乙脑病毒PrME基因在杆状病毒表达系统中的表达

本试验将乙型脑炎病毒（ＪＥＶ）的ＰｒＭ／Ｅ基因的ＨｉｎｄⅢ－ＢｇｌⅡ片段（２．１ｋｂ）插入到杆状病毒载体ｐＡｃＵＷ３１的ＢａｍＨＩ位点,使外源基因置于多角体蛋白启动予下游,构建成转移载体ｐＡｃＵＷ３１ＪＥ,以Ｌｉｐｏｆｅｃｔｉｎ作为共转染试剂,将纯化的ｐＡｃＵＷ３１ＪＥ与Ｂｓｕ３６Ｉ线性化的杆状病毒ＡｃＭＮＰＶ．ＬａｃＺＤＮＡ共转染昆虫ｓｆ９经病毒蚀斑纯化技术和Ｘ－ｇａｌ与中性红双重染色技术,随机     

杆状病毒非必需基因的结构与功能

本文主要以苜蓿丫纹夜蛾核型多角体病毒（ＡｃＭＮＰＶ）为例,概述了迄今展报道的杆状病毒非必需基因的结构与功能,为杆状病毒的基因工程研究提供参考。     

宿主域扩大的苜蓿尺蠖核多角体杂交型病毒

将家蚕核型多角体病毒 (BombyxmoriNuclearPolyhedrosisVirus,BmNPV)的解链酶基因DNA和苜蓿尺蠖核型多角体病毒 (AutographacalifonicaMultipleNuclearPolyhedrosisVirus,AcMNPV)的基因组DNA共转染到秋粘虫细胞系Sf 2 1,经过累代的筛选获得既能感染家蚕细胞 ,又能感染秋粘虫细胞的宿主域扩大的杂交型病毒HyNPV。     

Biological activity and characterization of nucleopolyhedrovirus isolates ofSpodoptera litura

Geographical isolates ofSpodoptera litura (Fabricius) (Noctuidae: Lepidoptera) nucleopolyhedrovirus (SpltNPV), collected from different parts of India and maintained at Tamil Nadu Agricultural University, were compared for their biological activity and subjected to Restriction Endonuclease (REN) analysis. Neonate and second instar bioassay studies revealed similarity in biological activity as shown by the overlapping fiducial limits of LC₅₀ values. However, there were differences in yield among isolates: significantly higher yields were obtained from isolates UAS and CBE than from the BARC isolate. REN analysis of the four isolates withPst I,Hind III,Bam HI andEco RI enzymes indicated genotypic variation among the isolates. Based on the commonality of the bands, the isolates could be broadly divided into two groups: isolates AU and CBE formed one group, and the other group comprised UAS and BARC based on genetic relatedness. http://www.phytoparasitica.org posting Nov. 21, 2005.     

Bac-to-Bac系统的研究进展及在家蚕中的应用

Bac-to-Bac策略是目前昆虫杆状病毒表达系统获取重组杆状病毒最为方便、快捷的途径之一。以Bac-to-Bac系统面向的苜蓿银纹夜蛾杆状病毒(AcMNPV)表达系统、家蚕杆状病毒(BmNPV)表达系统、棉铃虫杆状病毒(HaNPV)表达系统等3个主要子系统为对象,从宿主域逐渐扩大、蛋白的表达水平不断提高、筛选和纯化更为简便高效以及在家蚕中应用的状况等角度,对Bac-to-Bac系统的发展及功能、特点等进行了阐述。     

伪狂犬病病毒gE/gI基因在昆虫细胞中的表达及鉴定

为获得具有生物活性的伪狂犬病病毒(Pseudorabies virus,PRV)gE/gI蛋白,建立PRV抗体快速检测方法。将含有PRV gE、gI基因的质粒pFastBacdual-GP67-gE/gI转化至宿主菌,经位点特异性重组和蓝白斑筛选后获得重组杆粒rBacmid-gE/gI,转染Sf9细胞,获得重组杆状病毒。采用悬浮的Sf9细胞进行发酵并纯化产物,SDS-PAGE和Western blot分析镍柱纯化后的重组蛋白。结果显示,重组杆粒在4800 bp处克隆出预期大小的条带,表示重组杆粒构建成功。SDS-PAGE和Western blot表明,在50 ku和65 ku处出现预期大小的条带,能与PRV标准阳性血清特异性反应,不与PRV gE/gI缺失疫苗免疫血清反应。结果表明gE/gI重组蛋白具有良好的反应原性,为PRV抗体快速检测及区分PRV野毒感染和疫苗免疫奠定了基础。     

Comparison of biological activities of natural and recombinant chicken interferon-gamma

In recent years, chicken interferon-gamma (ChIFN-γ) has been identified and cloned from a chicken T cell line. In this study, recombinant ChIFN-γ produced in the baculovirus and prokaryotic (Escherichia coli) expression systems were characterized and their activity was compared to that of naturally ChIFN-γ produced by mitogen-activated splenic T cells. The baculovirus-derived ChIFN-γ protein (Bac-ChIFN-γ) proved to have physiochemical properties and biological activities similar to those of natural ChIFN-γ. Indeed, Bac-ChIFN-γ was able to inhibit the replication of cytolytic viruses in chicken embryo fibroblasts and to activate macrophages, as was determined by nitric oxide production. Levels ranging between 100 and 300 μg/ml of BacChIFN-γ could be obtained in the supernatants of infected insect cells. On the other hand, yields of the E. coli produced ChIFN-γ rarely exceeded 100 μg/ml after purification steps and although it was also able to activate the HD11 macrophage cell line in a specific manner, no anti-viral activity could be demonstrated. Therefore, the baculovirus expression system is an appropriate system for the high-level expression of biologically active ChIFN-γ and will allow further studies of the immunomodulatory and therapeutic effects of this cytokine in vivo.     

重组马传染性贫血病病毒核心蛋白抗原在 A GID 和ELISA中应用的评价

用昆虫杆状病毒表达系统获得的马传染性贫血病病毒（ Ｅ Ｉ Ａ Ｖ） 核心蛋白（ Ｇａｇ） 和 Ｐ２６ 蛋白, 作为免疫琼脂双扩散（ Ａ Ｇ Ｉ Ｄ） 和酶联免疫吸附试验（ Ｅ Ｌ Ｉ Ｓ Ａ） 抗原。对７６ 份已知马传贫非特异性血清进行检查, 同时与市售 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒作比较。证明, 用表达蛋白作抗原的 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 检测结果均为阴性反应, 而用市售 Ａ Ｇ Ｉ Ｄ 试剂盒检查有５４ 份马血清出现非特异性反应, 市售 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒检查也出现了非特异性反应, Ｏ Ｄ 值比表达抗原 Ｅ Ｌ Ｉ Ｓ Ａ 高３５ 倍。初步证明在 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 法中, 表达抗原优于常规马传贫病毒抗原。     

Detection of monodon baculovirus and white spot syndrome virus in apparently healthy Penaeus monodon postlarvae from India by polymerase chain reaction

The simultaneous presence of monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in apparently healthy postlarvae of Penaeus monodon from different hatcheries in India was studied by nested polymerase chain reaction (PCR). MBV could be detected in 54% of the samples. However, only 15% of samples were positive by non-nested reaction. WSSV could be detected in 75% of samples, 19% being positive by non-nested reaction. The results show simultaneous presence of WSSV and MBV in many samples at various degrees of infection. Only 14% of the samples analysed were negative for both viruses.     

猪瘟病毒石门株E2蛋白在杆状病毒系统中的表达

以杆状病毒Bac-to-Bac表达系统表达猪瘟病毒(CSFV)石门株的E2蛋白,将CSFV石门株的E2囊膜糖蛋白基因亚克隆到杆状病毒转移载体pFastBacHTA中,获得重组转移质粒pFastBacHTA-E2。转化大肠埃希菌DH10Bac感受态细胞,获得重组Bacmid质粒后转染Sf9昆虫细胞,获得重组病毒。传毒3代后对表达蛋白进行Western blot和间接免疫荧光试验检测。结果显示,E2蛋白获得高效表达,能被抗E2蛋白的单克隆抗体2B10和6×His-单克隆抗体特异性识别,表明CSFV石门株E2囊膜糖蛋白在Sf9昆虫细胞中得到成功表达,具有良好的抗原反应性。