腺病毒壳蛋白抗原提取,鉴定及其抗血清的制备

本文报道作者于１９９２－１９９３年在澳大利亚应用ＤＥＡＥＳｅｐｈａｄｅｘＡ－５０层析法对腺病毒群特异性抗原－六面体壳蛋白进行提取、纯化并免疫动物获得高效价的腺病毒群特异性抗体。在抗原洗脱过程中分别在０．０５Ｍ、０．２Ｍ和０．４８Ｍ出现了３个波峰，壳蛋白的洗脱浓度为０．４８Ｍ。为明确其生物学特性。作者应用ＳＤＳ－ＰＡＧＥ电泳，证实其分子量大约为４５ＫＤ。本文的实验结果为解决腺病毒的诊断抗体提供了新的     

肉桂醛抗腺病毒作用研究

目的: 研究肉桂醛抗腺病毒(ADV)作用。方法: 采用噻唑蓝(MTT)比色法和病毒增殖抑制实验观测肉桂醛抗ADV作用;免疫荧光法检测肉桂醛对ADV六邻体(hexon)蛋白表达的影响。结果: (1)MTT和病毒增殖抑制实验结果表明肉桂醛在3种作用方式(除细胞保护作用方式)下能够增强宿主细胞存活率,降低病毒滴度,且细胞存活率与药物浓度呈正相关,肉桂醛对宿主细胞无保护作用,不能阻断病毒进入细胞。(2)肉桂醛(除细胞保护作用方式)组与病毒组比较hexon蛋白的表达明显降低(P<0.05)。结论: 肉桂醛有抗ADV作用,但却对宿主细胞无保护作用,肉桂醛抗ADV作用可能与调节hexon蛋白的表达有关。     

Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon.     

人3型腺病毒六邻体基因表达质粒的构建及表达

目的表达人腺病毒3型六邻体蛋白,为制备基因工程疫苗打下基础。方法BarnHⅠ和．SolⅠ双酶切重组克隆质粒pUcl9Plexon和载体pQE3l,将得到的目的片段定向连接到表达载体pQE31中,对目的片段进行DNA测序鉴定,IPTG诱导高效表达。结果构建了表达重组质粒pQE31 Hexon,测序鉴定了克隆序列的准确性。在大肠杆菌中成功地表达了Ad3六邻体蛋白。结论成功表达了Ad3的六邻体蛋白。     

壳蛋白免疫法测定重组腺病毒滴度的方法学评价

目的：介绍壳蛋白免疫法（hexon immunoassay），并同传统细胞病变（CPE）法比较，检测其可靠性和可重复性。 方法： 3种重组腺病毒（Ad-lacz、Ad-hEndo和dl1520）扩增、提纯后，用壳蛋白免疫法、CPE法测定病毒滴度及A260值法测定病毒总颗粒数。 结果： 壳蛋白免疫法和CPE法同时测定3种同批次病毒滴度，结果一致，P>0.05；对不同批次3种腺病毒滴度测定，结果仍相似，平均差值±s=0.1527±0.3270，P>0.05，两法有很好的相关性，r＝0.913；壳蛋白免疫法对同批dl1520测定19次，变异系数仅为1.42%。 结论： 壳蛋白免疫法是测定腺病毒滴度的有效方法，系统误差小、重复性好、结果客观可靠，可避免CPE法中由于细胞退化和工作人员主观判断造成的系统误差，比CPE法更精确、更省时。     

A novel budgerigar-adenovirus belonging to group II avian adenovirus of Siadenovirus

Five budgerigars in the same breeding facility died or showed ruffled feathers. To determine the cause, five dead or euthanized budgerigars were examined. Splenomegaly was observed at necropsy in all birds examined. Histopathology of the spleen revealed a slight-to-moderate deletion of lymphocytes and increase of macrophages. Concurrent congestions in several tissues such as liver, lung, kidney, and/or brain and basophilic intranuclear inclusion bodies in the epithelial cells of renal tubules were found in all the birds examined. Psittacine adenoviral DNA was detected in the kidney of one of the five budgerigars by PCR. Sequencing and phylogenetic analysis of the hexon gene revealed that the adenovirus gene detected in the budgerigar was derived from an unknown adenovirus belonging to the genus Siadenovirus. Using a new pair of primers based on the obtained sequence, we confirmed the presence of the newly found adenovirus in all five birds. The newly found unknown adenovirus is designated as Budgerigar Adenovirus 1.     

人腺病毒3型六邻体基因的克隆与鉴定

目的 克隆人腺病毒3型(Ad3)六邻体-L1(Hexon-L1)、六邻体-L2(Hexon-L2)区基因，构建含有该基因的重组质粒，并进行测序鉴定，为进一步构建表达载体，制备基因工程疫苗打下基础。方法 从Ad3感染的HeLa细胞中提取Ad3DNA，用PCR方法扩增Hexon—L1、Hexon-L2基因，将得到的片段定向克隆到克隆载体pUC19质粒中，对克隆片段进行DNA测序鉴定。结果 构建了重组质粒pUC19Hexon，测序结果与Genebank中该序列进行同源性比较证明克隆的片段为。Ad3六邻体序列。结论 成功克隆了Ad3的Hexon-L1、Hexon-L2区基因。     

Neutralizing epitopes mapping of human adenovirus type 14 hexon

Human adenoviruses 14 (HAdV-14) caused several clusters of acute respiratory disease (ARD) outbreaks in both civilian and military settings. The identification of the neutralizing epitopes of HAdV-14 is important for the surveillance and control of infection. Since the previous studies had indicated that the adenoviruses neutralizing epitopes were likely to be exposed on the surface of the hexon, four epitope peptides, A14R1 (residues 141–157), A14R2 (residues 181–189), A14R4 (residues 252–260) and A14R7 (residues 430–442) were predicted and mapped onto the 3D structures of hexon by homology modeling approach. Then the four peptides were synthesized, and all the four putative epitopes were identified as neutralizing epitopes by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally we incorporated the four epitopes into human adenoviruses 3 (HAdV-3) vectors using the “antigen capsid-incorporation” strategy, and two chimeric adenoviruses, A14R2A3 and A14R4A3, were successfully obtained which displayed A14R2 and A14R4 respectively on the hexon surface of HAdV-3 virions. Further analysis showed that the two chimeric viruses antiserum could neutralize both HAdV-14 and HAdV-3 infection. The neutralization titers of anti-A14R4A3 group were significantly higher than the anti-KLH-A14R4 group (P = 0.0442). These findings have important implications for the development of peptide-based broadly protective HAdV-14 and HAdV-3 bivalent vaccine.