[Construction and activities of suilysin mutants].

AIM: To construct the suilysin mutant without hemolytic activity and evaluate its functions. METHODS: The proline in 353 site of suilysin was site-directed mutated to alanine, leucine and valine, respectively. The recombinant mutants were renaturated and purified by immobilized metal ion affinity chromatography, and the purified proteins were evaluated in the hemolytic activity and immunogenicity. RESULTS: We obtained three mutants, SLY(P353A), SLY(P353L) and SLY(P353V). The SLY(P353V) mutant had non-hemolytic activity. Western blotting and animal experiments showed that SLY(P353V) mutant still had immunogenicity. CONCLUSION: Suilysin mutant SLY(P353V) has no hemolytic activity but remains immunogenicity.

[Expression, purification and activity assay of Streptococcus suis serotype 2 cell wall protein SSU1664].

AIM: To amplify the SSU1664 gene from Streptococcus suis serotype 2 strain 05ZY, to express the gene in E.coli, and to evaluate the activities of the recombinant protein. METHODS: SSU1664 gene was amplified by PCR using primers according to 05ZY genome sequences and cloned into the expression vector. The recombinant protein was purified by affinity chromatography and its immunogen activities were tested by Western blot and ELISA. RESULTS: SSU1664 gene could solublely express in E.coli BL21(DE3). Western blot analysis showed that the recombinant protein could react with rat serum immunized with Streptococcus suis, but not with non-immunized rat serum. ELISA assay showed that anti-SSU1664 IgM content in Streptococcus suis-infected patient was significantly higher than that in healthy donors. CONCLUSION: The recombinant SSU1664 protein has immunogen activity and might be one promising Streptococcus suis vaccine candidate and diagnosis marker of Streptococcus suis early infection.

[Purification and biological activities analysis of streptococcus suis Serotype 2 suilysin].

AIM: To explore the purification methods of wild-type and recombinant suilysin and to evaluate their biological activities. METHODS: Wild-type suilysin was purified by ammonium sulfate precipitation, anion-exchange chromatography and hydrophobic chromatography in turn, while recombinant suilysin was first refolded and purified by immobilized metal ion affinity chromatography, and further purified by Thiopropyl Sepharose 6B. The biological activities were evaluated by hemolysis test, cytotoxicity assay. RESULTS: Both prepared wild-type and recombinant suilysin, with purify over 90%, have hemolysis activity and could injure target cells at high concentration while cholesterol could completely inhibit their activities. CONCLUSION: Recombinant suilysin has similar biological activities with wild-type suilysin, and this work contributed to further study the functions of suilysin on pathogenesis of steptococcus suis.

In vivo and in vitro antitumor effects of a staphylococcal enterotoxin A mutant (SEA-H61D).

In this study, we evaluated SEA-H61D, a staphylococcal enterotoxin A mutant without emetic activity, as an antitumor agent in vitro and in vivo. It showed that SEA-H61D could significantly inhibit the growth of many cancer cell lines in vitro at very low concentrations by activating human peripheral blood mononuclear cells (PBMCs). CD4+ and CD8+ T lymphocytes could be activated at a dose between 125 and 500 µg/kg. Systemic administration of SEA-H61D in vivo significantly inhibited tumor growth, with the treated group undergoing tumor necrosis and showing a strong infiltration of lymphocytes to the tumor area.

Development of colloidal gold-based immunochromatographic assay for rapid detection of Streptococcus suis serotype 2.

To develop colloidal gold immunochromatographic strips for the direct detection of the Streptococcus suis serotype 2 antigen, colloidal gold was prepared by reduction of a gold salt with sodium citrate and coupled with polyclonal antibody against S. suis serotype 2. The optimal concentrations of the capture antibody and the coating antibody were determined to be 22mug/mL and 2.0mg/mL, respectively, and that of the blocking buffer was determined to be 1.5% bovine serum albumin. Different serotypes of S. suis and other related bacteria were used to evaluate the sensitivity, specificity, and stability of the immunochromatographic strips. The detection sensitivity was found to be as high as 10(6)CFU/mL. There was no cross-reaction of the antibodies with other serotypes of S. suis (except with SS1/2, which shares some common sugar residues or antigenic determinants with serotype 2) and other related bacteria. In conclusion, we developed colloidal gold immunochromatographic strips that had high sensitivity and specificity. This method proved to be feasible, convenient, rapid, and effective for detecting S. suis serotype 2.

Targeting of hepatoma cell and suppression of tumor growth by a novel 12mer peptide fused to superantigen TSST-1.

Hepatocellular carcinoma (HCC), one of the most common and malignant tumors worldwide, is unresponsive to any of the available therapies. Using intact HCC cells as therapeutic targets, we isolated a novel peptide, denoted HCC79 (KSLSRHDHIHHH), from a phage display peptide library. HCC79 can bind to hepatoma cell membranes with high affinity and specificity. Remarkably, competitive binding assays demonstrated that HCC79 competed with HAb25, a specific antibody for HCC, in binding to hepatoma cells. The corresponding synthetic peptide did not inhibit tumor proliferation directly, but repressed tumor invasion significantly in a cell migration assay. Moreover, we explored the potential of the selected peptide to deliver a superantigen (SAg) to cancer cells, to attain a significant cell-targeting effect. When the peptide is fused to the TSST-1 SAg, the resulting fusion protein could bind to hepatoma cells with high affinity in vitro and improved the tumor inhibition effect by activating T lymphocyte cells in vitro and in vivo, compared with TSST-1 alone. Taken together, our results indicate that this peptide and its future derivatives may have the potential to be developed into highly specific therapeutic agents against cancer.

Epidemiological analysis of group A streptococci recovered from patients in China.

Since the mid-1980s, there has been a resurgence of severe forms of invasive group A streptococcal (GAS) disease in many countries and regions. However, there has not been any systemic epidemiologic analysis of GAS disease reported in mainland China. To analyse the molecular epidemiology of GAS disease, 86 strains from patients in different regions of mainland China were collected. The collection sites included blood, pus, wounds, the epipharynx and other sites. A total of 21 different emm types were identified in the isolates. In both invasive and non-invasive isolates, M1 (29.1%) and M12 (23.3%) were the most prevalent types, a different distribution to M type distributions reported in other countries. Furthermore, minor emm gene sequence alterations were noted for six types. Several important GAS virulence factors were detected by PCR using specific primers. The speB and slo genes were detected in all isolates and were species specific. Four superantigen genes, speA, speC, smeZ and ssa, were found in 52% (45/86), 51% (44/86), 82% (71/86) and 23% (27/86) of isolates, respectively. M1 isolates harboured more speA (84%) and fewer speC genes (44%), while M12 isolates had fewer speA (35%) and more speC genes (100%). There was also an association between some virulence genes and isolation sites, perhaps due to the correlation between the emm type distribution and virulence gene occurrence. For two important virulence genes related to necrotizing fasciitis, the sil gene was only carried by 11 of 86 isolates, and no sil gene contained the start codon ATA. The sla gene rarely occurred in GAS isolates, only four of 86 GAS strains being positive, including two isolates obtained from blood. In antimicrobial susceptibility tests, the overall rate of drug resistance in GAS isolates was higher than reported rates in other countries, and the resistance rates to erythromycin, tetracycline and clindamycin were 91.8, 93.4 and 80%, respectively. This epidemiological study may help to understand the pathogenesis of GAS disease and aid in vaccine development.

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.

[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.