以新型杆状病毒载体在昆虫、哺乳动物细胞中表达克里米亚-刚果出血热病毒核蛋白基因

目的 构建可以在哺乳动物细胞中高效表达的新型杆状病毒载体，并利用其将克里米亚—刚果出血热病毒(CCHFY)中国分离株(新疆出血热病毒，xHFY)BA88166的核蛋白(NP)基因在昆虫和哺乳动物细胞中进行表达。方法 将人巨细胞病毒(CMv)立即早期(IE)启动子连接至杆状病毒载体PFastBacl多角体启动子下游形成新载体PCB1，然后将xHFY NP基因克隆至该载体，通过重组质粒转染和病毒感染，检测其在哺乳动物细胞(COS—7和vero)及昆虫细胞中的表达。结果 连接至PCB1的xHFV NP基因均能在相应的细胞中获得良好表达；以重组杆状病毒感染的vero细胞可以作为抗原检测xHF血清，与ELISA的检测结果完全一致，并与临床诊断有很好的平行性。结论 新型杆状病毒载体能够驱动外源基因在昆虫和哺乳动物细胞中高效表达，不仅能方便快速地制备诊断抗原，还具有发展重组病毒疫苗和基因治疗的潜力。     

新疆出血热的研究进展

克里米亚-刚果出血热（Crimean Congo hemorrhagic fever,CCHF）于1944年发现于俄国的克果米亚,它是由克里米亚-刚果出血热病毒（Crimean Congo hemorrhagic fever virus,CCHFV）引起的,是一种流行于中国新疆南部、俄国北部、中东、南部欧亚大陆及非洲撒哈拉地区的烈性传染性疾病,其平均病死率在10％～50％之间。     

新疆出血热病毒核蛋白多克隆抗体的制备及其免疫学评价

目的 原核细胞中表达、纯化新疆出血热病毒BA88166毒株核蛋白(NP)并制备及鉴定抗NP蛋白的多克隆抗体.方法 采用逆转录-聚合酶链式反应(RT-PCR)扩增出BA88166毒株S基因的cDNA片段,将其构建到原核表达载体pET-32a上,形成重组原核表达质粒pET-88166S.构建好的质粒在大肠杆菌BL21(DE3)中进行诱导表达,经镍柱亲和层析法纯化NP-His融合蛋白,SDS-PAGE分析蛋白相对分子质量(Mr).用该纯化蛋白免疫新西兰白兔制备抗血清,ELISA和Western blot法检测血清效价和特异性.结果 双酶切鉴定和DNA测序证实构建的pET-88166S重组表达载体正确,目的基因序列与GenBank中公布的序列一致,在E.coli BL21中表达的NP-His融合蛋白经SDS-PAGE分析,其Mr约为66000.ELISA检测抗体效价高于1:25600,蛋白免疫印迹实验结果表明抗体能特异性识别新疆出血热病毒YL04057毒株的NP蛋白及其截短蛋白.结论 成功获得新疆出血热病毒NP-His融合蛋白,得到了特异性兔抗NP蛋白多克隆抗体.     

克里米亚—刚果出血热的诊断与治疗进展

克里米亚－刚果出血热（ＣＣＨＦ）是由克里米亚－刚果出血热病毒（ＣＣＨＦＶ）引起的以发热，出血为典型特征的病死率极高的急性烈性传染性疾病。我国发生的ＣＣＨＦ称为新疆出血热（ＸＨＦ）。本文综述了该病的病原诊断、抗体检测及抗病毒治疗方面的进展。     

三株克里米亚-刚果出血热病毒中国分离株糖蛋白基因的全序列测定与比较分析

目的 测定克里米亚－刚果出血热病毒（CCHFV）中国分离株（即新疆出血热病毒，XH－FV）BA66019、BA8402及BA88166糖蛋白（M）基因的全部序列并分析各毒株之间的相互关系。方法 病毒RNA在只与其末端互补的特异引物PCM－Tag和随机引物的共同引发下逆转录成cDNA，以单一PCM－Tag和高保守末端互补的特异引物PCM－Tag和高保真DNA聚合酶扩增出完整的M基因。扩增产物纯化后作为模板进行序列测定，并将所得序列经计算机辅助分析其种系发生与编码策略。结果 XHFV代表株BA6019的M基因与国际原型株IbAr10200的M基因比较多个5个碱基，为5365bp；比BA8402、BA88166多个4个碱基，即5364bp。3株XHFV长ORF起始于M基因的第78位碱基，比IbAr10200株提前15bp。编码的前体蛋白共1689个氨基酸，比IbAr10200株多6个。4株病毒之间核苷酸序列的同源性分别为：80.9%（IbAr10200-BA66019），80.2%（IbAr10200-BA8402),80.2%(IbAr10200-BA88166），83.7%（BA66019－BA8402),83.6%(BA66019-BA88166),99.0%(BA8402-BA88166）；对应的氨基酸序列的同源性分别为85.1%,86.3%,86.6%,87.8%,88.0%,98.8%。它们与Dugbe病毒在核苷酸和氨基酸的同源性较低，大约是55％和37.7%。结论 XHFV BA66019、BA8402和BA88166与国际原型株IbAr10200的M基因在进化上形成各自单独的分支，人源分离株BA88166可能是来自蜱的BA8402株的变异株，提示20世纪80年代在新疆疫区可能只流行一种XHFV。     

新疆地区克里米亚刚果出血热病毒M片段的遗传分析

目的 为进一步了解克里米亚刚果出血热病毒（CCHFV）M基因的特征，对新疆地区4侏克里米亚刚果出血热病毒分离株亚东璃眼蜱的部分M片段核苷酸序列进行测定及分析。方法 从感染CCHF病毒的鼠脑中提取RNA，应用逆转录聚合酶链反应扩增，测定CCHF病毒的部分M片段核苷酸序列，与GenBank中的已登录的部分CCHF病毒M基因的同源序列进行比较分析和种系进化分析。结果 经测序得到了4株病毒的3962～4385nt位点的424bp核苷酸序列（位点依据IbAr10200的M基因序列），该序列共编码139个氨基酸。前3株病毒序列有相当高的核苷酸同源性（99．5％～99．8％），将所得到的序列与已发表的中国、尼日利亚、巴基斯坦、乌兹别克斯坦、塔吉克斯坦和俄罗斯病毒株相比，中国株之间的核苷酸同源性为78．1％～99．8％，而非中国株则为42．9％～94．8％。但它们所编码的氨基酸序列变异不大，同源性非常高。系统发生树表明，所有的毒株被分为5个进化支（所比较的M基因长度为3962～4385nt核苷酸），来自新疆南部和东部的YT05099、YL04041和YL05035共处于同一分支。结论 相比于新疆其他分离株，YT05099、YL04041和YL05035的部分M基因特征有极高的相似性。新疆株CCHF病毒M基因与中东和远东病毒株之间有较近的亲缘关系。     

新疆出血热病毒重组核蛋白及糖蛋白片段能诱导小鼠免疫应答

目的研究重组表达的新疆出血热病毒(XHFV)核蛋白和糖蛋白截短片段在刺激机体免疫应答中的作用。方法采用基因重组技术和亲和层析技术对XHFV截短的核蛋白(NP2)、糖蛋白片段(Gc和Gn)进行表达和纯化,并通过蛋白免疫及核酸初次免疫、蛋白加强免疫的方式免疫BALB/c小鼠,通过ELISA检测鼠血清抗体效价,采用T淋巴细胞增殖试验检测细胞免疫应答的效果,对上述表达产物刺激小鼠免疫应答的作用进行初步评价。结果利用原核表达系统成功表达纯化出XHFV核蛋白和糖蛋白截短片段。重组蛋白免疫BALB/c小鼠后能刺激机体产生Ig G(效价为1∶12 800以上),并能与相应的抗XHFV核蛋白及糖蛋白的多克隆抗体发生特异性反应。各组均能有效地激发较强的细胞免疫应答,其中p VAX1-Gc联合r Gc实验组小鼠脾脏淋巴细胞增殖明显,与对照组相比,差异具有统计学意义(P0.01)。结论获得的重组XHFV核蛋白、糖蛋白片段能有效刺激小鼠的体液和细胞免疫应答。     

新疆出血热病毒S基因在真核系统的克隆和表达

目的　在真核系统表达XHFVS基因片段 ,制备安全、特异、便于操作的S蛋白。方法设计引物 ,扩增得到S基因编码片段 ,用杆状病毒表达载体pFastBacHTb克隆S基因 ,并在昆虫细胞中表达S基因。结果　地高辛探针杂交和PCR扩增结果表明成功地构建了含XHFVS基因片段的pBac Sxhf质粒。SDS PAGE分析表达产物在相对分子质量 (Mr)为 6 6× 10 3 下方出现 1条明显的蛋白条带 ,Westernblot发现该蛋白带可以与抗XHF病毒核蛋白单克隆抗体发生反应。结论　构建的含XHFVS基因的重组杆状病毒可以在昆虫细胞表达S蛋白 ,该蛋白可与特异性抗体相结合     

新疆出血热病毒S基因片段的测序和分析

目的　认识新疆出血热（ ＸＨＦ） 病毒不同分离株基因区别的本质,从分子水平揭示其基因结构与功能的关系,寻找传播及流行的原因。方法　对１９６５ 年自我国首例病人及１９８４ 年自蜱分离的两株ＸＨＦ 病毒进行了Ｓ 基因片段的克隆测序和分析。结果　两株病毒核苷酸序列同源性为９６ ．７ ％ ,与已经发表的１９６８ 年分离自我国蜱（ＨＹ１３） 和羊（Ｃ６８０３１） 的两株病毒核苷酸序列同源性均较高（９６ ．７ ％ ～９９ ％ ） ;与其它ＣＣＨＦ 病毒相比Ｓ 基因同源性为７７ ．４ ％ ～９２ ．７ ％ 。结论　计算机绘出的系统发生树状图显示我国分离的４ 株病毒形成一单个群体并进一步分为三组,提示流行源自我国。     

Array-ELISA 技术对肾综合征出血热病毒诊断抗原的评价

目的分段表达并纯化肾综合征出血热病毒核蛋白,应用array-ELISA技术评价重组表达的核蛋白片段的诊断价值。方法构建肾综合征出血热病毒核蛋白表达质粒pET-32a（＋）/Pn、pET-32a（＋）/Pc,在大肠杆菌中诱导表达并用亲和层析法纯化,运用array-ELISA方法鉴定重组蛋白的检测特异性,检测结果与商品化胶体金试剂盒进行比较。结果在大肠杆菌中正确表达了肾综合征出血热病毒的核蛋白,亲和纯化得到较高纯度的蛋白质;应用array-ELISA技术从16份临床疑似血清样本中检出13份阳性血清,与商品化胶体金试剂盒一致性达到94％。结论获得的重组蛋白His-Pn可作为肾综合征出血热特异性诊断抗原之一;array-ELISA方法可以作为检测病毒抗体的有效手段。     

Sequencing, Expression and Diagnostic Application of the Nucleoprotein Gene of Xinjiang Hemorrhagic Fever Virus

Crimean Congohemorrhagicfever(CCHF)wasasevere infectiousdiseasewithaveragemortalityof10% 50%. Thecausativeagent,thetick borneCCHFvirus,which causedthefirsthumancasein1945inCrimeanpeninsula, wasfirstisolatedin1956fromafebrilechildinformer BelgianCongo(nowZ…     

Human defined antigenic region on the nucleoprotein of Crimean-Congo hemorrhagic fever virus identified using truncated proteins and a bioinformatics approach

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. In this study, amino acid sequence data for the CCHFV nucleoprotein (NP) was used to identify potential linear epitopic regions which were subsequently included in the design of large and small truncated recombinant NP antigens and peptide libraries. Two truncated recombinant CCHFV NP antigens were prepared based on results of prediction studies to include epitopic regions and exclude hydrophobic regions that could influence protein expression and solubility. Serum samples were collected from acute and convalescent patients. An IgG antibody response was detected in 16/16 samples tested using the large recombinant NP-based ELISA and in 2/16 using the small recombinant NP-based ELISA. A total of 60 peptides covering predicted epitopic regions of the NP were synthesized and peptide NRGGDENPRGPVSR at amino acid position 182–195, reacted with 13/16 human serum samples. In summary, functional assays are required to determine the biological activity of predicted epitopes for development of peptide based assays for antibody detection. Bacterially expressed complete NP antigens have previously been shown to be useful tools for antibody detection. Truncation of the antigen to remove the hydrophobic C terminus had no impact on the ability of the antigen to detect IgG antibody in human sera. The results indicate that the region from amino acids 123 to 396 includes a highly antigenic region of the NP with application in development of antibody detection assays.     

Serum thrombin-activatable fibrinolysis inhibitor levels and its relation with pathogenesis and bleeding and prognosis in patients with Crimean Congo hemorrhagic fever

Crimean-Congo hemorrhagic fever (CCHF) is a viral hemorrhagic fever, which is common in Turkey and globally. The pathogenesis of coagulation disorders, which is seen in viral hemorrhagic fevers remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) has a key role in this process In this study, we aimed to evaluate whether TAFI levels contributed to bleeding and whether it is related to prognosis in CCHF patients. Eighty-four patients older than 15 years of age, who were admitted to our hospital who had positive immunoglobulin M (enzyme-linked immunosorbent assay [ELISA]) and/or polymerase chain reaction test results for CCHF between 2009 and 2010, were included in the study. The control group included 30 healthy adults. The plasma TAFI levels were compared between patients and controls, and also between patients with bleeding and no bleeding, and between patients with mild-moderate and severe disease. The mean TAFI levels were lower in patients (mean: 87.82 ng/ml, median: 61.69 ng/ml (interquartile range [IQR] 30.49–537.95) than controls (mean: 313.5 ng/ml with a median: 338.5 ng/ml (IQR 182–418). However, median TAFI levels were significantly higher in patients with bleeding compared to those without bleeding (78.99 and 50.28 ng/ml, respectively; p = 0.032). Median IQR TAFI levels were similar between patients with mild-moderate and severe disease (64.72 (41.37–113.85), and, 58.66 (42.44–118.93) ng/ml, respectively; p = 0.09) and survivors and nonsurvivors (86.14 ± 77.98 and 103.48 ± 69.92, respectively; p = 0.3). Although TAFI levels were lower in the patients with CCHF compared to healthy controls, it does not seem to be a major player in the prognosis.     

Role of actin filaments in targeting of Crimean Congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells

Crimean-Congo hemorrhagic fever virus is the causative agent of a severe disease throughout Africa, Europe, and Asia. Like other members of the genus Nairovirus, the Crimean-Congo hemorrhagic fever virus contains three genomic RNA segments, the small (S), medium (M), and large (L) segments. The S segment encodes the viral nucleocapsid protein (NP), while the M and L segments encode the glycoproteins and the RNA-dependent RNA polymerase, respectively. In this study, the site of expression and assembly of Crimean-Congo hemorrhagic fever virus NP in mammalian cells have been investigated. It was found that the NP is localized in the perinuclear region of infected cells. By using the Semliki forest virus expression system, it was shown that the Crimean-Congo hemorrhagic fever virus NP is targeted to the perinuclear region of cells in the absence of native RNA segments and virally encoded glycoproteins. It was also demonstrated that the Crimean-Congo hemorrhagic fever virus NP was not expressed as a Golgi-membrane associated protein. By using Cytochalasin D, an agent that disrupts actin filaments, it was found that actin filaments are involved in targeting the viral NP to perinuclear regions. We also demonstrated that disruption of actin filaments reduced the assembly of infectious Crimean-Congo hemorrhagic fever virus up to 97%. Furthermore, we showed that the NP of Crimean-Congo hemorrhagic fever virus NP interacts with actin.