人HSV-1 UL40基因重组质粒的构建及其在siRNA筛选中的应用

目的： 快速筛选出高效沉默人HSV-1 UL40基因的siRNA，为进一步应用RNA干扰的相关研究奠定基础。方法: 应用RT-PCR扩增人HSV-1 F株UL40基因，将其连接到pEGFP-N1构建pEGFP-N1-UL40融合蛋白表达质粒。将化学合成的针对UL40基因的siRNA与重组质粒共转染Vero细胞，通过观察绿色荧光蛋白报告基因的表达筛选高效沉默人HSV-1 UL40基因的siRNA。结果: 倒置荧光显微镜观察发现设计的4对siRNA中siRNA3和siRNA4能明显降低绿色荧光蛋白的表达，流式细胞仪检测证明siRNA3和siRNA4对目的基因沉默效率分别达到76.99％和84.00％。结论: 成功构建了人HSV-1 UL40基因融合表达载体pEGFP-N1-UL40，并利用该载体筛选出2对高效沉默人HSV-1 UL40基因的siRNA。     

Aβ单链抗体基因腺相关病毒的包装和纯化

目的 包装和纯化携带Aβ单链抗体基因的腺相关病毒，为阿尔茨海默病的基因治疗创造条件。 方法 用Lipofectamine 2000将pSNAV2.0-Abeta-scFv质粒转染BHK-21细胞并用G418筛选稳定细胞系。培养稳定细胞系用辅助病毒HSV1-rc/ UL2感染包装携带Aβ单链抗体基因的腺相关病毒。用氯仿-PEG/NaCl-氯仿法和离子交换层析法纯化重组腺相关病毒，SDS-PAGE和PCR方法检测纯化的病毒。用地高辛标记探针测定重组腺相关病毒的物理滴度。利用水迷宫测试重组病毒对转基因阿尔茨海默病小鼠模型的治疗效果。结果 带Aβ单链抗体基因的腺相关病毒的纯度为98%，物理滴度为1×1012vg/ml。经过治疗的小鼠模型潜伏期明显缩短。 结论 利用HSV1系统成功包装和纯化了携带Aβ单链抗体基因的腺相关病毒，动物实验表明对阿尔茨海默病具有治疗作用。     

rAAV2/1-Acrp30病毒的纯化、扩增与活性检测

目的 将大鼠脂联素基因(Acrp30)克隆到腺相关病毒(AAV)载体中,经包装、纯化、扩增后获得rAAV2/1-Aerp30病毒,并进行活性检测.方法 筛选阳性克隆获得pSNAV2.0-Acrp30,EcoR Ⅰ/Sal Ⅰ双酶切鉴定,并行测序.转染BHK21细胞,G418筛选培养,获得抗药克隆细胞株.HSV1-re/△UL2感染,包装此细胞株并收获病毒载体rAAV2/1-Acrp30.行DNA斑点杂交法测定病毒滴度,SDS-PAGE分析判断病毒纯度,Western blot法检测目的 蛋白脂联素在HEK293细胞中的表达活性.结果 PCR电泳及酶切鉴定表明,pSNAV2.0-Acrp30重组成功,基因测序显示装入pSNAV2.0质粒中的Acrp30基因正确.rAAV2/1-Acrp30病毒的大致滴度为1.0×1012μg/ml,HEK293细胞分泌蛋白浓度为50 ng/ml,病毒载体纯度在95%以上.结论 实验获得的脂联素病毒载体滴度高、感染性好,可试用于GK(Goto-Kakizaki)大鼠脂联素转基因治疗.     

放射免疫沉淀试验鉴定单纯疱疹病毒糖蛋白C

用放射免疫沉淀试验和单纯疱疹病毒1型、2型(HSV—1/HSV-2)型间重组株作物理谱图的方法.鉴定出抗HSV的单克隆抗体(McAb) 1A12、Mad-2、2D11、CM-D3、2A8和1C_4的靶抗原是一组分子量在105～130Ka之间的糖蛋白,其编码基因定位于长单一序列(UL)上,在0.536～0.682遗传单位之间,与gC的编码基因部分重叠。故认为这些McAbs的靶抗原为gC,其中,Mad-2和CM-D3分别为HSV-1和HSV-2型特异性,其靶抗原分别为gC-1和gC-2。ELISA阻断试验结果表明,4株型共同性McAbs 1A12、2D11、2A8和1C4.抗gC上4个独立的抗原位点。     

柯萨奇病毒B3VP1重组腺病毒载体疫苗rAd/VP22-L-VP1的构建及表达**

背景：VP22是单纯疱疹病毒1型(Herpes simplex virus type 1,HSV-1)UL49基因编码的碱性蛋白质,具有蛋白转导结构域(protein transduction domain, PTD),能够把与之融合的蛋白或与之结合的DNA 等大分子跨膜送递到邻近细胞,在基因靶向预防中表现出优势。 目的：构建表达单纯疱疹病毒1型VP22与柯萨奇病毒B3主要中和抗原VP1融合蛋白的重组腺病毒载体疫苗,观察外源基因在HEK293细胞中的良好表达。 方法：PCR法扩增目的基因HSV-1 VP22和CVB3 VP1,经Linker连接,将VP22-L-VP1插入腺病毒穿梭载体pAdTrack-CMV,构建重组穿梭质粒AdTrack-CMV/VP22-L-VP1。再将此载体与腺病毒骨架载体pAdEasy-1在大肠杆菌BJ5183中进行同源重组,生成重组腺病毒质粒pAd/VP22-L-VP1,脂质体介导pAd/VP22-L-VP1转染HEK293细胞包装重组腺病毒rAd/VP22-L-VP1。HEK293细胞上进行病毒扩增和滴定并检测外源基因的表达。 结果与结论：构建的重组腺病毒载体pAd/VP22-L-VP1经过第4轮扩增,其滴度达到6.77×107 pfu/mL,体外感染293细胞可见VP22和VP1融合蛋白的表达。说明实验成功构建并包装重组腺病毒rAd/VP22-L-VP1。     

质粒型HSV载体介导的GDNF和GFP基因在BHK细胞和骨骼肌细胞中的转移和表达

为了进一步探索治疗中枢神经损伤的新途径 ,本实验以质粒型单纯疱疹病毒 ( H SV)为载体 ,分别构建了含有胶质细胞源性神经营养因子 ( GDN F)和绿色荧光蛋白 ( GFP)基因的重组载体 H SV - GDNF和 H SV- GFP,用包装出的混合毒株分别感染体外培养的 BHK细胞及原代培养的大鼠骨骼肌细胞。 2 d后 ,对感染 HSV - G DNF组的细胞进行 G DNF免疫组织化学反应 ;感染HSV- GFP组的细胞在荧光显微镜下进行观察。结果发现 :质粒型 H SV载体可成功地将外源性 GDN F和 GFP基因导入 BH K细胞和原代培养的大鼠骨骼肌细胞。提示 :质粒型 H SV载体可以作为转基因 GDN F治疗中枢神经系统损伤的转移载体 ,为中枢神经系统损伤的治疗展示了广阔的前景。 GFP作为报告基因 ,也可因其适用广谱、观察简便等特性而得到更加广泛的应用     

单纯疱疹病毒Ⅰ型基因治疗载体的快速构建

背景：单纯疱疹病毒Ⅰ型载体因具有独特的优点目前被广泛应用,但其构建尚缺乏一种快速有效的方法。 目的：利用Cre/Loxp高效重组系统构建单纯疱疹病毒Ⅰ型载体。 方法：分离单纯疱疹病毒HSV-1,将含Cre重组酶的c66-SV40-cre质粒转染Vero细胞,构建一株带有Loxp位点的重组HSV-1框架载体HSVLoxp。构建穿梭载体pShuttle- SV40-Cre-Loxp-IRES及重组单纯疱疹病毒Ⅰ型载体HSV-GDNF,用HAT培养基筛选出阳性毒株后用GDNF引物做PCR鉴定,扩增培养后测定滴度。 结果与结论：成功构建pHV-TK-GFP质粒,并在Vero细胞内发生重组,分离出缺失了Us3基因的重组病毒HSVtk-Loxp-GFP01。成功构建HSV-1框架载体HSVLoxp及穿梭载体pShuttle- SV40-Cre-Loxp-IRES,成功获得GDNF基因,并将其转移到了HSV-1基因组上,成功构建了表达GDNF的单纯疱疹病毒HSV-1载体,测定其滴度约为2.25×10⁶ IU/mL。     

腺相关病毒载体pAGX(+)的构建

目的构建腺相关病毒(AAV)载体并进行鉴定,以介导真核细胞的基因传递和表达.方法以基因重组技术构建AAV载体,用Southern杂交、狭缝杂交以及激光共聚焦显微镜等鉴定构建的AAV载体.结果成功地获得了重组AAV表达载体pAGX(+),藉报告基因EYFP鉴定pAGX(+),见pAEYFP经包装的重组AAV储存液的感染滴度达1.39×107TU(transmissionunit)/ml、复制滴度达1.94×1011颗粒/ml.Southern杂交表明EYFP基因获得成功拯救.rAAV/EYFP颗粒成功转导COS7细胞,EYFP获得表达,并整合入COS7细胞基因组,而藉质粒载体pEYFPCMV介导的转移,EYFP未能整合.结论成功地构建了一个AAV载体,并成功地介导了基因的转移、表达和整合.     

粒-巨噬细胞集落刺激因子与绿色荧光蛋白融合基因慢病毒载体构建

目的：构建粒-巨噬细胞集落刺激因子(GM-CSF)与绿色荧光蛋白（GFP）融合基因慢病毒载体。 方法： 采用RT-PCR方法获得GM-CSF全外显子片段, 使之克隆到带GFP慢病毒表达载体质粒 FUGW中，经限制性酶切、PCR扩增和测序鉴定重组载体。在脂质体介导下将慢病毒包装系统的包装结构基因pCMV.△8.9、包膜基因VSVG、目的基因FUGW-GM-CSF导入病毒包装细胞293T，荧光显微镜检测基因的表达，包装成病毒后，收集病毒上清，浓缩，鉴定。用酶联免疫吸附实验（ELISA）检测转染FUGW-GM-CSF细胞上清液 GM-CSF表达水平，电子显微镜观察293T细胞中的慢病毒。 结果： 限制性内切酶酶切和DNA测序分析证实RT-PCR获得的GM-CSF/PCR产物与GenBank中的数据完全吻合; GM-CSF准确克隆入FUGW的多克隆位点。慢病毒的3种质粒可高效转染入293T细胞，荧光显微镜下观察可见大量绿色荧光。转染 FUGW-GM-CSF包装细胞上清液 GM-CSF酶联反应阳性。电子显微镜下见慢病毒颗粒位于293T细胞胞浆的内质网中。 结论： 成功构建了GM-CSF与GFP融合基因慢病毒载体，为诱导DC提供了适合的稳定转染的载体。     

同源重组构建表皮葡萄球菌附属基因调节子(agr)阴性突变株

目的　构建表皮葡萄球菌agr阴性突变株 ,以期得到除agr基因以外相同遗传背景的突变株与野生株。方法　首先构建同源重组质粒pBT2 △agr,后电转入金黄色葡萄球菌RN4 2 2 0 ,再转入表皮葡萄球菌 32 98。通过pBT2载体对温度敏感的特点 ,含重组质粒的表皮葡萄球菌 32 98在 4 0℃多次传代 ,最终筛选出agr阴性的突变株。结果　同源重组质粒pBT2 △agr通过酶切鉴定证明构建成功 ,表皮葡萄球菌 32 98接受来自金黄色葡萄球菌RN4 2 2 0的重组质粒 ,经酶切鉴定正确。 4 0℃多次传代后 ,经抗生素抗性筛选 ,并经斑点杂交及序列分析 ,证明获得表皮葡萄球菌 32 98 agr阴性突变株。结论　用同源重组的方法完成表皮葡萄球菌 32 98 agr阴性突变株的构建 ,使agr基因被大部分剔除掉。为今后研究表皮葡萄球菌agr基因在调节其生物膜形成的功能提供实验资料     

Helper functions required for wild type and recombinant adeno-associated virus growth

The human parvovirus Adeno-Associated virus (AAV-2) has been classified as a Dependovirus because it requires the presence of a helper virus to achieve a productive replication cycle. Several viruses such as Adenovirus (Ad), Herpes Simplex Virus (HSV), Vaccinia virus, and human papillomaviruses (HPV) can provide the helper activities required for AAV growth. The studies on the helper activities provided by adenovirus have provided useful information not only to understand the AAV-2 biology but also to develop tools for the production of recombinant AAV particles (rAAV). This review will focus on the current knowledge about the helper activities provided by the most extensively studied helper viruses, Ad and HSV-1, and also illustrate the methods used to supply the helper functions rAAV assembly.     

Chimeric herpes simplex virus/adeno-associated virus amplicon vectors

Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 pre-integration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome.     

Second generation adeno-associated virus type 2-based gene therapy systems with the potential for preferential integration into AAVS1

Adeno-associated virus type 2 (AAV-2) is a non-pathogenic human parvovirus that is being developed as a gene therapy vector for the treatment of numerous diseases. One property of wild-type AAV-2, that is highly desirable in a gene therapy vector, is its ability to preferentially integrate its DNA into a 4 kilobase region of human chromosome 19, designated AAVS1. One disadvantage of AAV-2 is its relatively small packaging capacity, approximately 4.7 kilobases. Because of this size limitation, the AAV-2 rep and cap genes were removed from first-generation AAV-2-based gene therapy vectors to make room for the therapeutic or marker gene. It was later discovered that the rep gene, or at least one of its products, the Rep68 or Rep78 protein, is required for preferential integration of AAV-2. Recent developments in AAV-2 gene therapy vector construction allow the inclusion of the rep gene into a second generation of AAV-2-based gene therapy systems. These new systems fall into four major categories: plasmid-based systems, co-transduction with multiple AAV-2 vectors, incorporation of the AAV-2 vector into a larger virus, and in vitro packaging. These systems not only allow the inclusion of the rep gene, they also allow the delivery of larger therapeutic genes.     

Gene delivery GAD 500 autoantigen by AAV serotype 1 prevented diabetes in NOD mice: transduction efficiency do not play important roles

We previously found that adeno-associated viral vector serotype 2 (AAV-2) muscle gene delivery of GAD 500-585 autoantigen efficiently prevented autoimmune diabetes in NOD mice. Recent reports suggest that AAV vectors based on serotype 1 (AAV-1) transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2 to 1000-fold. To determine whether this increased efficacy of AAV-1 could result in increased therapeutic effects in mice, we constructed rAAV1/GAD 500-585 vectors and compared their effects in preventing autoimmune diabetes in NOD mice with those of rAAV2/GAD 500-585 after muscle injection. rAAV(1)/GAD(500-585) gene therapy prevented diabetes in NOD mice. However, although much higher level of GAD 500-585 expression was found in mice using AAV-1 as gene delivery vector than those using AAV-2, no increased efficiency of AAV-1 vectors were found in their capability to prevent autoimmune diabetes, as higher titers of rAAV1/GAD 500-585 virus (3x10(11)v.g./mouse) were needed to obtain therapeutic effects in NOD mice, a titer not different from that of AAV-2. Protection resulted from rAAV1/GAD 500-585 gene therapy were marked by enhanced Th2 immune response and up-regulated CD4+ Foxp3+T regulatory cells, which might actively suppress effector T cells in NOD mice. As here we found that the therapeutic effects of AAV1 were not positively correlated to it's transduction efficiency, our data suggested that the safety and other factors besides efficiency should be considered when use different AAV serotype to treat autoimmune disease.     

Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions

Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.     

Adeno-associated viruses inhibit SV40 DNA amplification and replication of herpes simplex virus in SV40-transformed hamster cells

Coinfection with HSV-1 and any of the three defective parvoviruses AAV-2, AAV-3, or AAV-5 reduces the HSV-1-induced amplification of SV40 DNA in the SV40-transformed hamster cell lines CO631 and Elona. Moreover, all three viruses significantly decrease HSV-1 replication. The effects are measurable to the same extent in both cell lines and are dependent on the quantity of AAV DNA molecules rather than on infectious AAV particles. It seems unlikely that the inhibitions are due to simple competition but result from complex interactions that involve the terminal sequences of AAV DNA.     

Adeno-associated virus DNA replication is induced by genes that are essential for HSV-1 DNA synthesis

Adeno-associated virus (AAV) DNA replication is not detectable unless cells are coinfected with a helper adenovirus (Ad) or herpesvirus or unless AAV infection is carried out in certain established cell lines that have been treated with various metabolic inhibitors or uv irradiation. In helper-dependent infections, it has been shown that AAV DNA synthesis depends on one or more early Ad genes, whereas little is known concerning any herpesvirus gene that promotes AAV DNA synthesis. In this study we tested the ability of four cloned Xbal fragments of herpes simplex virus type 1 (HSV-1) DNA to induce AAV DNA synthesis in Vero cells. Cotransfections, which were carried out with pAV1 (an infectious AAV2 plasmid), revealed that AAV DNA synthesis could be optimally induced by three of these clones (C,D, and F) plus a clone of the HSV-1 ICP4 (IE 175) gene. ICP4, an immediate early gene, was presumably required to activate expression of other HSV genes. To help identify the additionally needed HSV genes, we tested Xbal C,D, and F subclones that contain genes previously found necessary for origin-dependent HSV DNA synthesis and found that at least five of these genes (UL 5, 8, 9, 29, and 30) contributed to the induction of AAV DNA synthesis. In contrast to their absolute requirement for HSV DNA synthesis, none of these genes were strictly necessary for AAV DNA replication. Because they are all known to specify proteins that are directly involved in HSV DNA synthesis, our results suggest that some or all of their products also may directly participate in the replication of AAV DNA.     

Evidence that the gene for herpes simplex virus type 1 DNA polymerase accounts for the capacity of an intertypic recombinant to spread from eye to central nervous system

HSV-1(17) replicates 100-fold more efficiently than HSV-2(186) within trigeminal ganglia following ocular infection. In order to identify the nucleotide sequences responsible for the differences in the capacity of the two HSV strains to grow within the peripheral nervous system, an intertypic recombinant was generated by infecting neuroblastoma cells with HSV-2(186) and a HSV strain possessing nucleotide sequences from HSV-1(17). The genome of the intertypic recombinant was composed entirely of HSV-2(186) DNA except for 2.0 kb of HSV-1(17) DNA positioned between m.u. 0.413 and 0.426. Following corneal infection of mice, the intertypic recombinant grew to higher titers in both ocular tissues and trigeminal ganglia than did the HSV-2 parent. Most significantly, the intertypic recombinant could spread into the brain from the trigeminal ganglion and kill the host whereas mice inoculated with the HSV-2(186) parent survived infection. The 2.0 kb of HSV-1(17) DNA inserted into the genome of the intertypic recombinant encodes the 5' terminus of the HSV-1 gene for DNA polymerase. Thus, the results suggest that the difference in the capacity of two HSV strains to replicate within the trigeminal ganglion of its host and to spread into the brain is determined by nucleotide sequences within the gene for DNA polymerase.     

Sequence-based methods for identifying epidemiologically linked herpes simplex virus type 2 strains

Traditional methods for confirming the identity of herpes simplex virus (HSV) isolates use restriction fragment length polymorphism (RFLP). However, RFLP is less amenable to high-throughput analyses of many samples, and the extent to which small differences in RFLP patterns distinguish between different viral strains remains unclear. Viral HSV type 2 (HSV-2) DNA isolates from 14 persons experiencing a primary HSV-2 infection and from their sexual partners were analyzed by RFLP and heteroduplex mobility assays. We also compared the HSV-2 sequences from seven regions, including noncoding regions between UL19 and UL20, UL24 and UL25, UL37 and UL38, and UL41 and UL42 and coding segments of the gC, gB, and gG genes. Although the resulting RFLP patterns of the couples were almost identical, minor banding differences existed between the source and susceptible partners in five couples. Heteroduplex mobility assays were unable to distinguish between unrelated strains. Overall, 22 sites of sequence variation were found in 1,482 bp of analyzed sequence. The DNA sequences differentiated between all unrelated infections, and epidemiologically related isolates had identical sequences in all but two pairs. Our results suggest that a multilocus assay based on several DNA sequences has the potential to be an informative tool for identifying epidemiologically related HSV-2 strains.