单纯疱疹病毒1型载体

本讨论单纯疱疹病毒１型的特点和作为基因治疗载体的单纯疱疹１型载体的构建方法及其应用。     

牛疱疹病毒I型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活…

由含有ＢＨＶ－１（Ｂｏｖｉｎｅ Ｈｅｒｐｅｓ Ｖｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＩＣＰＯ（ＢＨＶ－１Ｉｎｆｅｃｔｅｄ Ｃｅｌｌ Ｐｒｏｔｅｉｎ Ｏ）的ＤＮＡ序列至表达载体ｐＳＶＤ３,构建质粒ｐＳＶ２．９。将该质粒与ＰＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶ ＬＴＲ启动子控制下的荧光素酶基因的表达。根据ＰＳＶ２．９与含有Ｂ     

携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定

利用细菌人工染色体技术将串联的HIV-1 gp160、gag和protease基因以及表达元件插入1型单纯疱疹病毒(Herpes simplex virus type 1,HSV-1)内部反向重复序列区,以获得携带HIV-1抗原的单纯疱疹病毒载体疫苗。首先将HIV-1 gp160(B型和C型)、gag和protease基因串联克隆入pc DNA3获得重组质粒pc DNA/g Bgp和pc DNA/g Cgp,重组质粒转染293FT细胞,Western blotting检测HIV抗原表达。继而将pc DNA/g Bgp和pc DNA/g Cgp中包括HIV-1抗原基因和表达元件的表达框克隆入p KO5/BN获得重组穿梭质粒p KO5/BN/g Bgp和p KO5/BN/g Cgp,穿梭质粒电转含BAC-HSV的大肠杆菌,筛选重组菌,提取重组DNA并转染Vero细胞,挑取病毒蚀斑纯化重组病毒,Southern blotting鉴定重组病毒DNA,Western blotting检测重组病毒感染细胞中HIV抗原表达,并分析病毒的增殖特性。结果表明,Western blotting在pc DNA/g Bgp和pc DNA/g Cgp转染的293FT细胞中检测到表达的gp160和gag蛋白。p KO5/BN/g Bgp和p KO5/BN/g Cgp分别电转获得重组菌,并从重组DNA转染的Vero细胞中纯化获得重组HSV,Southern blotting检测表明重组HSV基因组发生特异性重组,重组病毒感染细胞中检测到gp120和gp41,且重组HSV保留了在哺乳动物细胞中的复制能力。本研究获得携载HIV-1 gp160、gag和protease基因的重组HSV,并保留了在哺乳动物细胞中的复制能力,可作为HIV-1复制型病毒载体疫苗。     

活载体疫苗的研究进展

免疫学与生物工程等领域取得的巨大进步,为发展基因工程疫苗提供了更多的理论和技术支持。活载体疫苗(Live Vector Vaccine,LVV),是以细菌或病毒作为外源抗原和治疗因子的载体系统,是目前最具发展潜力的基因工程疫苗之一。不同疫苗载体、抗原呈递方式和对宿主细胞免疫系统启动机制等研究的深入,开拓了活载体疫苗的实用价值。对国际上相关研究予以重点综述,以期为深入开展该领域研究提供依据。     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.     

以痘苗病毒天坛株为载体的表达单纯疱疹病毒I型糖蛋白D的重组活疫苗…

从我国分离到的一株单纯疱疹病毒Ｉ型病毒基因组中,分离出含有糖蛋白Ｄ基因的１．２ｋｂ片估,插入带有痘苗病毒天坛株ＴＫ区的持粒ｐＪＳＢ１１７５Ｐ７．５ｋ启动子下游,转染无白血病鸡胚原供细胞,获得带有ＨＳＶ－１－１６８ｇＤ基因的重组痘苗病毒。此株重组病毒在感染细胞膜上表达ＨＳＶ－１－１６８ｇＤ糖蛋白抗原,能与特异性单克隆抗体反应。     

猫疱疹病毒1型分子生物学研究进展

猫疱疹病毒1型(Feline herpesvirus-1,FHV-1)是引起猫科动物以上呼吸道感染和眼部溃疡为主要特征的猫传染性鼻气管炎的病原。该病毒在全球范围内流行,对宠物猫及虎、豹等野生猫科动物的健康造成了严重的威胁。本文介绍了FHV-1的基因组结构、编码蛋白及其生物学功能、病毒融合宿主细胞机制,以期为该病致病机制的深入研究与防控策略的制定提供参考。     

单纯疱疹病毒I型扩增子系列载体的构建

我们先前已报道了构建成一种能在ＨＳＶ ｔｓＫ株辅助下进行复制和包装,并表达β－半乳糖苷酶基因ｌａｃＺ的ＨＳＶ－１扩增子质粒ｐＨＳＬ,以及它的应用。该质料中依次含有ＨＳＶ－１复制起点ｏｒｉＳ序列及ＩＥ６８启动子、ｌａｃＺ基因、ＳＶ４０ｐｏｌｙＡ、ＨＳＶ－１包装信号‘ａ’序列和大肠杆菌质粒骨架。然而,该质粒中的报告基因ｌａｃＺ无法用简单的酶切方法卸载下来,继而装入目的基因。本研究在此基础上,新构建了一     

The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil-DNA Glycosylase

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence downstream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl β-D -thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.     

牛疱疹病毒Ⅰ型VP22和猪繁殖与呼吸综合征病毒GP5融合基因DNA疫苗的免疫效应

为了提高表达GP5的猪繁殖与呼吸综合征病毒（PRRSV）DNA疫苗的免疫效应,将具有蛋白转导功能的牛疱疹病毒1型（BHV-1）VP22基因插入到经过修饰具有更好免疫原性的PRRSV修饰型ORF5基因（ORF5M）上游,构建VP22和ORF5M融合表达的真核表达质粒pCI-VP22-ORF5M。经间接免疫荧光试验（IFA）和Westernblot检测证实体外表达后,免疫BALB/c小鼠,检测小鼠免疫后的GP5特异性ELISA抗体、抗PRRSV中和抗体和脾淋巴细胞增殖反应,并与非融合的真核表达质粒pCI-ORF5M进行比较。结果显示,融合表达VP22-GP5的DNA疫苗 pCI-VP22ORF5M诱导的体液免疫和细胞免疫反应均明显高于非融合表达的DNA疫苗pCI-ORF5M,表明蛋白转导相关蛋白BHV-1 VP22能显著增强表达GP5的PRRSV DNA 疫苗的免疫效应,有效发挥了基因免疫佐剂效应;这为研制PRRSV高效DNA疫苗奠定了基础,同时也为其它疾病的高效新型疫苗研究提供了思路。     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

Serum-free produced Bovine Herpesvirus type 1 and Bovine Parainfluenza type 3 virus vaccines are efficacious and safe

The studies described in this report were performed to determine, whether it is possible to produce live virus vaccines without serum or fractions thereof used during any cell or virus passage, thus completely serum-free. Two viruses were included in the experiments: Bovine Herpesvirus 1 (BHV-1) and Bovine Parainfluenza type 3 virus (PI3). Both viruses were found to grow to satisfactory titers, and to be stable after freeze-drying and subsequent storage at temperatures of +4 °C and −20 °C for at least one year. Moreover, a vaccine containing serum free produced BHV-1 was tested in a vaccination-challenge experiment. For comparison, a vaccine batch with BHV-1 grown in serum-containing cell culture medium was included in the study. Both vaccine preparations performed equally well and both met the strict requirements as laid down in the European Phamacopeia. Moreover, in two separate experiments the safety of serum-free produced BHV-1 and PI3 after overdose and repeated administration even in very young calves and even after four administrations has been demonstrated. This report is the first, which to our knowledge demonstrates the safety and efficacy of serum-free produced live vaccines in the target animal as well as the stability of these products. This revised version was published online in September 2006 with corrections to the Cover Date.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases Bovine Herpesvirus type-1 (BHV-1) replication in Madin-Darby Bovine Kidney (MDBK) cells in vitro

Dioxin-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental toxin of current interest. In the last years, higher levels of TCDD than those permitted in UE [European Commission. 2002. European Commission Recommendation 2002/201/CE. Official Gazette, L 67/69] were detected in milk samples from cow, water buffalo, goat, and sheep raised on some areas of Campania Region (South Italy). Dioxin often causes immunosuppression and might render the animal liable to viral infections. In addition, viral infections are able to alter the pattern of dioxin distribution in different organs of the exposed animals. Bovine Herpesvirus type-1 (BHV-1) is a widespread pathogen, which causes infectious rhinotracheitis and infectious pustular vulvovaginitis in cattle. Herein, we have studied the effects of TCDD and BHV-1 infection, in Madin-Darby Bovine Kidney (MDBK) cells, alone as well as in association, so as cellular proliferation, apoptosis, and virus replication. We have observed an increase in cell viability of confluent monolayers at low TCDD concentrations. TCDD treated cells demonstrated increased viability compared to controls as evaluated by MTT test. TCDD exposure increased cell proliferation but induced no changes on apoptosis. Cells exposed to TCDD along with BHV-1 showed a dose-dependent increase in cytopathy, represented by ample syncytia formation with the elimination of the cellular sheets and increased viral titer. These results suggest that TCDD increases viral replication in MDBK cells while BHV-1 further decreases viability of TCDD exposed cells. Since very low concentrations (0.01 pg/ml) are sufficient to augment BHV-1 titer, TCDD may contribute to reactivate BHV-1 from latency, leading to recurrent disease and increase virus transmission.     

Molecular and in vitro Characterization of Field Isolates of Bovine Herpesvirus-1

Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and to characterize (molecular and biological characterization) BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates, each from a different animal, such as from the respiratory and reproductive tracts. In some cases the cytopathic effect was visible 12 hours post-inoculation, and became characteristic after 36-48 hours. Biological characteristics were evaluated and compared with Iowa and Colorado-1 reference strains, and differences were found in plaque size, virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results showed that some isolates had a highly virulent-like behavior in vitro, compared to the reference strains, with shorter eclipse periods, faster release of virus into the supernatants, and higher burst size and viral titer. There were no differences in glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers. Moreover, using restriction endonucleases analysis, most of the viruses were confirmed as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to develop new monovalent vaccines.     

牛泡沫病毒（BSV）对牛免疫缺陷病毒（BIV）的激活作用

用瞬时表达分析等方法,证明牛泡沫病毒（ Ｂ Ｓ Ｖ）３０２６ 中国毒株能在体外激活牛免疫缺陷病毒（ Ｂ Ｉ Ｖ） 基因表达, Ｂ Ｓ Ｖ３０２６ 编码的反式激活因子 Ｂｏｒｆ１ 行使这种激活作用。缺失突变分析表明, Ｂｏｒｆ１ 在 Ｂ Ｉ Ｖ Ｌ Ｔ Ｒ 上靶序列位于－ ４１０／ － １１５（ ＋ １ 为转录起始位点） 区域,但其中的 Ｎ Ｆκ Ｂ 位点（ － ３６７／ － ３１９） 与这种激活作用无关,包括转录起点下游（ Ｒ Ｕ５ 区） 在内的－ １１５／ ＋ ２０４ 区域也与这种激活作用无关。该结果对研究 Ｂ Ｉ Ｖ 致病机理及防治 Ａ Ｉ Ｄ Ｓ 有重要意义。     

融合表达牛疱疹病毒1型VP22及猪繁殖与呼吸综合征病毒GP5重组伪狂犬病毒TK-/gE-/VP22GP5+的构建

猪繁殖与呼吸综合征(porcine reproductive andrespiratory syndrome,PRRS)和伪狂犬病(pseudora-bies,PR)是两种严重危害养猪业的重要病毒性传染病。而引发PR的伪狂犬病病毒(pseudorabiesvirus,PRV)是构建基因工程疫苗优良的活病毒载体[1]。已有报道将表达猪瘟病毒(hog cholera virus,HCV)囊膜蛋白E1基因的重组PRV(rPRV)二价基因工程疫苗免疫猪后,能同时抵抗HCV和PRV的强毒攻击[2],显示了PRV作为活病毒载体的可行性和“一针防两病”的独特优点。由ORF5基因编码的囊膜糖蛋白GP5是猪繁殖与呼吸综合征病毒(porcine reproductive …     

牛泡沫病毒中国株感染兔的研究

将牛泡沫病毒(BFV3026)感染的细胞经耳缘静脉注射兔子,并以正常细胞注射的兔为对照。1年后处死,病毒挽救实验及PCR检测显示：兔经一次注射即可被BFV3026感染,病毒广泛分布于感染兔的多种脏器中,通过共培养可从感染兔血、肝、脾、肺、肾中拯救出相应感染性病毒颗粒,并在脑、骨髓、心、胰、肠系膜中检到高拷贝BFV原病毒DNA存在。同时,血清学检测表明：感染兔在接受注射一个月后即产生高滴度抗病毒蛋白抗体,并维持该滴度水平直至实验终止,兔未表现任何可观病变。     

牛泡沫病毒中国株感染兔的研究

将牛泡沫病毒(BFV3026)感染的细胞经耳缘静脉注射兔子,并以正常细胞注射的兔为对照.1年后处死,病毒挽救实验及PCR检测显示兔经一次注射即可被BFV3026感染,病毒广泛分布于感染兔的多种脏器中,通过共培养可从感染兔血、肝、脾、肺、肾中拯救出相应感染性病毒颗粒,并在脑、骨髓、心、胰、肠系膜中检到高拷贝BFV原病毒DNA存在.同时,血清学检测表明感染兔在接受注射一个月后即产生高滴度抗病毒蛋白抗体,并维持该滴度水平直至实验终止,兔未表现任何可观病变.