A practical and cost-effective mutation scanning-based approach for investigating genetic variation in Cryptosporidium

In the present study, we used a mutation scanning-targeted sequencing approach to assess variation in part (pgp60) of the 60 kDa glycoprotein (gp60) gene among Cryptosporidium samples from humans in Victoria, Australia. Two nuclear ribosomal loci (the small subunit rRNA gene and the second internal transcribed spacer) were used to identify the samples as Cryptosporidium hominis (n = 74), Cryptosporidium parvum (n = 23) or Cryptosporidium meleagridis (n = 1). In total, nine distinct pgp60 sequences were identified (three C. hominis, five C. parvum and one C. meleagridis). Phylogenetic analyses of the pgp60 sequence data, employing well-defined reference sequences for comparison, allowed the genotypic and subgenotypic classification of samples. The C. hominis samples were classified as Ib A10G2R2, Id A15G1R2, and a new genotype, designated Ib2, was identified subgenotypically as A18G1R4. The C. parvum samples were classified as IIa A18G3R1, IIa A20G3R1, IIa A22G3R1, IIa A23G3R1 and IIc A5G3R2. These findings suggested that the C. hominis metapopulation is largely homogeneous, consisting of a single dominant genotype, Ib A10G2R2, whereas the C. parvum metapopulation is considerably more heterogeneous, with no single dominant genotype. The greater level of genetic heterogeneity found among the C. parvum samples, despite the smaller sample size, may relate to the zoonotic infection pattern of this species, which would be reflective of a greater number of possible infection sources. The present mutation scanning approach, coupled with targeted sequencing of genetically distinct representatives, is a practical, cost-effective tool for large-scale population genetic and epidemiological studies of Cryptosporidium and other eukaryotic organisms.     

Isolation and characterization of a new subunit of phycocyanin from Chroomonas placoidea

A new phycocyanin（PC） fluorescent subunit namedβ₂（18kDa） was isolated and characterized by both SDS-PAGE and isoelectric focusing（IEF） from a species of cryptophytic alga Chroomonas placoidea.PC was separated and purified by ammonium sulfate sedimentation followed by two steps of Sephadex G-100 chromatography.After denatured in 4 mol/L urea for 48 h,PC was divided into two fractions by passing through a Sephacryl S-100 chromatography column twice.The blue fraction（S-1） containedβsubunits with a maximal absorbance at 595 nm in visible light region.While the green fraction（S-2） enriched inαsubunits showed a characteristic long wavelength absorbance at 680-700 nm region and exhibited a relatively low molecular weight of 9.4（α₁） and 8.5 kDa（α₂）.Fraction S-1 also consisted of two different fluorescent subunits with molecular weight of 20.1 kDa（β₁） and 18 kDa （β₂） and differed from each other on isoelectric points of pH 5.7（ft） and 6.0（ft）,respectively.Further investigation of peptide sequence will help a lot in elucidating the new subunit ft that was smaller in size and more neutral than the known ft subunit,and may provide an alternative explanation in structure of cryptophytic phycobiliproteins.     

An Optical Biosensing System Based on Interference-Enhanced Reflection with Aptameric Enzyme Subunits of Thrombin

The aptameric enzyme subunit (AES) is an artificial enzyme subunit that can allosterically control partner enzyme activity. By means of an AES, target molecules can be detected by measurement of enzymatic activity in a homogeneous solution. We have developed a thrombin aptamer-based AES that can detect several targets by measuring clotting time in a fibrinogen solution. Measurement of the clotting activity in the fibrinogen solution is not suitable for a convenient biosensor. However, kinetics measurement of clotting activity is suitable for convenient detection of thrombin activity. The interference-enhanced reflection (IER) method is a simple, real-time technique to detect the thickness and/or refractive index of a thin film formed on a glass substrate surface. We demonstrated that clotting activity on a glass substrate with immobilized thrombin can be monitored in real time by IER. By means of the IER method, we were able to detect the target molecules of an AES. IER-based sensors have already been commercialized as portable sensors for the detection of organic compounds. Thus, portable biosensors could be developed by combination of the IER and AES technologies.     

Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.     

Expression of CTB-human Insulin(BA) Fusion Protein in Gynostemma Pentapyhllum Makino Callus Cells and Its Hypoglycemic Effect in Mice

The plant expression vector of choleratoxin B subunit(CTB)-human insulin(BA) fusion protein pBI121/(CTB-BA) was constructed first and then the Gynostemma Pentapyhllum Makino callus cell line that could express CTB-human insulin fusion protein was constructed and its hypoglycemic effect was evaluated in mice. The plant expression vector pBI121/(CTB-BA) was digested with both BamI and SacI. Agrobacterium tumerfaciens strain LBA4404 was transformed with previously constructed recombinant plasmid pBI121/(CTB-BA) via the freeze tha-wing method, then CTB-BA gene was integrated to G Pentapyhllum Makino callus cells by co-culturing the cells with the transformed LBA4404 strain. The transformed G Pentapyhllum Makino callus cells were identified by DNA se-quence assey and RT-PCR. The expressed product was identified by western-blot and its amount was tested by ELISA kit and its blood sugar decreasing effect was tested in mice.The sequences of synthetic CTB and human insulin genes(BA) were completely identical to those designed. Restriction map proved that the length of gene fragment in-serted into expression vector pBI121 was consistent with that expected. The sequence of genomic DNA of expressed product was completely identical to that designed. The result of RT-PCR was consistent with that expected. The ex-pressed product showed a specific band with a relative molecular mass of 17000 by Western-blot. The human insulin expression amount was 6.03 μIU/mL according to the ELISA result.The animal test showed that only the G Penta-pyhllum Makino callus cell line itself showed activity in decreasing the blood sugar of mice, however, the activity of the transformed G Pentapyhllum Makino callus cells was much higher. The plant expression vector pBI121/(CTB-BA) was constructed and expressed in the G Pentapyhllum Makino callus cells successfully for the first time. The trans-formed G Pentapyhllum Makino callus cells showed high activity in decreasing the blood sugar of mice. This study developed a new way for the development of oral administration insulin.     

Expression of Shiga Toxin B Subunit at Cell Surface in E. coli K-12

The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.     

用液相等电聚焦电泳纯化藻蓝蛋白亚基

以纯藻蓝蛋白(C-phycocyanin, C-PC)为材料, 采用Rotofor系统进行液相等电聚焦(Liquid-phase isoeletric focusing, LP-IEF)电泳纯化C-PC的α, β亚基, 探讨蛋白质亚基纯化的制备电泳(Preparative eletrophoresis)技术. 结果显示, 样品经2次等电聚焦电泳后, C-PC 的α, β亚基分别浓集在pH=4.9和pH=4.1附近, 平板超薄等电聚焦(Slab ultra thin IEF)和SDS-PAGE电泳鉴定表明分别为高纯度的C-PC α, β亚基. 提示LP-IEF是分离纯化等电点差异蛋白质活性亚基的简便有效的方法.     

Lateral gene transfer in phylogeny of azoreductase enzyme

This paper attempts to reconstruct the phylogeny of azoreductase enzyme from different organisms and compare it with the small subunit rRNA-based phylogeny of the organisms. The two phylogenies were found to be incongruent, indicating several events of lateral transfer of azoreductase gene between phylogenetically diverse organisms. However, the phylogenetic analysis methods have several limitations and a single method may not give the true pattern. Hence, it is necessary to corroborate the results with other complementary analysis tools. We used several tools to test our hypothesis of lateral transfer and found that it was supported not only by the analysis of the whole sequences, but also by the conserved motifs detected in these sequences. There were ample evidences for lateral transfer of azoreductase gene among enteric bacteria. There were also indications that azoreductase probably evolved in prokaryotes and then it was laterally transferred to eukaryotes in multiple events, resulting in some sequence variation among eukaryotic azoreductases. Finally, profile HMMs and conserved motifs extracted from these azoreductase sequences were found to provide sensitive tools for identifying potential azoreductases from the database. The analysis techniques used in this study can be extended to other gene trees to verify their evolutionary histories.