Expression and biological activity identification of recombinant Hap protein of NTHi / 南方医科大学学报

<p><b>OBJECTIVE</b>To express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity.</p><p><b>METHOD</b>Hap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>SDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05).</p><p><b>CONCLUSION</b>Highly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.</p>

Effects of ST6Gal I antisense oligonucleotide-mediated gene silencing on cell adhesion and invasiveness of hela cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effects of antisense oligonucleotide (ASODN) targeting ST6Gal I on cell adhesion and invasiveness of human cervical carcinoma cell line HeLa which over-expressed ST6Gal I .</p><p><b>METHODS</b>ASODN and sense oligonucleotide (SODN) targeting ST6Gal I were designed and constructed, and transfected into a cervical cancer cell line, HeLa, by lipofectmine 2000. HeLa cells were cultured and divided into 4 groups: blank control group, liposome group, SODN group and ASODN group. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion and invasiveness to extracellular matrix ( ECM) were analyzed by using CytoMatrixTM kit and cell invasion assay kit, respectively.</p><p><b>RESULTS</b>The expression of ST6Gal I mRNA in HeLa cells at 48 hrs after transfection in the ASODN group was significantly decreased in comparison with that in the blank control group, liposome group, and SODN group(P <0. 01). The amount of alpha2,6-sialylation on cell surface in ASODN group was significantly lower than that of the other 3 groups ( P <0. 05). The adhesion and invasiveness of the cells in the ASODN group decreased remarkably, both significantly lower than those of the other 3 groups ( all P < 0. 05).</p><p><b>CONCLUSION</b>Specific ASODN targeting ST6Gal I effectively inhibits HeLa cell ST6Gal I expression, decreases the amount of alpha2,6-sialylation on cell surface and leads to a decline of cell adhesion and invasiveness to ECM. This result also established a fine base for further studying on anti-tumor treatment with antisense oligonucleotide.</p>

Preparation and characterization of a monoclonal antibody against human SOCS3 / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare and characterize the monoclonal antibody (mAb) against human SOCS3.</p><p><b>METHODS</b>BALB/c mice were immunized with recombinant GST-SOCS3 protein, from which the spleen cells were isolated and fused with Sp2/0 cells. After several rounds of screening and cloning, the hybridoma cell strain secreting anti-SOCS3 mAb was obtained, whose specificity was evaluated using ELISA and Western blotting, and the titer, immunoglobulin subtype and affinity of the mAb were also measured.</p><p><b>RESULTS</b>The hybridoma cell strain secreting anti-SOCS3 mAb was identified to belong to IgG2a subtype. The mAb titers in cultural supernatant and acetic fluid were 1:640 and 1:25600, respectively, as determined by ELISA with affinity reaching 4.84x10(6) L/mol.</p><p><b>CONCLUSION</b>The success in anti-SOCS3 mAb preparation provides the basis for further study of the negative regulation of cytokine signal transduction and the immunoregulation in microorganism infections.</p>

Optimized isolation and purification of non-typeable Haemophilus influenzae Haps protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To optimize the isolation and purification conditions for Hap(s) protein of non-typeable Haemophilus influenzae.</p><p><b>METHODS</b>Hap(s) protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap weak cation exchange columns of CM Sepharose Fast Flow. The condition of the elution was optimized for pH and ionic strength, the absorbance at 280 nm of the elution samples were detected, and the targeted protein band in the collected samples was observed by SDS-PAGE electrophoresis.</p><p><b>RESULTS</b>The Hitrap ion exchange column was eluted with buffer 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths.</p><p><b>CONCLUSION</b>The pH of the buffer only affect the elution of the irrelevant proteins rather than the Hap(s) protein, and elution with the buffer containing 100 mmol/L NaCl can be optimal for eluting the Hap(s) protein.</p>

ST6Gal I siRNA and antisense oligonucleotide-mediated gene silencing lowers the invasiveness potential of colonic carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting ST6Gal I on adhesion and invasiveness of human colonic carcinoma cell line SW480 over-expressing ST6Gal I.</p><p><b>METHODS</b>siRNA and ASOs targeting ST6Gal I were constructed and transfected into SW480 cells via lipofectmine 2000. SW480 cells were cultured and divided into 7 groups, namely the blank control group, liposome group, siRNA group (transfected with ST6Gal I siRNA), ASO(1) group (transfected with ST6Gal I ASO whose target site is different from the siRNA), ASO(2) group (transfected with ST6Gal I ASO targeting the same site as siRNA), siRNA+ASO(1) group (transfected with siRNA and ASO(1)), siRNA+ASO(2) group (transfected with siRNA and ASO(2)). RT-PCR was used to examine ST6Gal I mRNA expression following the treatment. Flow cytometry was used to examine the amount of alpha2,6-sialylation on SW480 cell surface. SW480 cell adhesion and invasiveness to the extracellular matrix (ECM) were analyzed using CytoMatrix kit and cell invasion assay kit, respectively.</p><p><b>RESULTS</b>The expression of ST6Gal I mRNA, the amount of alpha2,6-sialylation on the cell surface and cell adhesion and invasion to ECM decreased remarkably in groups siRNA, ASO(1), ASO(2), siRNA+ASO(1) and siRNA+ASO(2), all significantly lower than those of the blank control and liposome groups (all P<0.05), especially in siRNA+ASO(1) group. Significant difference was noted between siRNA+ASO(1) and siRNA groups (P<0.05), but not between siRNA+ASO(2) and siRNA groups, or between blank control and liposome groups (all P>0.05).</p><p><b>CONCLUSION</b>Chemically synthesized specific siRNA targeting ST6Gal I effectively inhibits SW480 cell ST6Gal I expression and leads to diminished cell adhesion and invasiveness to ECM, suggesting a combined effect of siRNA and ASO with different targeting sites.</p>