[Damage analyse of X-ray radiated cellobiohydrolase II molecule from Trichoderma viride].

After X-ray treatment at 1.82 keV and 40 mA and 4 hours, the cellobiohydrolase II (CBH II) aqueous solution of Trichoderma viride was analysed, and the damage state of the irradiated molecule was detected using some cysteine residue relative parameters in Raman spectroscopic methods. The results show that S-H stretch modes of CBH II exhibited some shift, which means that the hydrogen proton donor state of sulfhydryl groups was stronger and weaker, respectively. The 2 554 cm(-1) peak of irradiated sample was wide. The -S-S- construction of disulfide bonds was not broken, and the geometrical conformation types did not change either, but its bond length was somewhat shortened. Before irradiation, C-S isomer mode content of cysteine residue was Pc type slightly more than both P(N) and P(H) types, while P(N) and P(H) types increased after irradiation. Besides, CH2 rocking mode of cysteine residue was weakened remarkably after the treatement, and the protein molecule structure did not show important damage in sulfhydryl and disulfide bonds, but showed some change because of the X-ray irradiation condition.

Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety.

Bacterial superantigens (SAg) are the most potent activators of human T lymphocytes and recombinant immunotoxin using bacterial SAg shows promising clinical values. To engineer superantigen for immunotherapy of hepatocellular carcinoma, we genetically fused the superantigen staphylococcus enterotoxin A (SEA(D(227)A)) to the single-chain disulfide-stabilized Fv (scdsFv) of anti-hepatoma monoclonal antibody HAb25 through a short peptide GGGSGGS. We expressed this recombinant protein in Escherichia coli and extract it from inclusion bodies. We found purified scdsFv-targeted SAg contains equivalent binding affinity with disulfide-stabilized Fv (dsFv) targeted SAg and single-chain Fvs (scFv) targeted SAg, but more stable and more suitable for large scale production. The MTS(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, inner salt) assay shows that the scdsFv-targeted SAg also shares the ability to activate a large number of T lymphocytes and has cytotoxic activity on human hepatoma cell line SMMC-7721. Therefore, this novel generation of recombinant immunotoxins using scdsFv has a high potential in hepato cancer treatment and the same strategy may also be applied to other cancer treatments.

[Hydrogen-bond state analysis of cellobiohydrolase I molecule from Trichoderma viride].

Hydrogen-bond states on cellobiohydrolase II (CBH II) of Trichoderma viride were analysed by laser Raman spectroscopic method used to reveal molecular normal modes of vibration character. The results indicated that hydrogen-bond ability of carbonyl oxygen-atom of amide I was raised in two types of aqueous solution samples (6.0 and 8.0 of pH value) compared to the solid sample. The variation trend of beta structure modes was similar between amide II and amide I. As far as the ability of hydrogen-bond forming is concerned, tryptophan (Trp) and tyrosine (Typ) residues are strong hydrogen proton donor in pH 6.0 and solid sample, thus proving the results of previously spatial structure analysis. By analysis of free S-H character, the -S-S- construction in Trichoderma viride mature peptide has been proved to be the same as that of Trichoderma reesei.

[Effects of Corbicula fluminea bioturbation on the community composition and abundance of ammonia-oxidizing archaea and bacteria in surface sediments].

To better understand the effects of Corbicula fluminea bioturbation on the ammonia-oxidizing microorganisms in the surface sediment, sediment-water microcosms with different densities of Corbicula fluminea were constructed. Clone libraries and real-time qPCR were applied to analyze the community composition and abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in the surface sediments. The results obtained indicated that the bioturbation of Corbicula fluminea accelerated the release of nitrogen from the surface sediment. In the amoA gene clone libraries, the identified AOA amoA gene sequences affiliated with the two known clusters (marine and soil clusters). The identified AOB amoA gene sequences mostly belonged to the Nitrosomonas of beta-Proteobacteria. The abundance of the bacterial amoA gene was higher than that of the archaeal amoA gene in all treatments. With increasing density of Corbicula fluminea, decreased abundances of the bacterial amoA gene were observed. At the same time, the diversity of AOA and AOB reduced in the Corbicula fluminea containing microcosms. In conclusion, the bioturbation of Corbicula fluminea could affected the community composition and abundance of ammonia-oxidizing microorganisms in surface sediments.

[Distribution and diversity of Legionella spp. in Lake Taihu in the winter].

The Legionella spp. are ubiquitous in aquatic environment and could cause certain risks on human health. In order to investigate the distribution and diversity of Legionella spp. in Lake Taihu during winter time, water samples were collected from 32 sites of the whole lake in February 2010. The presence of Legionella spp. was screened by nested-PCR and their phylogenetic diversity was determined by denaturing gradient gel electrophorese (DGGE) and sequencing analysis of excised DGGE bands. The Legionella spp. was detected from 21 out of 32 sites in particle-associated bacterial samples and 11 out of 32 sites in free-living bacterial samples, which accounted for 65.63% and 34.38% of each type of bacteria, respectively. In total, 40 and 36 unique bands were identified among those particle-associated and free-living bacterial DGGE profiles, respectively. Community characteristic indices of different euthrophic areas showed that the diversity of Legionella spp. in mild eutrophic areas was higher than that in the moderate eutrophic areas. In total, thirty-four DGGE bands were excised and sequenced, which could be classified into 12 OTUs by 97% similarity. Among all the sequences, three showed a high similarity with two pathogenic Legionella species Legionella feeleii and Legionella longbeachae. This revealed potential healthy risks to the people lived around the Lake Taihu.

[Expression, purification, and characterization of single-chain disulfide-bond Fv (ScdsFv) antibody fused with targeted superantigen SEA (D227A)].

AIM: To express, purify, and characterize scdsFv antibody fused with superantigen SEA(D227A). METHODS: The expression plasmid of scdsFv-SEA(D227A) was constructed by standard molecular cloning procedures. The recombinant protein was induced to express in E. coli BL21plusS by IPTG and purified by Q Sepharose HP column and Hiprep 26/60 Sephacryl S-200 HR column. Formation of the intramolecular disulfide bond of the purified protein was analysed by AMS alkylation and PAGE electrophoresis. The binding activity, stability and killing activity of the purified protein were assayed by ELISA and MTS, respectively. RESULTS: The recombinant protein was expressed as inclusion body, accounting for more than 30% of total bacterial protein. After purification by Q Sepharose HP and Hiprep 26/60 Sephacryl S-200 HR, the yield of the purified protein was 60 mg per liter of induced culture. AMS alkylation and PAGE electrophoresis analysis showed that intramolecular disulfide bond formed correctly in the recombinant protein. The purified protein had similar binding affinity as dsFv fused SEA and scFv fused SEA have and similar killing activity as native SEA has to human hepatoma cell line, but more stable, in vitro, as compared with dsFv fused SEA and scFv fused with SEA. CONCLUSION: The scdsFv fused with SEA, as a novel form of immunotoxin, might be used in cancer treatment.