Highly efficient expression, purification of recombinant LTB protein and its activity against mucosal immunoadjuvant by nasal immunization / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To develop an efficient expression, purification system of recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization.</p><p><b>METHODS</b>A recombinant, pMMB68-LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60. The relevant target protein was identified using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Sephacryl S-100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60. BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration. After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA.</p><p><b>RESULTS</b>rLTB protein was highly expressed in VSP60. After gel filtration with Sephacryl S-100, the purity of rLTB reached 98.1%, the yield rate was about 52%. After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL + rLTB groups were significantly increased, compared with the HEL alone group (P < 0.001).</p><p><b>CONCLUSIONS</b>A set of protocols for large-scale rLTB preparation has been established, which is simple, efficient and applicable. The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co-administered antigen.</p>

Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro.</p><p><b>METHODS</b>Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation.</p><p><b>RESULTS</b>Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC.</p><p><b>CONCLUSIONS</b>Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.</p>

IN VITRO CO-STIMULATORY ACTIVITY OF HUMAN B7.2(IgV+C)PROTEIN PRODUCED BY ENGINEERED BACTERIA / 药物分析学报

Objective To express human B7.2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-α. The fusion protein consisted of GST and hB7.2(IgV+C) was identified by SDS-PAGE and Western blotting.T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated by anti-CD3 antibody. Results The fusion protein GST-hB7.2 (IgV+C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7.2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.

THE EUKARYOTIC EXPRESSION OF HUMAN PERFORIN PROTEIN AND THE ESTABLISHMENT OF HYBRIDOMAS PRODUCING ANTI-HUMAN PERFORIN PROTEIN / 药物分析学报

Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombinant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hybridoma cell lines secreting McAbs against human perforin were established, and the three McAbs showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as lgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.

CONSTRUCTION OF HUMAN INTERLUEKIN-18 DNA VACCINE AND IT'S EXPRESSION IN MAMMALIAN CELLS / 药物分析学报

Objective To construct the human interleukin-18(hIL-18) DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos-7 and D5.Methods Gene recombinant technique was used to construct hIL-18 eukaryotic expression vectors.Calcium phosphate method was performed to transect recombinant hIL-18 eukaryotic expression vectors into Cos-7 and D5 cells.In situ hybridization and Western Blot were implemented to verify the transient expression of recombinant hIL-18 in Cos-7 and D5.Results The eukaryotic expression plasmid pVAX1-IL 18 was constructed successfully.hIL-18 was transiently expressed in Cos-7 and D5.Conclusion The eukaryotic expression plasmid pVAX1-IL 18 was constructed.In situ hybridization and Western Blot results proved the successful transient expression of pVAX1-IL 18 in Cos-7 and D5.Therefore,the work has settled the foundation for further biological research on hIL-18,including immunogene therapy through hIL-18.

ANALYSIS OF TYPE I AND TYPE Ⅱ CYTOKINES PROFILE OF LYMPHOCYTE IN PLEURAL EFFUSION OF NON-SMALL CELL LUNG CANCER PATIENTS / 药物分析学报

Objective To analyze mRNA expressions of 7 cytoklnes which influence the immune response in lym- phocytes in pleural effusion of non-small cell lung cancer patients to evaluate the effect of local tumor microenviron- ment on anti-tumor immune response and to explore the mechanism of tumor escape. Methods Detecting the mRNA expression of IL-2,INF-γ,IL-12,IL-18,IL-10,IL-4 and TGF-β 1 in lymphocytes in pleural effusion of non-small cell lung cancer patients and tuberculotic pleurisy patients on the single cell level by using in situ hybridization. Results In the pleural effusion of non-small cell lung cancer, the mRNA expressions of IL-10,TGF-β1 and IL-4 were signifi- cantly higher than those of IL-2,IL-12,IL-18 and INF-γ,as well as these of control group. The cytokine expression levels of tuberculotic pleurisy patients were very Iow, and there were no significant differences between different cy- tokines. Conclusion Type 2 cytokines are expressed predominantly in the pleural effusion of non-small cell lung can- cer. The increased co-expression of IL-10 and TGF-β1 indicates that they might act Jointly and play a critical role in the immunosuppression of non-small cell lung cancer.

Experimental Study on the Anti-tumor Mechanism of Interleukin-6 / 中国肿瘤生物治疗杂志

Segregated Kunming mice bearing S180 sarcoma were used as tumor models and treated with rhIL-6. The tumor regressed in most of the treated mice (8/10) in which there was no tumor growth when rechallenged with the same tumor cells, whereas 9/10 of controls died as the tumor progressed. Morphologically, in the treated group, the mice had enlarged spleen (4 times larger than that of control group)with hyperplastic white pulp consisted predominantly of activated lymphocytes. Cytotoxicity of spleen cells from IL-6 treated group to autologous tumor cells was higher than that of control group (907 ?318: 387 ?144, P=0.003) and so did L929 cell line used as target (1145?164: 186?251).We also set in vitro experiment using human PBL and human melanoma and colon carcinoma cell lines (A375,LS174). These cells were treated with IL-6 respectively and the cytotoxicity was assayed. Although PBL stimulated with IL-6 killed the LS174 more efficiently,the higher cytotoxicity to LS174 is because of the increased sensitivty of LS174 to the effector cells .On the contrary,IL-6 showed no effect on the A 375 cell line. It is assumed that this difference might result from the discrepancy of the recognition molecules existed on the cell surface between these two cell lines.