Identification of Novel Residues Involved in Nuclear Localization of a Baculovirus Polyhedrin Protein

A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin.     

Genome Organization of Xestia c-nigrum Granulovirus

In order to characterize the genome organization of Xestia c-nigrum granulovirus (XcGV), mapping of putative XcGV genes was performed by construction of lambda and M13 phage libraries followed by Southern blot and nucleotide sequencing analyses. Mapping of the lambda (32 clones covering the entire XcGV genome) and M13 (133 clones made by random cloning) phage library clones was carried out by hybridization of the labeled lambda phage clone DNAs to 1) Southern blotted XcGV genomic DNA fragments cleaved with EcoRI, BamHI, or HindIII, and 2) dot blotted M13 clone DNAs. All 133 M13 clone DNAs were sequenced, and coding possibilities were investigated by computer-assisted homology search; in total, about 43 kb of the genome was sequenced. Amino acid sequence homology searches of 67 M13 clones suggested that these GV DNAs coded for previously characterized genes identified in nucleopolyhedroviruses (NPVs) and GVs. These 67 M13 clones were classified into 25 gene homolog groups (including 29 putative genes) based on their homologies to NPV and GV genes. The remaining M13 clones, except one that encoded a putative metalloproteinase, did not possess deduced amino acid sequences with significant homology to proteins in gene databases. Complete nucleotide sequences of the putative XcGV DNA polymerase and Ac144 homolog genes confirmed the reliability of our speculation of putative genes based on the M13 clones sequencing analysis. In a comparison of relative locations of putative XcGV genes with locations of their homologs in NPVs, most XcGV genes were mapped close to the corresponding locations in NPV genomes. These results suggested that XcGV, compared to NPVs, had relatively conserved gene arrangements, although about 22 kb of 43 kb of DNA sequenced randomly in the XcGV genome consisted of sequences/genes non-homologous to those of previously characterized NPVs. This revised version was published online in August 2006 with corrections to the Cover Date.     

抗癌胚抗原单链抗体基因与Mn-SOD基因的融合及在昆虫细胞中的表达

将抗癌胚抗原单链抗体基因与锰 超氧化物歧化酶基因融合 (ScFv Mn SOD )插入昆虫杆状病毒供体质粒pFastBacHTb中 ,经大肠杆菌DH1 0Bac体内转座 ,产生重组杆状病毒pBacHTb Mn SOD ScFv。将其转染粉纹夜蛾Tn 5B1 4细胞 ,经扩增后在细胞内进行表达。SDS PAGE分析结果表明 ,融合基因得到高效表达 ,其表达产物相对分子质量为 40 0 0 0单体和 1 60 0 0 0左右的四聚体。Western印迹分析结果 ,以 6×His单克隆抗体为一抗进行蛋白印迹在相对分子质量 40 0 0 0单体和 1 60 0 0 0四聚体处可见表达条带 ,放射免疫分析表明重组杆状病毒表达ScFv Mn SOD融合蛋白能与CEA有较高的结合力 ,并且此融合蛋白具有特异SOD酶活性 ,酶比活可达 32 6 5u/mg。     

Generation of a Recombinant Anticarsia Gemmatalis Multicapsid Nucleopolyhedrovirus Expressing a Foreign Gene under the Control of a Very Late Promoter

A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing -galactosidase under the control of the polyhedrin promoter was generated in our laboratory. To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions. Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh ^–/LacZ ⁺ plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. The kinetics and levels of expression of -galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.     

Soluble thymic stromal lymphopoietin receptors are absent in murine sera--detection with anti-mTSLPR monoclonal antibodies

Two monoclonal antibodies, termed nnIE11 and nnIG11, were generated against the murine thymic stromal lymphopoietin receptor, mTSLPR, using traditional hybridoma technology. The antibody-producing hybridoma clones were obtained by fusing P3X63-Ag8.653 myeloma cells with splenocytes from Balb/c mice immunized with anti-FLAG M2 affinity-purified FLAG-tagged mTSLPR from pSVL-mTSLPR-FLAG-transfected COS cells and Ni-NTA-purified his-tagged mTSLPR from recombinant FastBacHisB-mdelta1 baculovirus-infected Sf9 cells. Several monoclonal anti-mTSLPR-specific hybridoma clones were obtained and two of these clones are further characterized here. The generated antibodies could in an immunoblotting identify baculovirus-expressed mTSLPR proteins with a molecular weight corresponding to 50 kDa. Both immunoblotting and ELISA with recombinant mouse TSLPR/Fc chimera as antigen, having only the N-terminal domain of mTSLPR present, indicated that the generated monoclonal antibodies identify the C-terminus of mTSLPR. Although sandwich ELISAs performed with a goat anti-mTSLPR antiserum as capture antibody and nnIE11 as indicator antibody were able to detect mTSLPR in the range of 5 ng/ml, no souble mTSLPR could be observed in serum samples from CBA/H, Balb/c and C57Bl/6 mice.     

Seroepidemiology of human group C rotavirus in the UK

The gene coding for the major inner capsid protein VP6 of human group C rotavirus was cloned into baculovirus using the pBlueBac2^TM vector and expressed in insect cells. When cultured in High Five^TM cells, VP6 was expressed at a high level and exported to the cell culture medium. Purified VP6 was used to immunise rabbits. Hyperimmune rabbit serum, which reacted with native human group C rotavirus in infected cells, was used to develop and optimise an EIA for the detection of antibodies to group C rotavirus using the recombinant VP6 as a source of antigen. In a local epidemiological survey of 1000 sera grouped by age, an average of 43% of samples were found to have antibodies to human group C rotavirus with the highest proportion (66%) in the 71–75 year age group. In comparison, 97% of adults and 85% of children had antibodies to recombinant VP6 from the bovine RF strain of group A rotavirus. These results suggest that infection with human group C rotavirus is a common occurrence despite the apparent rarity of reports of human group C rotavirus in clinical samples from patients with gastroenteritis. J. Med. Virol. 52:86–91, 1997. © 1997 Wiley-Liss, Inc.     

大肠癌新型CEA疫苗重组杆状病毒的表达与鉴定

目的利用Bac-to-Bac杆状病毒表达系统,构建新型CEA融合表位疫苗(以HPV16L1为蛋白载体)的重组杆状病毒.方法构建含有HPV16L1片段和CEA-PADRE合成片段的表达载体pFastBacl-Ⅴ,转化DH1OBac感受态菌.利用其含有的细菌Tn7转座系统,将该基因重组至杆状病毒穿梭质粒bacmid中,转染昆虫细胞sf9,获得新型CEA疫苗的重组杆状病毒.结果限制性内切酶酶切和DNA序列分析表明目的片断正确克隆到杆状病毒转移载体pFastBac1中;重组杆状病毒感染昆虫细胞后,PCR检测可以扩增出相应大小的片段.结论利用杆状病毒表达系统,成功构建了CEA融合表位疫苗的重组杆状病毒,为进一步的研究奠定了基础.     

Global Protein Synthesis Shutdown in Autographa californica Nucleopolyhedrovirus-Infected Ld652Y Cells Is Rescued by tRNA from Uninfected Cells

Global protein synthesis arrest occurs in Autographa californica nucleopolyhedrovirus (AcNPV)-infected Ld652Y cells at late times postinfection (p.i.). A Lymantria dispar nucleopolyhedrovirus gene, hrf-1, precludes this protein synthesis arrest. We used in vitro translation assays to characterize the translation defect. Cell-free lysates prepared from uninfected Ld652Y cells, AcNPV-infected cells harvested at early times p.i., and cells infected with vAchrf-1, a recombinant AcNPV bearing hrf-1, all supported translation. Lysates prepared from AcNPV-infected Ld652Y cells at late times p.i. did not support translation, but activity was restored by adding small RNA species from mock-, vAchrf-1- (24 or 48 h p.i.), and AcNPV- (6 h p.i.) infected cells. Small RNA species (24 and 48 h p.i.) from AcNPV-infected cells did not rescue translation. Assays of RNA species further fractionated by ion exchange chromatography demonstrated that tRNA rescued translation. Although specific defective tRNA species were not revealed by comparative two-dimensional gel analysis, analysis of ³²P-labeled tRNAs showed a reduction in de novo synthesis of small RNA isolated from AcNPV-infected cells compared with mock- and vAchrf-1-infected cells. This study suggests a mechanism of translation arrest involving defective or depleted tRNA species in AcNPV-infected Ld652Y cells.