沈阳市人类免疫缺陷病毒的基因序列分析

从10份沈阳地区人类免疫缺陷病毒1型(HIV-1)血浆标本中提取核糖核酸(RNA),经逆转录聚合酶链反应(RT-PCR)和套式聚合酶链反应(nest-PCR)扩增HIV-1的p17与p24交界部分的基因片断并进行测序.将所测序列与各亚型国际参考株及亚洲流行参考序列进行比对,确定被检标本的亚型,并进行基因序列分析.同时将所得亚型结果与经过亚型特异性引物的复合套氏PCR鉴定基因亚型的方法所获结果进行比较.结果表明,10份HIV-1病毒株分属B'、CRF07-BC和CRF01-AE 3种基因亚型.本文所研究样本的p17区段的ks/ka值小于1,而p24区段的ks/ka值大于1;p24部分的基因同源性高于p17部分,即我国HIV-1 B'、CRF07-BC和CRF01-AE 3种亚型毒株的p17区段的基因变异较大,而p24区段相对较为保守.提示上述3种亚型HIV-1病毒株的p24区段更适合于HIV-1疫苗的研制.     

B'亚型人类免疫缺陷病毒的基因序列分析

目的研究沈阳市人类免疫缺陷病毒1型(HIV-1)B'亚型毒株抗原表位的变异特征.方法从确诊的HIV-1感染者的全血样本中提取基因组DNA,经套式聚合酶链反应(PCR)扩增、产物纯化和测序分析后,将所得病毒核苷酸序列翻译成蛋白质的氨基酸序列,比较和分析我国人群中较常见的HLA型别限制的CTL表位的突变情况.结果在HIV-1 gag蛋白P24编码区,有4个抗原表位相当保守,且P17部分的抗原表位的变异率高于P24部分.结论 HIV-1 B'亚型毒株P24部分的4个抗原表位适合于抗原表位疫苗的研制.     

CRF01-AE亚型人类免疫缺陷病毒的基因序列特征研究

从确诊的HIV-1感染者的全血样本中提取基因组DNA,经套式聚合酶链反应(PCR)扩增其gag蛋白P17/P24交界区基因片断后,将扩增产物进行纯化和测序,分析其氨基酸序列。进而了解所检出的病毒基因变异和分子流行病学特征。结果发现,HIV-1 CRF01-AE亚型病毒分别与3株不同来源的国际参考毒株具有紧密的亲缘关系,表明这些毒株可能分别由不同的传播路线进入我国大陆境内。     

B′亚型人类免疫缺陷病毒的基因序列分析

目的 研究沈阳市人类免疫缺陷病毒1型（HIV-1）B′亚型毒株抗原表位的变异特征。方法 从确诊的HIV-1感染者的全血样本中提取基因组DNA,经套式聚合酶链反应（PCR）扩增、产物纯化和测序分析后,将所得病毒核苷酸序列翻译成蛋白质的氨基酸序列,比较和分析我国人群中较常见的HLA型别限制的CTL表位的突变情况。结果 在HIV-1 gag蛋白P24编码区,有4个抗原表位相当保守,且P17部分的抗原表位的变异率高于P24部分。结论 HIV-1 B′亚型毒株P24部分的4个抗原表位适合于抗原表位疫苗的研制。     

人类免疫缺陷病毒1型耐药基因的监测

2001年12月在美国举行的第41届ICAAC年会上公布的研究结果显示,美国78％的成人人类免疫缺陷病毒1型(HIV-1)感染者中存在着对一种或多种抗反转录病毒(ARV)药物耐药的病毒株。其中,对核苷类反转录酶抑制剂(NRTI)的耐药率最高,为……     

人类免疫缺陷病毒1型逃避RNA干扰的研究

用RNA干扰(RNAi)技术来阻断特定基因的表达,为治疗诸如人类免疫缺陷病毒1型(HIV—1)等慢性病毒感染提供了一种新的手段。虽然,通过小干扰RNA(siRNA)以降解序列特异性的mRNA可选择性地抑制一些在HIV—1复制过程中起关键作用的病毒蛋白,但RNAi技术仍然存在着许多问题亟待解决。本文就HIV—1特异性的RNAi技术作用的潜力和持久性作一研究。     

北京地区部分人类免疫缺陷病毒基因型分析

我国于1985年发现第1例AIDS患者，据估计到2001年底我国艾滋病病毒(Human immunodeficiency viruses，HrV)感染者和患者累计数已达84万人，而且我国艾滋病的传播呈快速增长的态势，2001年报告艾滋病病毒感染数较上年增长了58％。HIV主要通过静脉吸毒、输血或使用血液制品以及不良性行     

我国首次发现的HIV-1和HIV-2双重感染样品中病毒部分基因的序列特征分析

上海市卫生检疫局送检了一例HIV - 1和HIV - 2抗体检测均呈阳性的双重感染样品 ,对其感染的HIV前病毒的 gag和env基因区进行了序列分析 ,首次阐明我国发现的HIV双重感染样品的HIV部分基因特征。从HIV感染者淋巴细胞 (peripheralbloodmononuclearcells,PBMC)中提取前病毒DNA ,分别使用HIV 1和HIV 2特异性引物用套式PCR扩增HIV 1和HIV 2的部分基因区。PCR产物不经克隆直接测序 ,经GenBank检索并使用GCG软件包进行序列分析。结果表明 ,其中感染的HIV 2毒株中gag基因区与德国株HI2PEI2KR相似 ,基因离散率仅为8 1% ,env基因C2 -V3区与来自几内亚比绍的HIV 2U0 5 35 8株最近 ,离散率为 13 0 4% ,在其 gp36区发现与HIV 2U0 5 35 8基因离散率为 10 6 3% ,两个毒株均属HIV 2中的A亚型。而其HIV 1型毒株在 gag和env区都与从尼日利亚分离的H92NG0 83株相似 ,属HIV 1的G亚型。本文首次对我国发现的HIV 2型毒株进行了主要基因区的序列分析 ,表明在非洲较常见的HIV 2A亚型和HIV 1G亚型毒株 ,已随援外劳工传入我国。     

乙型流感病毒河北株的HA1基因序列及种系发生的分析

对１９８９年春在河北保定分离的两株乙型流感病毒进行了抗原性、ＨＡ＿１基因序列和种系发生分析,与不同期的国内外代表株比较结果表明,自１９８８年以来乙型流感病毒变异较快,Ｂ／河北／５３／８９株的ＨＡ＿１基因序列与Ｂ／挪威／１／８５株相比,其氨基酸的同源性为９４．５２％,与同期的Ｂ／香港／２０／８９株同源性为９７．１２％。种系发生分析结果,从１９８８至１９８９年乙型流感病毒出现了５个支系,同期在保定分离的两株病毒,在同源替代中分别属于两个支系。日本国基本每隔两年出现一次乙型流感的流行优势型,其发生频度与甲型流感相似。国内少见乙型的流行优势型,可能和使用的分离病毒材料有关,日本用ＭＤＣＫ细胞比国内用鸡胚对乙型流感病毒的分离阳性率高。     

HIV—1融膜肽的编码DNA序列比较

通过分析GenBank中的全部95个HIV－1完整基因组序列,设计融膜肽“探针序列”,对所获得的95段融膜肽的编码DNA序列进行了翻译、对准和分析。得到融膜肽及其编码序称的“优势序列”及突变分布。     

靶向HIV-1 vif的高效siRNA的筛选及慢病毒介导的体外抗病毒研究

RNAi技术在艾滋病治疗研究中已展现出巨大的潜力,兼具高效抑制特性和保守性的siRNA靶位是其获得成功应用的重要基础.本研究选择以HIV-1 vif基因为靶区筛选高效保守的RNAi序列,共选择设计了30个识别不同位点的siRNA序列,以pSUPER为载体构建了相应的shRNA表达质粒.通过与pNL4-3质粒在293FT细胞中进行共转染抑制实验,以及对初筛获得的高效序列进行保守性分析显示siRNA-vif37序列具有高效抑制效率和较好的保守性特征.通过与pGL3-vif报告质粒的共转染实验证明siRNA-vif37具有vif基因抑制特异性.带有shRNA-vif37表达元件的重组慢病毒转导后的MT-4细胞在HIV-1NL4-3体外攻毒实验中可显示出较有效的抑制病毒复制的能力,本研究进一步对转导后细胞进行克隆化筛选,获得稳定整合shRNA-vif37表达元件的MT-4-vif37细胞克隆,该细胞具有显著的抑制病毒复制的能力,在高攻毒剂量下仍可获得良好的抑制效果.本研究为进一步应用RNAi技术进行新型艾滋病治疗方法研究提供了重要基础.     

siRNA抑制HIV-1基因表达的研究

RNA干扰(RNAinterfering,RNAi)现象是动植物体内的一种序列特异性的、转录后基因沉默的过程.在哺乳动物细胞中,19～25个核苷酸长短的双链siRNA(smallinterferingRNA)可有效地抑制基因表达.利用pBS/H1SP载体,它表达的双链RNA的转录产物可以用来抑制特异性HIV-1基因的表达.在siRNA实验中,为了比较由H1启动子转录的siRNA在细胞内的作用,使用以绿色荧光蛋白(EGFP)为报告基因的载体——pEGFP-C1质粒,在荧光显微镜下,很容易地看到EGFP在细胞中表达.将HIV-1siRNA表达载体与表达相应EGFP-HIV融合基因的质粒,共转染人胚肾293细胞,结果表明,和对照质控载体相比,转染了pHIV-siRNA质粒的细胞中EGFP-HIV的表达得到显著抑制.通过该途径,筛选出能抑制HIV-1基因表达的有效siRNA.此外,还在同一载体上表达两种或三种siRNA,分别针对不同的HIV基因,获得了良好的抑制效果.     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Repfication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 （HIV-1）-based defective lentiviral vectors （DLV） and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV （0.5-1.2 × 107IU/mL）, which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)