斜纹夜蛾核型多角体病毒不同分离株基因序列的同源性分析

研究SpltMNPV不同分离株及SpltMNPV分离株与SpliNPV间基因序列的同源性,为SpltMNPV分离株的利用提供理论基础。根据已发表的斜纹夜蛾核型多角体病毒（SpltMNPV）中国株（Zh）基因组全序列（AF527603）和海灰翅夜蛾核型多角体病毒（SpliNPV）Not I-D片段序列（AF527603）设计引物,PCR方法扩增得到SpltMNPV日本福冈株（Fu）、埃及株（Eg）和小笠原株（Og）的ORF39～ORF42和ORF119～ORF124编码区全序列。SpltMNPV不同分离株及SpltMNPV分离株与SpliNPV间基因序列的相似性比较, Zh株和Og株,Eg株、Fu株和SpliNPV的相似性高,而Zh株和Eg株、Fu株或SpliNPV,Og株和Eg株、Fu株或SpliNPV的相似性都比较低。亦即SpltMNPV 3种基因型,B型和C型的同源性高,A型与B型或C型的同源性比较低,但A型与SpliNPV的同源性高;同一基因型内不同分离株（Eg株和Fu株）的同源性高。ETG分子进化分析表明Eg株、Fu株和SpliNPV处于一个分支,而Eg株、Fu株和SpliNPV与Zh株和Og株则处于不同的分支。因此推断Eg株和Fu株为SpliNPV的分离株,而Og株为SpltMNPV的分离株。     

EBV LMP1通过诱导c-myc表达活化端粒酶hTERT

利用原代人胚鼻咽上皮细胞和Tet on LMP1HNE2等良好的细胞体系,采用报道基因法和Westernblot法等,分别检测Epstein Barr病毒(EBV)潜伏膜蛋白1(LMP1)诱导的c myc反式激活活性和蛋白表达水平,从LMP1诱导细胞内c myc表达的角度,探讨LMP1诱导端粒酶表达的分子机制。结果表明,LMP1促使细胞内c myc反式激活活性增强,c Myc蛋白表达量升高;导入反义LMP1表达质粒阻断LMP1表达后,c myc反式激活活性下降。将端粒酶hTERT启动子上c myc结合位点突变后,LMP1不能诱导端粒酶hTERT表达。因而认为,EB病毒LMP1通过诱导c myc表达而活化端粒酶hTERT。     

鸡贫血病毒衣壳蛋白基因克隆、测序和比较

VP1 gene of chicken anemia virus in liver of infected chicken from Harbin was amplified by polymerase chain reaction and cloned into pUC18The recombinant vector was identified by restriction digestionThree clones were sequenced with sequencing kit ABI PRISM by TaKaRa CoLtdThe open reading frame of the VP1 gene is made up of 1,350bpIt encodes 449 amino acid residuesIt was found that there is an abundant Arg region from position 3 to 46Its isoelectric point is up to 107 because of its abundant basic amino acidsComparing this Harbin isolate with 26p4,Cux-1,82-2,Del-rose,A2,Connb,L-028,TR20,CIA-1,CAV15,2A9 showed there were 168 nucleoside differences which lead to 26 amino acid changes of the VP1The amino acid changes may influence the antigenic character of different VP1sIt was also found that there is a hyper-mutation region from amino acids position 139-157,its mutation frequency is up to 42% among these CAV's VP1s.It is necessary to research the function of this region     

乙型脑炎病毒毒力基因定位的研究进展

乙型脑炎病毒(Japanese encephalitis virus,JEV)属于黄病毒科(Flaviviridae),简称乙脑病毒.该病毒能引起人类的急性脑炎,致死率在20%左右,另有50%的患者会遗留神经系统方面的后遗症.乙脑病毒流行于亚洲及太平洋地区,有季节性的特点,一般蚊虫滋生的7、8、9月是其发病高峰期.每年在亚洲的乙脑病例有50 000例左右,其中致死的有上万人之多,是亚洲公共卫生的一个大问题[1],因而对乙型脑炎病毒功能与结构的研究,尤其是对影响其毒力的功能域的定位,多年以来都是研究的热点.     

HPV16型E7复制型DNA疫苗诱发的抗肿瘤免疫反应

为了研制有效的疫苗 ,用于HPV16型重度感染和与其感染相关的宫颈癌晚期病人手术后的免疫治疗 ,用复制型DAN疫苗载体 pSCA1,在其CMVIE启动子之下插入修饰的HPV16型E7基因mE7- 3( 2 4G、2 6G、6 7R) ,构建成 pSCA1mE7- 3复制型DNA疫苗。以重组质粒免疫C5 7BL/ 6小鼠 ,检测诱发的特异性CTL活性 ;将免疫后的小鼠用TC - 1肿瘤细胞攻击 ,观察免疫保护效果。实验结果显示 :pSCA1mE7- 3复制型DNA疫苗能诱导小鼠产生针对TC - 1肿瘤细胞的特异性CTL反应 ;复制型DNA疫苗免疫后的小鼠能耐受 1× 10 4 TC - 1细胞的攻击 ,成瘤时间推迟 ,并且成瘤率明显下降 ,部分小鼠得到保护能免受肿瘤攻击。因此pSCA1mE7- 3复制型DNA疫苗可作为HPV16相关肿瘤的癌前病变及中晚期病人术后免疫治疗的候选疫苗。     

表达鸡传染性支气管炎病毒JS/95/03株S1蛋白的重组杆状病毒构建

The recombinant baculovirus expressing S1 glycoprotein of nephropathogenic strain JS/95/03 of infectious bronchitis virus was generated by using the Bac-to-Bac baculovirus expression system.The BamH I-Sal Ⅰ fragment containing S1 gene from the recombinant plasmid pMDJS9503S1 was purified and cloned in frame into the baculovirus transposing vector pFASTBAC HTa under the polyhedrin gene promoter.The recombinant transposing plasmid pFASTJS9503S1 was screened and then transformed into Escherichia coli DH10BAC.The resulting recombinant bacmid rBacmidJS9503S1 was transfected into cells of the insect Spodoptera frugiperda(Sf9)and the recombinant baculoviruse rAcJS9503S1 was obtained.The lysates of cells infected with rAcJS9503S1 were analyzed by SDS-PAGE and the expressed product of S1 gene was detected by Western bloting and immunofluorescence assay(IFA).The results showed the recombinant baculovirus was fully capable of expressing S1 glycoprotein of JS/95/03.Maybe owing to the incomplete glycosylation in insect cells,the S1 gene product had a Mr of only 61000.In immunofluorescence test and Western blotting,the expressed product could react with polycolonal antibody against IBV M41 strain,indicating it possessed the antigenic properties specific for native S1 glycoprotein.     

EBV LMPl通过诱导c-myc表达活化端粒酶hTERT

利用原代人胚鼻咽上皮细胞和Tet—on—LMP1 HNE2等良好的细胞体系,采用报道基因法和Western blot法等,分别检测Epstein—Barr病毒(EBV)潜伏膜蛋白1(LMPl)诱导的c—myc反式激活活性和蛋白表达水平。从LMPl诱导细胞内c—myc表达的角度,探讨LMPl诱导端粒酶表达的分子机制。结果表明,LMPl促使细胞内c-myc反式激活活性增强,c-Myc蛋白表达量升高;导入反义LMPl表达质粒阻断LMPl表达后。c—myc反式激活活性下降。将端粒酶hTERT启动子上c—myc：结合位点突变后,LMP1不能诱导端粒酶hTERT表达。因而认为,EB病毒LMPl通过诱导c—mvc表达而活化端粒酶hTERT。     

重组腺相关病毒2型转导人外周血单核细胞来源树突状细胞的研究

To find out the infection efficiency of recombinant adeno-as sociated virus 2-mediated exogenou s genes in human peripheral blood monocyte-derived dendritic cells(D Cs),the process of transfection wa s investigated by FITC-labeled rAA V2,and observed under confocal mic roscope.Newly separated dendritic cells were tranfected by rAAV2-luc and rAAV2-GFP at different MOI,and transfection efficiency were detec ted by luminometer for rAAV2-luc a nd flow cytometry for rAAV2-GFP.Th e results were elucidated in four different assay systems:(1)60min w as needed for rAAV2 to bind on den dritic cells,and got into cells in the following 10min;(2)the express ion of luc could be detected at th e MOI as low as 1×10~5v.g/cell,and the expression plateau was reached by the MOI of 10~6～10~7v.g/cell,furt her increase of MOI had no functio n on expression level;(3)transgene expression was detected after 48h, and maintained a higher expression level from 96h to 240h after infec tion;(4)7days postinfection of rAA V2-GFP,5%～18% dendritic cells were GFP positive. These data suggest th at rAAV2 vector can efficiently in fect monocyte-derived dendritic ce lls and mediate exogenous gene exp ression,and that the application o f rAAV2 as vector may be useful fo r gene transfer to dendritic cells in ex vivo immunotherapy.     

反向遗传学技术及其在FMDV研究中的应用

反向遗传技术是一种新兴的分子生物学技术,已广泛应用于生命科学研究的各个领域。综述反向遗传技术研究进展,并讨论该技术在口蹄疫病毒研究中的应用。     

一株中华绒螯蟹呼肠孤病毒RNA1 cDNA文库构建及其RNA聚合酶基因部分序列分析

在中华绒螯蟹体内分离一株呼肠孤病毒（命名为EsRV905株）,采用Trizol试剂提取病毒核酸,经聚丙烯酰胺凝胶电泳,碎胶法回收基因组各节段。随机引物法合成第一节段的cDNA文库,胶回收试剂盒去除小片段,平端连接于载体,化学转化,利用蓝白斑筛选阳性克隆子,酶切鉴定重组质粒。从基因组第一节段的重组质粒中选择2个插入片段为1.5kb的质粒测序,结果得到包括RNA聚合酶主要特征性结构的一段序列。结果说明,这株蟹呼肠孤病毒的RNA聚合酶定位于基因组第一节段。