV-1、DENV-2和DENV-4的血清型特异抗体。以所制备的DENV NS1抗体为基础,采用双抗体夹心ELISA可快速鉴别不同血清型DENV。DENV血清型特异单克隆抗体的制备和甄别DENV血清型ELISA方法的建立为快速鉴别感染DENV血清型的临床诊断奠定了基础。     

研究报告重组Ⅱ型登革病毒NS1的表达及其免疫原性的研究

目的：表达制备重组Ⅱ型登革病毒非结构蛋白NS1,检测该重组表达蛋白的免疫原性,为检测试剂盒的研制提供基础。方法：根据Ⅱ型登革病毒NS1蛋白基因序列（GenBank 登录号：NC_001474）,对基因序列进行编码密码子优化后进行全基因合成,随后,经双酶切将目的片段克隆到表达载体pET28a上,通过IPTG诱导表达;表达产物经Western blot鉴定后,进一步进行蛋白纯化和透析复性,制备获得重组Ⅱ型登革病毒NS1蛋白。收获的NS1蛋白免疫小鼠,制备多克隆抗体,通过间接酶联免疫学方法测定其效价,检测其免疫原性。结果：成功构建了Ⅱ型登革病毒NS1蛋白的表达载体。Western blot显示表达的重组蛋白能够被NS1单抗特异性的结合,纯化复性后的重组蛋白在小鼠体内表现出良好的免疫原性。结论：重组表达的NS1蛋白有良好的免疫原性,为以后的NS1单克隆抗体的制备和登革病毒检测试剂盒的研制提供了良好的基础。     

登革病毒2型E蛋白基因真核表达和DNA免疫的研究

将编码登革病毒2型(DV2)氨基末端80％的E蛋白的DNA片段克隆到真核表达载体pCXN2 AG强启动子下游,构建成DV2E重组真核表达质粒pCXN-E。间接免疫荧光显示其可在COS-7细胞中表达。ELISA法检测pCXN2-E DNA免疫BALB/c鼠血清中的E抗体变化和维持规律,结果显示三次免疫后2周已有抗体产生,15周时仍维持较高的水平;血清空斑减数中和实验显示其中和滴度高于1:640;流式细胞计数仪(FACS)检测DNA免疫鼠CD4~+、CD8~+T淋巴细胞变化情况,与注射空载体pCXN2的阴性鼠相比,CD4~+淋巴细胞水平略有上升。CD8~+细胞水平有较大升高(p<0.01);动物保护性实验结果显示,当用致死剂量登革病毒攻击免疫鼠时,其保护率为60％。以上结果表明:pCXN2-E在实验动物内表达出的DV2E蛋白可以诱导免疫动物的体液免疫和细胞免疫应答,尤其是MHC-I限制性杀伤性CD8~+T淋巴细胞水平的提高对清除病毒是十分有利的。因此,DV2 E DNA免疫为登革病毒DNA疫苗的发展进行了有益的探索。     

国内首次自患者标本中同时分离的登革2型和3型病毒的鉴定

采用间接免疫荧光方法 ,检测患者血清标本中的抗登革病毒IgM和IgG抗体 ;同时将病人急性期血清接种C6 36细胞进行病毒分离。从分离的病毒悬液中提取RNA ,进行RT PCR扩增和序列测定。结果显示 ,该患者血清中存在抗登革病毒的IgM和IgG抗体。从病人血清中分离的病毒 ,经RT PCR和序列测定证实为登革 2型和 3型病毒的特异序列。表明该患者为登革 2型和 3型病毒混合感染     

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.     

Structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga

A sulfated polysaccharide, named fucoidan, from the marine alga Cladosiphon okamuranus is comprised of carbohydrate units containing glucuronic acid and sulfated fucose residues. Here we found this compound potently inhibits dengue virus type 2 (DEN2) infection. Viral infection was inhibited when DEN2, but not other serotypes, was pretreated with fucoidan. A carboxy-reduced fucoidan derivative in which glucuronic acid was converted to glucose did not inhibit viral infection. Elimination of the sulfated function group from fucoidan significantly attenuated the inhibitory activity on DEN2 infection with <1% fucoidan. DEN2 particles bound exclusively to fucoidan, indicating that fucoidan interacts directly with envelope glycoprotein (EGP) on DEN2. Structure-based analysis suggested that Arg323 of DEN2 EGP, which is conformationally proximal to one of the putative heparin binding residues, Lys310, is critical for the interaction with fucoidan. In conclusion, both the sulfated group and glucuronic acid of fucoidan account for the inhibition of DEN2 infection.     

水稻矮缩病毒非结构蛋白基因S6的表达和抗血清的制备

经RT-PCR扩增出水稻矮缩病毒（RDV）中国分离物非结构蛋白基因S6,并克隆至pGEM-Teasy载体上,序列分析表明该基因与日本株具有高度同源性,并且含有较高比例的稀有密码子。将S6基因克隆到表达载体pGEX-6P-1,并转化大肠杆菌,该基因在大肠杆菌以包涵体形式大量表达,以表达的融合蛋白作抗原免疫家兔,制备抗S6蛋白的抗血清,ELISA测定表明,该血清与抗原共价特异性反应,抗血清的效价为1：3000.Western blot印迹实验表明该抗血清能特异性检测RDV感染的水稻组织中的S6蛋白,因而可作为感染RDV的水稻植株的分子手段。     

Expression and immunoaffinity purification of recombinant dengue virus 2 NS1 protein as a cleavable SUMOstar fusion

Dengue virus (DENV) encoded nonstructural one (NS1) is a 352 amino acid protein that exists in multiple oligomeric states and is conserved within the flavivirus family. Although NS1 has been heavily researched for its diagnostic utility, there is a gap in the understanding of its role in a range of viral processes, including replication and development of clinical pathologies such as vascular leakage. Many of these functions involve unknown interactions with viral and host proteins. This study describes the generation of a mouse monoclonal antibody (mAb 56.2) that reacts with NS1 from DENV1 and 2, and the expression of recombinant SUMOstar-tagged DENV2 NS1 (DENV2 S^∗-NS1) in baculovirus. This is the first time dengue NS1 has been produced as a SUMOstar fusion with the S^∗-tag increasing protein solubility and secretion compared with a non-S^∗-tagged NS1 construct. The protein was readily purified using a mAb 56.2 immunoaffinity column and untagged NS1 was obtained by treatment with tobacco etch virus protease to remove the S^∗-tag. Size exclusion chromatography and glycosylation assays showed that both secreted S^∗-NS1, and cleaved NS1, are hexameric and glycosylated, and will be useful tools in elucidating dengue NS1 protein interactions and functions.     

Synthesis and biological evaluation of novel imidazole nucleosides as potential anti-dengue virus agents

In this work, we developed imidazole nucleoside derivatives with anti-dengue virus (DENV) activity was examined. First, compounds in a nucleosides library were screened to find lead compounds which inhibit replication of DENV. As a result, 5-ethynyl-(1-β-d-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) and its 4-carbonitrile derivative EICNR (2) were selected as promising antiviral compounds. However, both of them also exhibited cytotoxicity. In order to develop an effective and less toxic compound, 4′-thio and 4′-seleno derivatives of EICAR and EICNR 3–6 were prepared. The resulting 4′-thioEICAR and 4′-thioEICNR showed inhibitory effect on DENV replication without cytotoxicity as potent as ribavirin, a positive control.     

The fusion site of envelope fragments from each serotype of Dengue virus in the P64k protein,influence some parameters of the resulting chimeric constructs

To characterize the effect of the envelope fragment fusion site in the P64k protein from Neisseria meningitidis several chimeric constructs were obtained. One variant consisted in the insertion of the E fragment from each Dengue serotype within the lipoil binding domain of the P64k, whereas the other was based on the fusion of the envelope fragment at the C-terminus of the same meningoccocal protein. The results of the expression study revealed the majoritary levels with the C-terminus fusion variants of each serotype. In contrast, the highest proportion of soluble protein was reached with the insertion variants independently of the viral serotype. On the other hand, a significant level of degradation was detected for the semipurified forms of the insertion variants being remarkable in the Dengue 2 construct. Finally, the recognition by Dengue murine antibodies was similar independently of the fusion site. Regarding these results, we can affirm the suitability of the C-terminus fusion variants for further vaccine development as well as for a diagnostic system.     

鼠冠状病毒核衣壳蛋白的抗体制备与抗原决定簇分析

核衣壳(nucleocapsid,N)蛋白有稳定病毒基因组、调控病毒复制及细胞状态的特殊作用。鼠肝炎病毒(murine hepatitis virus,MHV)为乙型冠状病毒属的原型病毒,是研究冠状病毒N蛋白功能的经典模型。本研究用去污剂处理鼠冠状病毒粒子暴露N蛋白,另用原核表达纯化的重组N蛋白分别免疫小鼠,制备多克隆及单克隆抗体。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和蛋白免疫印迹分析结果显示两类抗体均具有高灵敏度和特异度,与甲型冠状病毒猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)的N蛋白无交叉反应。原核表达缺失突变的N蛋白分析结果显示,多克隆抗体与单克隆抗体2E6识别的鼠冠状病毒N蛋白抗原决定簇完全一致,位于N端结构域(N-terminal domain,NTD)C端与SR之间的58个氨基酸残基内。此外,基于单克隆抗体2E6的ELISA及免疫荧光法能检测到感染细胞中和培养上清液中的N蛋白组分,且其含量与病毒复制的滴度一致。这些结果表明,鼠冠状病毒复制过程中粒子与细胞中的N蛋白可能维持相似的结构,使NTD与SR之间的部分氨基酸残基一直暴露在表面,从而形成了优势抗原决定簇。