肠三叶因子在大肠杆菌中的表达、纯化及其抗体制备

目的:获得肠三叶因子(ITF)的原核表达产物及抗rITF抗体,为深入研究ITF的作用机制及其受体研究奠定基础。方法:常规提取人小肠组织总RNA,用RT-PCR获得ITF编码基因片段,克隆至质粒pET32a获得原核表达栽体,双酶切和测序后转化至Origami B(DE3)用IPTG诱导表达,优化条件获得最大表达产量;用SDS-PAGE、Western blot鉴定表达产物,亲和层析纯化获得的重组蛋白rITF皮下多点注射家兔,制备多克隆抗体,并用此抗体进行大鼠肠组织免疫组化研究。结果:测序证实PCR扩增获得ITF全长基因序列与基因文库中的完全一致,将该基因片段正确插入表达载体pET32a中、优化表达条件后,重组蛋白的表达量达到50mg/L;Western blot证明重组蛋白具有良好的抗原性和特异性;通过Ni-NTA亲和层析、超滤离心后,得到90%纯度的蛋白;收集兔血清,纯化后获得特异性良好的ITF抗体,免疫组化染色肠组织显示ITF表达的部位定位于杯状细胞。结论:成功构建了表达载体pET32a-ITF,在大肠杆菌中表达并纯化获得纯度较高的rITF,并获得了生物活性较高的ITF抗体,ITF主要在肠道杯状细胞分泌表达。     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.     

Proteome analysis of Rickettsia felis highlights the expression profile of intracellular bacteria

The proteome of Rickettsia felis, an obligate intracellular bacterium responsible for spotted fever, was analyzed using two complementary proteomic approaches: 2-DE coupled with MALDI-TOF, and SDS-PAGE with nanoLC-MS/MS. This strategy allowed identification of 165 proteins and helped to answer some questions raised by the genome sequence of this bacterium. We successfully identified potential virulence factors including two putative adhesins, four proteins of the type IV secretion system, four Sca autotransporters, four components of ABC transporters, some R. felis-specific proteins, and one antitoxin of the toxin-antitoxin system. Notably, the antitoxin was the first to be identified in intracellular bacteria. Only one protein containing rickettsia palindromic repeats was found, whereas none of the split genes, transposases, or tetratricopeptide/ankyrin repeats were detectably expressed. Comparison of the protein expression profiles of R. felis and 23 other bacterial species according to functional categories showed that intracellular bacteria express more proteins related to translation, especially ribosomal proteins. However, the remaining bacteria express more proteins related to energy production and carbohydrate/amino acid metabolism. In conclusion, this study reveals R. felis virulence factor expression and highlights the unique protein expression profile of intracellular bacteria.     

Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFβ, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

融和蛋白GST-SUMO-MT在大肠杆菌中的表达、纯化及其活性研究

将编码融合蛋白GST-SUMO-MT的DNA片段连接到大肠杆菌表达载体pET-28a中,构建重组表达质粒pET-GS-MT并转化到大肠杆菌Origami（DE3）中。20℃,1mM的IPTG诱导20h后,获得分子量约为43Kd的融合蛋白,表达量占菌体上清总蛋白的38.4％。利用谷胱甘肽交联琼脂糖（Glutathione Sepharose 4B）凝胶柱和Sephardex G-25分子筛联用可以得到纯度为95%以上的融合蛋白,得率约为70mg/L。该融合蛋白可与GST抗体产生阳性反应。融合蛋白GST-SUMO-MT可以显著提高宿主对Cd2+、Zn2+和Cu2+离子聚积的能力,其耐受能力比对照组分别提高4.2倍、4倍及1.6倍。此外,原子吸收光谱法测定,每分子GST-SUMO-MT可以结合2-3个Cd2+离子。     

A patched1 gene homologue participates in female differentiation of Cynoglossus semilaevis

Patched (Ptch) is a receptor in the hedgehog signaling pathway, essential for animal development. Our previous study showed that ptch1 gene participates in the maintenance of the male germline and spermatogenesis in Cynoglossus semilaevis (csptch1). In this study, we identified a patched1 gene homolog (csptch1 x1). The csptch1 x1 gene is 5761 bp long, with a 4638 bp coding sequence that encodes 1545 amino acids. The Csptch1 x1 protein has 12 transmembrane regions and sterol-sensing domains and is highly homologous to the csptch1 (91 amino acids difference). Expression pattern analysis showed that csptch1 x1 is expressed in eight different tissues of adult tongue sole, and the expression is significantly higher in tissues of female than that in male tissues. The expression pattern in developmental stages was also analyzed. csptch1 x1 could be detected at the 1-cell stage and was highly expressed at the blastocyst, somite, and blastopore closing stages, implying that it participates in cell differentiation. In ovarian development, the expression of csptch1 x1 was initiated at 20 days after hatching (dah) and was significantly high at 35–50 and 95–150 dah. In situ hybridization showed that csptch1 x1 was predominantly expressed in primordial germ cells, oocytes, and follicular cells, but the expression of the gene was lower in the testis. These results suggest that csptch1 x1 may be mainly involved in female differentiation and ovarian development, different from the role of csptch1 in spermatogenesis.     

ezQTL: A Web Platform for Interactive Visualization and Colocalization of QTLs and GWAS Loci

Genome-wide association studies (GWAS) have identified thousands of genomic loci associated with complex diseases and traits, including cancer. The vast majority of common trait-associated variants identified via GWAS fall in non-coding regions of the genome, posing a challenge in elucidating the causal variants, genes, and mechanisms involved. Expression quantitative trait locus (eQTL) and other molecular QTL studies have been valuable resources in identifying candidate causal genes from GWAS loci through statistical colocalization methods. While QTL colocalization is becoming a standard analysis in post-GWAS investigation, an easy web tool for users to perform formal colocalization analyses with either user-provided or public GWAS and eQTL datasets has been lacking. Here, we present ezQTL, a web-based bioinformatic application to interactively visualize and analyze genetic association data such as GWAS loci and molecular QTLs under different linkage disequilibrium (LD) patterns (1000 Genomes Project, UK Biobank, or user-provided data). This application allows users to perform data quality control for variants matched between different datasets, LD visualization, and two-trait colocalization analyses using two state-of-the-art methodologies (eCAVIAR and HyPrColoc), including batch processing. ezQTL is a free and publicly available cross-platform web tool, which can be accessed online at https://analysistools.cancer.gov/ezqtl.     

Gene microarray analysis of expression profiles in Suberoyllanilide hyroxamic acid-treated Dendritic cells

Objective

The purpose of this study is to provide a further theoretical basis for the role of Suberoyllanilide hyroxamic acid (SAHA) affect on Dendritic cells (DCs).