Publications (19)26.34 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: To construct the suilysin mutant without hemolytic activity and evaluate its functions. 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. 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. Suilysin mutant SLY(P353V) has no hemolytic activity but remains immunogenicity.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: To amplify the mrp gene of Streptococcus suis type 2 05ZYH33, express it in E.coli BL21 in order to acquire high purity recombinant protein MRP, then evaluate the protective antigen of recombinant protein MRP. Using PCR technology to obtain the product of mrp gene of 05ZYH33, and then cloned it into the expression vector pET28a(+). The recombinant protein was purified by affinity chromatography, later immunized New Zealand rabbit to gain anti-serum, then test the anti-serum titer by ELISA. The opsonophagocytic killing test demonstrated the abilities of protective antigen of MRP. The truncated of MRP recombinant protein in E.coli BL21 expressed by inclusion bodies, and purified it in high purity. After immunoprotection, the survival condition of CD-1 was significantly elevated. The survival rate of wild-type strain 05ZYH33 in blood was apparently decreased after anti-serum opsonophagocyticed, but the mutant delta; MRP showed no differences. MRP represent an important protective antigen activity.
- [Show abstract] [Hide abstract] ABSTRACT: 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. 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. 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. The recombinant SSU1664 protein has immunogen activity and might be one promising Streptococcus suis vaccine candidate and diagnosis marker of Streptococcus suis early infection.
- [Show abstract] [Hide abstract] ABSTRACT: To explore the purification methods of wild-type and recombinant suilysin and to evaluate their biological activities. 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. 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. 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.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Outbreaks in humans, caused by Streptococcus suis serotype 2 (SS2), were reported in 1998 and 2005 in China. However, the mechanism of SS2-associated infection remains unclear. For the first time, a 2-D gel approach combined with MS was used to establish a comprehensive 2-D reference map for aiding our understanding of the pathogenicity of SS2. The identification of 694 out of 834 processed spots revealed 373 proteins. Most of the identified proteins were located in the cytoplasm and were involved in energy metabolism, protein synthesis, and cellular processes. Proteins that were abundant in the 2-DE gels could be linked mainly to housekeeping functions in carbohydrate metabolism, protein quality control and translation. 2-DE of secretory proteins was performed using IPG strips of pH 4-7. Among the 102 protein spots processed, 87 spots representing 77 proteins were successfully identified. Some virulence-associated proteins of SS2 were found, including arginine deiminase, ornithine carbamoyl-transferase, carbamate kinase, muramidase-released protein precursor, extracellular factor, and suilysin. Enolase and endopeptidase have been proposed as putative virulence-associated factors in this study. The 2-D reference map might provide a powerful tool for analyzing the virulence factor and the regulatory network involved in the pathogenicity of this microorganism.
- [Show abstract] [Hide abstract] ABSTRACT: To express the HSP65-MUC1 VNTR(2) in E.coli and to evaluate its activity of inhibiting tumor growth in vivo. HSP65 and MUC1 VNTR(2) were generated by PCR method and sub-cloned to pET28a(+) to construct the recombinant expression vector HSP65-MUC1 VNTR(2)-pET28a(+). E.coli BL21(DE3) bearing the plasmid was induced with IPTG for protein production. Target protein was characterized by Western blot with monoclonal antibody and purified by Q-Sepharose ion-exchange chromatography and gel filtration. The murine cancer cell linejB16 that transfected by human gene MUC1 was utilized to construct the model of carcinoma, and the tumor growth inhibition activities of HSP65-MUC1VNTR(2) was evaluated in mice C57BL/6. The gene HSP65 and MUC1 VNTR(2) confirmed by sequence analysis matched respectively with BCG HSP65 and human gene MUC1 VNTRs in GenBank exactly. The reconstructed vector HSP65-MUC1 VNTR(2)-pET28a could express target protein stably in the soluble fraction of bacterial extract. The purity of HSP65-MUC1 VNTR(2) protein could be above 95% after purification by Q ion-exchange chromatography and gel filtration. The result of Western blot with monoclonal antibody showed positive. The results of prophylactic immunization with HSP65-MUC1 VNTR(2) fusion protein showed that experiment all groups had significantly higher tumor inhibition rates than that of control group. In summary, HSP65-MUC1 VNTR(2) fusion protein was solubly expressed in prokaryotic expression system and its tumor growth inhibition activity was evaluated primarily. The result indicated that the fusion protein could inhibit the MUC1 positive tumor growth significantly. It can be used in the future research as the cancer vaccine.
- [Show abstract] [Hide abstract] ABSTRACT: Superantigens tremendously activate T lymphocytes by recognizing the particular region on TCR Vbeta, by which cytotoxic T cells can be well armed to kill tumor cells. However, the obstacle exists in the fact that immunosuppression is induced adversely. Staphylococcal enterotoxins (SEs) are pyrogenic superantigens who invoke T lymphocytes cytotoxicity at a very low dosage where their endotoxic activity diminishes. Despite that the elaborate mechanisms are largely unknown, tumoricidal capacity of SEA and SEB has been well studied. In this study, we devoted our attention to evaluate Staphylococcal Enterotoxin C (SEC) regarding its tumoricidal activity versus immunosuppression. We proved with flow cytometry that SEC treatment on C57 mice resulted in a boost of the differentiation of T lymphocytes into CD4(+), CD8(+) subpopulations. In vitro, SEC causes increased IFNgamma release from human PBMC. Furthermore, in coculture SEC-treated human PBMC led to more death of cancer cell lines from a variety of origins. Systemic SEC treatment in mouse and rabbit models significantly decreases tumor growth. In tumor-bearing rabbits, tumor necrosis and strong infiltration of lymphocytes into tumor tissue were observed; the rabbits also benefit with less metastasic cancer cells in the lung. In the meantime, the induced cell immune responses, both T cell differentiation and PBMC IFNgamma release, declined as SEC concentration rose. Tumor growth data obtained from animal models are in accordance with the changes in immunity, in which tumor growth ceased to respond to high dosage SEC as it did to lower dosage. These observations on SEC investigation, particularly in aspect of dosage-related immunosuppression, are of significance to SEC therapeutic potential to cancer. Molecular mechanism underlying these findings warrants further intensive investigation.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Adhesins are considered the most important virulence factors during early phase Staphylococcus aureus infection. The present report describes a newly fusion protein, named Cna-FnBP that was constructed by fusion of the fnb and cna genes of S. aureus and expressed in E. coli. The recombinant protein was designed to broaden the function spectrum of block binding activity to S. aureus adhesion. Vaccination of the recombinant protein induced a strong and specific humoral response to Cna-FnBP in mice. In addition, splenocyte proliferation was provoked by in vitro stimulation with recombinant Cna-FnBP, thus, indicating direct implication of these cells in the immune response. These pre-incubated bacteria were phagocytosed by neutrophils at an increased level in vitro in a mouse model. Mice immunized with Cna-FnBP survived significantly longer following the challenge with S. aureus than nonimmunized mice did. These results indicate that Cna-FnBP is a promising vaccine for the prevention of S. aureus infections. Overall, the results suggest that fusion compounds which elicted from ECM-binding proteins (ECMBPs) were used to immunize against adhesins represents a valuable approach to combat S. aureus infections.
- [Show abstract] [Hide abstract] ABSTRACT: To construct the expression vector of a recombinant toxin composed of a disulfide stable single-chain antibody from mAb B3 and PE38 and examine the binding ability and cytotoxicity of the purified renatured products against the B3 positive carcinoma cells. The V(H) and V(L) fragments of the mAb B3 were ligated by overlaping PCR and the resulting product was cloned to the pET22b expression vector. The PE38 fragment was inserted into the B3dsscFv-pET22b expression vector which was digested by EcoR I and Hind III. The identified expression plasmid was tansformed into E.coli BL21(DE3) followed by IPTG induction. The inclusion body was purified through Q-Sepharose anion exchange column after denaturing and refolding. The binding and cytotoxic ability of the purified products were examined by cell-ELISA and Non-Radioactive Cell Proliferation Assay seperately. The stability assay was performed by incubating the protein sample at 37degrees Celsius. The expression vector B3dsscFv-PE38-pET was constructed successfully and the expression product existed mainly in the form of inclusion body, accounting for 45% of the totle protein. The refolding product remained the binding ability of the single-chain antibody and exerted cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37degrees Celsius. This genetically engineered B3dsscFv-PE38 fusion protein was stable and bifunctional, tumor targeting and tumor cell killing, supporting that it would be a promising candidate for tumor targeted immunotherapy.
- [Show abstract] [Hide abstract] ABSTRACT: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines. This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography. The expression vector B3 (scdsFv)-SEA-PET was constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 degrees. This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: To screen the antibibody mimic peptide binding to hepatocellular carcinoma (HCC) from the circular 7-mer peptide libraries. The enrichment of phage was achieved by repetitious biopanning. The affinity of peptide was evaluated by ELISA, and the binding site of the peptide was analysed by the competition ELISA. The output of phage gradually rose along with increasing of biopanning times. The obtained circular 7-mer peptide showed good binding activity to hepatocellular carcinoma cell lines SMMC-7721 and BEL-7402 (P<0.05), and its affinity to SMMC-7721 cells was superior to that of BEL-7402 (P<0.05). At the level of alpha=0.01, the screened 7-mer peptide monoclonal stocks could obviously compete with scFv to bind SMMC-7721 cells (0.005<P<0.01). As the concentration of scFv gradually fell, the competition inhibition rate dropped accordingly. Sequence analysis showed that the sequences of 7-mer positive monoclonal stocks were identical. The peptide obtained through biopanning was specific and effective, which may provide further support for target therapy of HCC.
- [Show abstract] [Hide abstract] ABSTRACT: In order to construct RGD-mSAK mutant with reduced immunogenicity, and identify its biological activity after purification, mSAK gene fragment was amplified by over-lapping extension PCR. Then the gene was inserted into the prokaryotic expression vector pBV220 with P(R)P(L) promoters after confirmed by DNA sequencing; the expression plasmid pBV220-RGD-mSAK was constructed, and then was transformed into E. coli. DH5alpha. After temperature induction, the mutant Staphylokinase was over-expressed and much of protein was in the supernate of lysate, which is over 50% of total protein in the host. The protein was isolated and purified in Q-Sepharose FF, Sephacryl S-200 and SP, high purity protein was obtained and its purity was over 98%. The thrombolysis activity of the RGD-mSAK protein is 1.68 x 10(5) u/mg by fibrin plate assay, which is slightly higher than that of the wild-type, and antiserum titers raised against this protein in guinea pigs were much lower than those of wild-type SAK, determined by ELISA. In anti-platelets aggregation assay in vitro, the RGD-mSAK protein has obvious inhibition activity of platelet aggregation in low concentration comparing to the control group and wild-type SAK group. So the RGD-mSAK protein is a low immunogenicity, bi-function molecular with both thrombolysis activity and anti-embolism activity. It provided the basis for further research of RGD-SAK.
- [Show abstract] [Hide abstract] ABSTRACT: To express, purify, and characterize scdsFv antibody fused with superantigen SEA(D227A). 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. 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. The scdsFv fused with SEA, as a novel form of immunotoxin, might be used in cancer treatment.
- [Show abstract] [Hide abstract] ABSTRACT: To prepare mutant Staphylococcal enterotoxin B(SEB) and observe its anti-tumor activity. The expressed mutant SEB-K172E in inclusion body was denatured and renatured, and then isolated and purified. The anti-tumor activity of the mutant SEB-K172E was compared with wild-type SEB. Renaturation and purification method of the mutant SEB-K172E was developed. The anti-tumor activity of acquired mutant protein was ten times higher than that of wild-type SEB. The anti-tumor activity of the mutant SEB-K172E is much higher than that of wild-type SEB, suggesting that the mutant SEB-K172E may become a potential anti-tumor drug.
Academy of Military Medical SciencesT’ien-ching-shih, Tianjin Shi, China
Nanjing Normal University
Nanjing, Jiangsu Sheng, China
- College of Life Sciences
Beijing Institute of Microbiology and EpidemiologyPeping, Beijing, China