7型腺病毒疫苗株基因克隆及其右侧末端Sma ⅠH片段的核苷酸序列分析

目的获得７型腺病毒（Ａｄ７）载体构建的基本元件，为构建Ａｄ７载体及阐明其基因组一级结构打下基础。方法用改良Ｈｉｒｔｓ法提取Ａｄ７ＤＮＡ，酶切后克隆于质粒载体，克隆了ＸｈｏⅠＡ＋Ｄ片段（１７０－７６５ｍｕ）、Ｄ片段（１７０－２２９ｍｕ）、Ｅ片段（１０８－１４１ｍｕ）、Ｆ＋Ｇ片段（１４１－１７０ｍｕ）、Ｇ片段（１５８－１７０），用ＥｃｏＲⅠ＋ＸｈｏⅠ双酶切，克隆了７６５－８７ｍｕ，用ＳａｍⅠ＋ＥｃｏＲⅠ双酶切，克隆了８７０－９７４ｍｕ。用腺病毒ＤＮＡ做为模板测定了Ａｄ７疫苗株右侧末端ＳｍａⅠＨ片段（９７４－１００ｍｕ）核酸苷序列，根据所测序列设计引物，用聚合酶链反应（ＰＣＲ）获得右侧末端，并对其再次测序证实。结果建立了Ａｄ７疫苗株部分基因文库（１０８－１００ｍｕ），完成Ａｄ７疫苗株ＳｍａⅠＨ片段核苷酸序列。结论这一结果为阐明Ａｄ７基因组一级结构及构建Ａｄ７载体奠定了基础。     

4型腺病毒载体的构建和β－半乳糖苷酶基因的表达

以４型腺病毒疫苗株感染Ａ５４９细胞，提取病毒ＤＮＡ，将ＥｃｏＲＩ消化的Ｄ片段（７０．５－８３．０基因图谱单位）克隆入ｐＵＣ１８质粒，得ｐＡｄ４（７０．５－８３）质粒，质粒ｐＡｄ４（７０．５－８３）和ｐＡｄ４Ｃ１－２５经酶切、系列亚克隆、加接头等方法得到大部分和部分缺失Ｅ３区的重组质粒。聚合酶链反应（ＰＣＲ）法获得ｐｏｌｙ（Ａ），构建约８００ｂＰ腺病毒晚期表达盒，在所构建的腺病毒重组质粒Ｅ３缺失区或Ｅ４与ＩＴＲ间插入表达盒，得到可同时表达２个或２个以上外源基因和保留了Ｅ３区编码分子量为１９３００糖蛋白基因的３种Ａｄ４载体。将ｌａｃＺ基因插入载体表达区，与Ｂｅｌｌ消化的Ａｄ４ＤＮＡＡ片段共转染Ａ５４９细胞，ＯＮＰＧ法检测证实所构建的载体和表达盒功能良好。本项工作对于在国内研究口服活疫苗及开展基因治疗均具有重要意义。     

4型腺病毒载体的构建和β—半乳糖苷酶基因的表达

以４型腺病毒疫苗株感染Ａ５４９细胞，提取病毒ＤＮＡ，将ＥｃｏＲＩ消化的Ｄ片段克隆入ｐＵＣ１８质粒，得ｐＡｄ４质粒，质粒ｐＡｄ４和ｐＡｄ４Ｃ１－２５经酶切、系列亚克隆、加接头等方法得到大部分和部分缺失Ｅ３区的重组质粒。聚合酶链反应（ＰＣＲ）法获得ｐｏｌｙ（Ａ），构建约８００ｂｐ腺病毒晚期表达盒，在所构宾腺病毒重组质粒Ｅ３缺失区或Ｅ４与ＩＴＲ间插入表达盒，得到可同时表达２个或２个以上外源基因和保留了Ｅ     

4型腺病毒疫苗株载体的构建及β-半乳糖苷酶基因的表达

目的构建缺失Ｅ３区７８．９－８６ｍｕ的４型腺病毒疫苗株载体。方法从４型腺病毒疫苗株（Ａｄ４）感染的人胚肺二倍体细胞２ＢＳ中提取病毒ＤＮＡ,经过多步亚克隆构建成功缺失７８．９－８６ｍｕ的载体。将含有ＣＭＶ早期启动子的β－半乳糖苷酶基因插入缺失部位,将这一重组质粒与Ａｄ４ＢｃｌⅠ大片段共转染２９３细胞,铺含有Ｘ－Ｇａｌ的营养琼脂,经蓝色蚀斑纯化三代,ＯＮＰＧ法测定β－半乳糖苷酶基因的表达量。结果所构建的载体能稳定地表达β－半乳糖苷酶基因。结论４型腺病毒疫苗株载体的构建为利用该病毒做为基因工程疫苗载体打下了基础。     

重组人肝细胞生长因子-NK4腺病毒的制备及初步鉴定

目的制备表达人肝细胞生长因子（HGF）-NK4蛋白的复制缺陷型重组腺病毒。方法酶切pcDNA3-hNK4质粒获得NK4基因编码区序列并克隆至穿梭载体构建重组pAdTrack—CMV—NK4载体，线性化后与pAdEasy-1共转化BJ5183，通过同源重组得到重组pAd—NK4病毒载体。将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒，用病毒悬液感染人肝癌细胞株HepG2。RT—PCR法检测感染肿瘤细胞中NK4mRNA表达。结果酶切鉴定得到阳性pAd—NK4重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装。在转染2d后能观察到绿色荧光蛋白（GFP）表达。制备的Ad—NK4在体外能有效感染HepG2细胞并获得NK4基因高水平表达。结论成功构建了NK4基因的重组腺病毒载体并制备重组腺病毒颗粒，为进一步研究NK4基因的功能及应用NK4进行基因治疗提供实验依据。     

大鼠GLUT1基因的扩增及其重组腺病毒载体的构建

目的:构建携带大鼠葡萄糖转运体1基因（GLUT1）的复制缺陷型重组腺病毒载体，为进一步研究脑缺血缺氧引起的神经元死亡机制奠定基础。方法:采用逆转录-聚合酶链反应（RT-PCR）的方法获取大鼠GLUT1基因全长cDNA，将其克隆至穿梭质粒pShuttle中构建穿梭质粒pShuttle-Glut1，经酶切后与线性化的腺病毒骨架质粒pAdeno-X体外连接并转化大肠杆菌DH5α，构建重组腺病毒质粒pAd-Glut1，酶切线性化重组腺病毒质粒后转染HEK293细胞包装成重组病毒颗粒，重组腺病毒在HEK293细胞中反复扩增数代后，分别用聚合酶链反应（PCR）及免疫印迹法（Western blotting）从基因和蛋白表达水平鉴定重组的腺病毒。结果:经PCR分析及DNA测序显示GLUT1 cDNA序列正确；筛选出重组腺病毒质粒pAd-Glut1后在HEK293细胞中成功包装出重组病毒，包装后冻融细胞行PCR及Western blotting检测表明重组腺病毒包装成功。结论:成功扩增了大鼠GLUT1基因并构建了携带大鼠GLUT1基因的复制缺陷型重组腺病毒载体，这有助于研究脑缺血缺氧引起的神经元死亡的机制。     

7型腺病毒疫苗株DNA左侧17.5mu片段克隆及4.8mu片？…

目的 获得７型腺病毒疫苗株ＤＮＡ左侧０－１７．５ｍｕ片段克隆,分析０－４．８ｍｕ片段（末端倒置重复序列,包装信号位点和Ｅｌａ区）核苷酸序列,。方法从Ａｄ７疫苗株感染的Ａ５４９细胞提取Ａｄ７ ＤＮＡ,将其０．３－１７．５ｍｕ片段克隆到质粒ｐＡｄ７Ｔ,并用自动和银染方法测序。结果 获得了Ａｄ７疫苗株左侧１７．５ｍｕ片段克隆,测定了左侧１７３７ｂｐ核苷酸序列,其中Ｅｌａ区所编码的６３００,２４０００,２     

小鼠Hsp27基因siRNA重组腺病毒载体的构建与鉴定

Hsp27是小分子量热休克蛋白亚家族的重要成员之一，主要参与细胞内蛋白质的折叠、抗凋亡、甾体激素合成、氧化应激及信号通路等生理过程。为进一步研究其生理与病理生理功能，本文构建了针对小鼠Hsp27基因的小分子干扰RNA（siRNA）重组腺病毒载体，并在AD-293细胞中与Hsp27超表达载体共同转染，观察其对Hsp27表达的影响。根据siRNA靶序列的设计要求，设计并合成针对小鼠Hsp27的siRNA靶DNA序列，克隆至穿梭载体pShuttle-H1中，与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组，转染AD-293细胞，包装得到含siHsp27的重组腺病毒，在体外与Hsp27超表达载体共转AD-293细胞。通过酶切鉴定、序列测定和Westernblot鉴定，确定小鼠Hsp27siRNA重组腺病毒载体构建成功，且该重组腺病毒能降低67％的Hsp27蛋白表达。本文构建的针对小鼠Hsp27基因的siRNA重组腺病毒载体，为进一步研究Hsp27基因的功能奠定了基础。     

人白介素24腺病毒载体构建及其生物学活性分析

目的：克隆人白介素24基因，构建其腺病毒载体，获取病毒重组子，并研究其生物学括性。方法：用密执毒素（Mezerein）诱导HeLa细胞表达IL-24，通过RT-PCR获取IL-24 cDNA，将其亚克隆至pAdTrack-CMV载体，经PmeⅠ线性化后，与腺病毒的骨架载体pAdEasy-Ⅰ在BJ5183菌中同源重组，HEK293细胞包装扩增，PCR、Western blot鉴定。AdIL-24处理宫颈癌CaSki细胞，通过MTT细胞存活实验检测其活性；Hoeehst33342染色、流式细胞仪检测凋亡；WesternNot检测Bax、Bd。2、p53蛋白的表达。结果：获取人IL-24的cDNA，序列与GeneBank公布序列完全一致，成功构建腺病毒载体，AdIL-24对CaSki细胞具有抑制生长、促进凋亡的作用，并上调Bax、p53蛋白表达，下调Bcl-2蛋白表达。结论：成功构建人IL-24的重组腺病毒载体，其病毒重组子具有生物活性。     

腺病毒伴随病毒载体介导的Ⅰ型单纯疱前病毒胸苷 …

目的 研究腺病毒伴随病毒（ＡＡＶ）载体介导杀肿瘤瘤基因的转移及其在肿瘤治疗方面的应用。方法 应用作者构建的腺病毒伴随病毒载体，克隆Ⅰ型单纯疱疹病毒胸苷激酶（ＨＳＶＩ－ＴＫ）基因，构建质粒ｐＡＣＴＫ－１９。用ｐＡＣＴＫ－１９转染５型腺病毒（Ａｄ５）感染的重组ＡＡＶ包装细胞系ＡＥ１２０１，获得重组病毒ｒＡＡＶ／ＡＣＴＫ。再用此重组病毒感染肺癌细胞系Ａ５４９，并联合现氧鸟苷（ＧＣＶ）作用，研究其体外对肺     

Adenovirus E4orf4 induces HPV-16 late L1 mRNA production

The adenovirus E4orf4 protein regulates the switch from early to late gene expression during the adenoviral replication cycle. Here we report that overexpression of adenovirus E4orf4 induces human papillomavirus type 16 (HPV-16) late gene expression from subgenomic expression plasmids. E4orf4 specifically overcomes the negative effects of two splicing silencers at the two late HPV-16 splice sites SD3632 and SA5639. This results in the production of HPV-16 spliced L1 mRNAs. We show that the interaction of E4orf4 with protein phosphatase 2A (PP2A) is necessary for induction of HPV-16 late gene expression. Also an E4orf4 mutant that fails to bind the cellular splicing factor ASF/SF2 fails to induce L1 mRNA production. Collectively, these results suggest that dephosphorylation of SR proteins by E4orf4 activates HPV-16 late gene expression. Indeed, a mutant ASF/SF2 protein in which the RS-domain had been deleted could itself induce HPV-16 late gene expression, whereas wild type ASF/SF2 could not.     

Adenovirus Type 4 and 7 Vaccination or Adenovirus Type 4 Respiratory Infection Elicits Minimal Cross-Reactive Antibody Responses to Nonhuman Adenovirus Vaccine Vectors

Antivector immunity may limit the immunogenicity of adenovirus vector vaccines. We tested sera from individuals immunized with adenovirus type 4 and 7 (Ad4 and Ad7, respectively) vaccine or naturally infected with Ad4 for their ability to neutralize a panel of E1-deleted human and chimpanzee adenoviruses (ChAd). Small statistically significant increases in titers to ChAd63, ChAd3, human Ad35, and human Ad5 were observed. Neutralizing antibodies elicited by Ad4 infection or immunization results in a small amount of adenovirus cross-reactivity.     

Adeno-Associated Virus in Adenovirus Type 3 Conjunctivitis

Although human infection with adenovirus-associated virus (AAV) has been demonstrated, there is no evidence that disease results from such infections. The proportion of adenovirus infections which are dual infections with AAV is virtually unknown, since special methods are required to demonstrate infection with AAV. To search for AAV, we re-examined a collection of specimens from 40 persons involved in an epidemic of pharyngoconjunctival fever associated with a swimming pool. Virological and serological studies indicated that the etiological agent was adenovirus type 3. When the 91 original eye, throat, and fecal specimens were re-examined, using methods suitable for detection of adenovirus and AAV, 37 strains of adenovirus type 3 and 35 strains of AAV type 3 (AAV3) were isolated. Surprisingly, 19 AAV3 but only 11 adenovirus isolates were found in eye specimens, whereas adenovirus isolates were equally distributed in all types of specimens. Four AAV3 strains were isolated from adults. Significant (fourfold or greater) rises in AAV3 complement-fixing antibody titers were seen in six of 14 persons shedding AAV3, whereas nine of 10 persons shedding adenovirus type 3 showed significant rises in adenovirus complement-fixing antibody. These results raise the question whether AAV persists better in eyes than adenovirus or that a possible association with conjunctivitis might be present. In contrast to the results in the specimens from the swimming pool epidemic, only one of 36 adenovirus strains isolated in other Seattle-based studies yielded AAV. Complement fixation tests on serial sets of sera collected from 60 children not involved in the swimming pool episode revealed nine AAV2 and 12 AAV3 infections during a 4-year period.     

Genomic DNA Analysis of Rat Retinal Tumor Induced by Adenovirus Type 12

To elucidate the pathogenesis of human retinoblastoma, we investigated the genomic expression in retinal tumors induced by human adenovirus type 12 in rats, using various DNA probes. Seven rats received a single intraocular inoculation of concentrated virus fluid within 24 hours after birth. Intravitreous tumors were induced in two out of seven animals (28.5%) within 30 to 64 days after the inoculation. A remarkably uniform histologic feature, i.e., neuroblastic cells in association with Homer-Wright pseudorosettes, was present in all cases. The adenovirusrelated oncoprotein gene E1A and human retinoblastoma susceptibility gene were detected in the tumors by Southern blot hybridization. In situ hybridization analysis demonstrated expression of adenovirus type 12 E1A gene in the inner granular layer of the retina. It was suggested that integration of adenovirus type 12 E1A fragment with the host genome and expression of the gene were required for induction of this tumor.     

Adenovirus infection in patients with Kawasaki disease

Two outbreaks of Kawasaki disease at different times and areas (Kyoto in 1982 and Sapporo in 1985) of Japan were studied retrospectively for the presence of antibodies to adenoviruses and herpesviruses. Only 2 of 12 (16.7%) consecutive acute phase sera of patients from the outbreak in 1982 and 1 of 10 (10.0%) sera from 1985 showed positive antibodies for the common adenovirus antigen by a complement fixation (CF) test, whereas 10 of 16 (62.5%) age- and sex-matched controls during the outbreak of Kawasaki disease in 1985 were seropositive by the CF test. In contrast, using a recently developed enzyme-linked immunosorbent assay (ELISA), 9 patients (75.0%) in 1982 and 9 patients (90.0%) in 1985 had antibodies to adenovirus type 2. In addition, 5 of 10 (50.0%) of the 1982 and 6 of 9 (66.7%) of the 1985 patients who were seronegative for CF antibodies were positive for IgM antibodies to adenovirus type 2. Fifteen (93.8%) controls were positive for antibodies to adenovirus type 2 by ELISA and only two sera showed negative CF antibodies with positive IgM antibodies to adenovirus type 2. Sequential sera from 4 patients in 1985 had either IgM or IgG antibodies by ELISA and eventually three became seropositive by the CF test in time. Additionally, no significant difference was noted with antibody status to herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), and cytomegalovirus (CMV) between patients and controls.(ABSTRACT TRUNCATED AT 250 WORDS)