HIV-1感染诱导丝氨酸/苏氨酸蛋白激酶Citron kinase上调机制研究

HIV-1感染可以改变宿主细胞的表达谱,上调和病毒转录复制翻译包装所需的宿主蛋白,使宿主变成更加适应病毒复制繁殖的环境。研究表明丝氨酸/苏氨酸蛋白激酶Citron kinase(citK)可以促进HIV-1病毒的包装释放,所以我们在本文中进一步探讨了HIV-1感染对Citron kinase在自然生理状态下的表达是否有调节作用。我们用含有荧光素酶报告基因的HIV-1假病毒感染外周血单个核细胞(PBMC)和HEK293T细胞系,检测Citron kinase表达的上调情况。此外,将Citron kinase的上游启动子克隆入含荧光素酶报告基因的载体上,检测HIV假病毒感染对Citron kinase启动子的影响。结果显示:HIV-1可以显著提高PBMC细胞中Citron kinase的表达量,而Citron kinase为HIV-1复制包装所需。在原代CD4+T细胞中过表达Citron kinase,HIV-1的复制可以提高2倍以上。沉默Citron kinase的表达,HIV-1病毒产生量显著降低。在HEK293T细胞系中,HIV-1假病毒感染可以使Citron kinase的mRNA的水平提高2.5倍,蛋白表达量提高2.7倍。我们通过将Citron kinase的启动子克隆到含有荧光素酶报告系统的载体上,感染HIV-1假病毒,发现荧光素酶的活性增加。这提示着HIV-1感染通过转录水平上调Citron kinase的表达,从而为病毒创造复制繁殖更有利的宿主环境。     

Scirpusin A和scirpusin B体外抗人类免疫缺陷病毒1型的作用

Scirpusin A和scirpusin B是从药用植物中发现的2种天然茋类二聚体,具有一定的抗病毒效应。本研究利用人类免疫缺陷病毒1型(HIV-1)包膜和水疱性口炎病毒G蛋白(VSV-G)包膜的HIV假病毒及实验室适应株HIV-1ⅢB,在体外评价2种药物的抗病毒效果并初步探讨其作用机制。研究发现,scirpusin A和scirpusin B不仅能在体外有效抑制HIV假病毒感染TZM-bl细胞(一种HIV-1易感细胞),还能抑制实验室适应株HIV-1ⅢB。Scirpusin B的作用优于scirpusin A。对实验室适应株HIV-1ⅢB,scirpusin B的半数抑制浓度(IC50)为1.33μmol/L,scirpusin A的IC50为4.77μmol/L。此外,scirpusin A和scirpusin B还能抑制VSV-G包膜假病毒,提示其作用可能与病毒在细胞内的复制过程相关。Scirpusin A在病毒进入细胞后仍可发挥抑制作用,但具体机制尚待深入研究。     

整合HA蛋白的HIV假病毒展示禽流感病毒感染宿主细胞机制

通过将高致病性禽流感病毒HA蛋白整合到HIV颗粒,包装成表达HA蛋白的假病毒粒子（命名为HIV/H5-HA）,并对所包装的假病毒的生物学功能进行了研究.通过RT PCR获得了H5N1亚型禽流感病毒完整的血凝素基因(HA)并克隆到真核表达载体pcDNA3.1(+)上,通过与假病毒构建体系的2种质粒pCMV△8.2和pHR′-CMVLacZ共转染293T细胞,包装成假病毒颗粒.利用LacZ染色和HA假病毒颗粒感染MDCK等6种细胞株并对标记基因LacZ进行检测.结果表明,HIV/H5-HA与天然的禽流感病毒相似,具有广泛的细胞嗜性; Western 印迹和FACS检测结果,和HA假病毒颗粒的电镜照片确认了HA基因在假病毒颗粒表面得到了表达;HIV/H5-HA能够凝集鸡红细胞,并且pH值依赖性测定表明,HA假病毒需要低pH值才能实现正确的入侵宿主细胞.本研究结果显示：禽流感病毒H5N1亚型的HA基因得到了有效的包装,并且所包装的假病毒颗粒能够表达具有高度生物活性的HA蛋白.同时,假病毒模型的建立为进一步研究禽流感病毒与宿主之间的免疫应答提供了一种新的途径.     

HIV-1抗体蛋白印迹确认与核酸检测复核对比研究

应用病毒核酸载量法NASBA和HIV-1 RNA的巢式逆转录PCR(nested RT-PCR)法与HIV抗体蛋白印迹(WB)方法,对经过初筛的44例HIV-1抗体阳性标本进行了对照检测研究。发现了2例(gp160、p24)和1例(gp160g、p120p、66、p24)的特殊阳性样本,经NASBA法和该RT-PCR法核酸检测为阴性;WB确认的4例gp160阳性带、1例p24、p17阳性带和13例p24阳性带,经NASBA法和该RT-PCR法核酸检测也为阴性;而WB确认的其余全部带型的抗体阳性标本经过NASBA法和该RT-PCR法检测均为阳性。该研究表明对只有gp160p、24和gp160、gp120p、66、p24的特殊阳性标本和以p24为主的抗体不确定标本需要用RT-PCR或NASBA方法进行核酸检测,以进一步确认。     

HIV-1感染上调细胞系中泛素样蛋白ISG15表达

利用体外细胞感染模型,分别检测HIV-1感染的细胞系中转录和翻译水平ISG15的表达及细胞上清中p24蛋白水平,探讨HIV-1感染对ISG15表达的影响及后者对前者的抑制效应。6种HIV-1易感细胞系中,以IFN-α 2b刺激作为阳性对照,利用HIV-1感染性克隆pNL4-3转染293T、TZM-bl和HeLa细胞;HIV-1假病毒感染Jurkat、MT-4和THP-1细胞,24h收细胞提取RNA,利用荧光定量PCR(qPCR)的方法检测转录水平ISG15的表达情况。48h后收细胞提取总蛋白,Western blot检测蛋白水平ISG15的表达情况。HIV-1感染或转染后明显上调ISG15转录水平,且THP-1和TZM-bl细胞中上调尤为显著。但除了TZM-bl细胞,其他5种细胞系中没有表现出ISG15蛋白水平的上调。ISG15真核表达质粒与HIV-1感染性克隆pNL4-3共转染结果显示,293T细胞中ISG15能直接抑制HIV-1子代病毒颗粒的产生和成熟,并且这种抑制作用具有时间剂量依赖性;TZM-bl中虽然也有显著的抑制作用,但是趋势不同。HIV-1感染明显上调ISG15转录但却无ISG15蛋白产生,预示该病毒能对抗固有免疫细胞的识别活化及其效应功能,具体机制还有待进一步阐明。     

利用报告基因比较悬浮细胞与贴壁细胞对HIV-1假病毒感染效率的差异

【目的】研究用人免疫缺陷病毒(Human immunodeficiency virus,HIV)-1假病毒感染带有β-半乳糖苷酶(β-galactosidase,β-gal)报告基因和HIV受体CD4+CCR5+的Tzmbl细胞,分析悬浮状态与贴壁状态对HIV-1假病毒感染Tzmbl细胞的影响,为进一步进行HIV生物学研究与中和抗体实验室评价提供实验基础。【方法】通过将pNL43 R-E-与编码HIV膜蛋白的质粒共转染293T细胞,收集上清,获得HIV假病毒。该假病毒感染悬浮的和贴壁的Tzmbl细胞后可表达β-gal报告蛋白,通过X-gal染色和仪器分析可测定表达β-gal报告基因的细胞数与细胞感染率。【结果】HIV假病毒感染悬浮细胞的效率高于其对贴壁的Tzmbl细胞感染的效率,且细胞的感染率的改变与病毒的型相关。【结论】该研究结果可为进一步利用具有单轮感染活性的HIV假病毒进行生物研究和中和抗体实验提供研究方法。     

HIV-1 p24和SIV p27两种ELISA试剂盒检测SIV p27抗原的比较

目的由于检测SIV p27抗原试剂盒来源困难,有时不稳定,鉴于HIV-1 p24与SIVp27有较强的交叉抗原,本研究比较HIV-1 p24和SIV p27两种ELISA试剂盒检测SIV p27抗原得出的结果是否存在一定的相关性。方法 HIV-1 p24和SIV p27两种ELISA试剂盒定性和定量检测样品中SIV p27抗原,并对检测结果进行回归和相关分析。结果 HIV-1 p24和SIV p27两种ELISA试剂盒检测SIV p27抗原的灵敏度分别是150 pg/mL和62.5 pg/mL。两种试剂盒检测病毒液和血浆中SIV p27抗原的定性结果一致。定量结果的统计分析得出病毒液的直线回归决定系数R2=0.857,直线相关系数r=0.926,P〈0.01,直线正相关程度较高;血浆的直线回归决定系数R2=0.512,直线相关系数r=0.716,P〈0.05,直线正相关程度较低。结论 HIV-1 p24 ELISA试剂盒能够替代SIVp27 ELISA试剂盒定性检测病毒液和血浆中SIV p27抗原,但只能定量检测病毒液中SIV p27抗原。     

中东呼吸综合征冠状病毒假病毒系统的建立及其在中和抗体检测中的应用

目的:为了避免中东呼吸综合征冠状病毒(MERS-CoV)感染与中和试验中操作活病毒带来的生物安全隐患,构建只具有一次感染能力而无复制能力的MERS假病毒,建立MERS假病毒系统,并应用于中和抗体检测。方法:构建含有MERS-CoV S基因的重组质粒pcDNA3.1-MERS-S,与缺失Env基因、含有萤光素酶报告基因的HIV-1骨架质粒pNL4-3.Luc.RE共转染293T细胞,收获含有假病毒的上清;通过Western印迹、细胞感染实验和血清中和试验,确定是否包装出MERS假病毒,及是否能有效应用于细胞感染与中和试验。结果:MERS假病毒pMERS-S培养上清经Western印迹鉴定出相对分子质量为25×103的HIV-1 P24蛋白和相对分子质量为180×103的MERS-CoV S蛋白;与阴性对照假病毒pEnv-相比,pMERS-S能有效感染MERS-CoV敏感细胞系Huh-7,在感染细胞中产生荧光信号,感染细胞的假病毒量与产生的荧光信号呈明显的量效关系;在MERS假病毒中和试验中,pMERS-S能被MERS-CoV中和抗体中和而失去感染力,反映抗体对MERS-CoV的中和活性。结论:建立了不依赖于BSL-3高等级生物安全条件的MERS假病毒系统,并有效应用于中和抗体检测,为MERS-CoV疫苗、药物评价及病毒致病机制研究提供了良好的技术支撑手段。     

野生冬虫夏草水提物体外抗HIV-1病毒作用的初步研究

研究分析野生冬虫夏草的水溶浸提物抑制人免疫缺陷病毒Ⅰ型(human immunodeficiency virus-1,HIV-1)的效果及其抗病毒作用的靶点。研究分别选取新鲜虫体、子座、全草、干燥虫体和子座5种不同组分,获得水溶性提取物,采用CCK-8实验检测水提物的细胞毒性;假病毒单周期感染实验评价水提物的体外抗病毒活性;体外检测水提物对逆转录酶活性的影响,应用表面等离子共振(SPR)技术检测水提物与HIV-1Vif蛋白的相互作用。这5种虫草水提物均具有显著的体外抗HIV-1病毒活性,抑制HIV-1逆转录酶的活性,新鲜子座水提物与Vif蛋白有良好的体外结合力。本实验明确了野生冬虫夏草的体外抗病毒活性,其作用机制可能与抑制逆转录酶活性和体外Vif蛋白有关,研究将为抗HIV-1病毒药物的研发提供实验基础。     

HIV-1 Gag p17-p24在痘苗病毒中的表达及性质研究

研究了重组痘苗病毒表达的HIV-1核心蛋白(Gag)p17-p24蛋白的一些生物学及免疫学特点。间接免疫荧光、Dot ELISA及Western blot结果表明,构建的两株重组病毒分别表达了HIV-1 Gag p24及p17-p24融合蛋白。电镜观察证实,Gag p24及p17-24重组蛋白均可形成病毒样粒子。重组病毒可诱导小鼠产生抗HIV-1 Gag p24抗体。重组病毒感染BHK21细胞后,可见由于细胞凋亡而致的染色体DNA断裂“梯子”电泳图。     

HIV—1 P24截短体与gp41截短体的融合蛋白在大肠杆菌中的表达、纯化和鉴定

用基因重组技术将截短的HIV－1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24－gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24－gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24－gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。     

博尔纳病病毒pEGFP-p24基因重组质粒的构建及鉴定

构建博尔纳病病毒pEGFP-p24基因重组表达质粒。通过PCR方法扩增获得博尔纳病痛毒p24基因的完整序列,将此片段定向克隆到pEGFP-N1载体多克隆位点区,筛选重组阳性菌株,提取重组质粒,利用PCR方法和核酸序列测定验证重组质粒构建的正确性。PCR及核酸序列测定证明博尔纳病病毒pEGFP-p24基因重组表达质粒构建成功。构建的重组质粒将为研究博尔纳病病毒p24基因在真核细胞中的功能和作用提供实验依据。     

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.     

Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins. 2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis

The capacity of a previously described plasmid vector pAZ to deliver bioactive proteins to targets in vivo has been studied. This vector molecule has a strong constitutive promoter, is extremely stable in cells of vaccinal S. choleraesuis strain, and encodes the synthesis of marker protein beta-galactosidase which helps monitor the vector's fate in the host. The gene encoding hepatitis B virus core antigen (HBcAg) has been inserted into pAZ under its constitutive promoter. The resultant recombinant plasmid p19-24 has been used to transform Enterobacteriaceae (E. coli and S. choleraesuis) cells. Transformed cells produce immunologically active HBcAg. p19-24 was stable in S. choleraesuis cells during their culturing and during this strain persistence in mice. Triple oral immunization of rabbits in a dose of 1 x 10(9) S. choleraesuis cells TC177 induced the production of virus-specific antibodies. Successful transformation of cells of another vaccinal strain S. abortus ovis by this plasmid extends the potentialities of the vector. The results demonstrate good prospects of using pAZ vector for the construction of live oral vaccines.     

HIV—1核蛋白p24在昆虫细胞中的表达

将完整的ＨＩＶ－１ ｐ２４基因克隆到杆状病毒转移质粒中,使用重组转移质粒与野生型杆状病毒ＤＮＡ共转染Ｓｆ９昆虫细胞,经筛选获得带有编码ｐ２４基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中表达了ＨＩＶ核蛋白ｐ２４。其重组蛋白的分子量为２４ｋＤ。此重组糖蛋白在免疫荧光,免疫印染和酶联免疫实验中都能被人ＨＩＶ－１阳性血清和单克隆抗体所识别。     

Efficient replication of human immunodeficiency virus type 1 requires a threshold level of Rev: potential implications for latency.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the expression of the structural genes of HIV-1. To determine whether a functional threshold level of Rev is required to allow efficient HIV-1 replication, CD4-positive HeLa cells, constitutively expressing a Rev-deficient provirus, were transfected with various quantities of a Rev-expressing plasmid. Compared with the quantity of the Rev-producing plasmid transfected, HIV-1 replication was distinctly nonlinear as measured by HIV-1 p24 antigen and HIV-1-specific RNA production. A quantitative RNA polymerase chain reaction (PCR) demonstrated that Rev mRNA expression was linearly correlated with the quantity of Rev-expressing plasmid which was transfected into these cells. These data suggest that a critical threshold of Rev is required for a highly productive HIV-1 infection. This threshold level of Rev may be involved in the generation and maintenance of HIV-1 proviral latency.     

The rolling-circle plasmid pTN1 from the hyperthermophilic archaeon Thermococcus nautilus

The hyperthermophilic archaeon Thermococcus nautilus carries a plasmid, pTN1, which encodes a rolling-circle (RC) replication initiator protein of 74 kDa (Rep74) and an orphan protein of 24 kDa (p24). The Rep74 protein is homologous to the Rep75 protein encoded by the RC plasmid pGT5 from Pyrococcus abyssi. Comparative analysis of Rep74 and Rep75 sequences shows that these proteins correspond to a new family of RC initiators formed by the fusion of a Rep domain with an N-terminal domain of unknown function. Surprisingly, the Rep domain of Rep74/75 is more closely related to transposases encoded by IS elements than to Rep proteins of other RC plasmids. The p24 protein contains a hydrophobic segment, a highly charged region and a zinc finger motif. A recombinant p24 protein lacking the hydrophobic segment binds and condenses both single- and double-stranded DNA, and forms DNA aggregates with extreme compaction at high protein to DNA ratio. In addition to encoding proteins of significant interest, pTN1 is remarkable by being the only characterized plasmid isolated from a Thermococcus strain, thus being useful to develop genetic tools in Thermococcus kodakaraensis for which gene disruption methods became recently available.     

Assignment to chromosome 12 of the gene coding for the human cell surface antigen CD9(p24) using the monoclonal antibody ALB6

An analysis of 20 independent man-mouse and man-hamster hybrids has shown that the gene coding for the cell-surface protein CD9(p24), a differentiation antigen recognized by the monoclonal antibody ALB6, is located on chromosome 12. A positive correlation was shown between CD9(p24) and chromosome 12 and all other chromosomes were excluded. In addition a synteny was observed between CD9(p24) and LDH-B, a well known marker of chromosome 12 (out of 27 hybrids, 15 were LDH-B+ ALB6+ and 12 were LDH-B-ALB6-). Expression of the antigen in hybrid CH-35K issued from parental fibroblasts possessing a balanced reciprocal translocation 46,X,Y,t(X,12)(q23,q12) indicated that the gene for CD9(p24) is probably localized on 12q12----pter. Monoclonal antibodies ALB6, Ba2 and 602/29 recognize the same protein of molecular weight 21 to 24 KD controlled by a gene located on chromosome 12. It is known that Ba2 and 602/29 recognize two different epitopes but the existence of a third epitope recognized by ALB6 remains to be shown.     

Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae

We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-mum plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-mum plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1). (c) 1996 John Wiley & Sons, Inc.     

Expression of the gag gene of human T-cell leukemia virus type I in Escherichia coli and its diagnostic use

An expression plasmid, pHY202, was constructed which directs the synthesis of a fusion protein encoded by the gag sequence of human T-cell leukemia virus type I (HTLV-I) inserted into the lacZ' gene. Escherichia coli cells harboring pHY202 produced the 43-kDal LacZ'-Gag fusion protein with a yield of approx. 0.3% of total soluble proteins. The fusion protein is specifically recognized by monoclonal antibodies against the Gag proteins p19 and p24, and could be applicable for the diagnosis of HTLV-I infection, because almost all sera from HTLV-I carriers gave a positive response in the enzyme-linked immunosorbent assay (ELISA) employing the LacZ'-Gag hybrid protein purified by immunoaffinity column chromatography.