Expression, purification and characterization of three overlapping immunodominant recombinant fragments from Bordetella pertussis filamentous hemagglutinin

Filamentous hemagglutinin [FHA] is one of the most important immunoprotective antigens of Bordetella pertussis [B. pertussis] and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated. Three overlapping coding sequences of FHA antigen were amplified from B. pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli [E. coli] BL21[DE3] strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells [PBMC] proliferation and IFN- gamma production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively. Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21[DE3]. SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN- gamma production. Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immunoprotective

Cloning, expression and characterization of recombinant human Fc receptor like 1, 2 and 4 molecules

The Fc receptor like [FCRL] molecules belong to the immunoglobulin [Ig] superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identified for the human FCRL1, 2 and 4 molecules. Cloning, expression, purification and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b [+] and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fine adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purified by metal affinity chromatography using Ni-NTA resin. Purified FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specific polyclonal antibodies. Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b [+] vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37[degree]C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% [FCRL1] to 25% [FCRL2 and 4] of the total bacterial lysate proteins. These purified recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specific mAbs for immunotherapeutic interventions.

Comparative expression profile of orphan receptor tyrosine kinase ROR1 in Iranian patients with lymphoid and myeloid leukemias

It has recently been shown that ROR1, a member of the receptor tyrosine kinase family, is overexpressed in leukemic B cells of Chronic Lymphocytic Leukemia [CLL] and a subset of Acute Lymphoblastic Leukemia [ALL]. In this comparative study the expression profile of ROR1 mRNA was investigated in Iranian patients with CLL and Acute Myelogenous Leukemia [AML] and the results were compared with those previously reported in our Iranian ALL patients. RT-PCR was performed on bone marrow and/or peripheral blood samples of 84 CLL and 12 AML patients. CLL samples were classified into immunoglobulin heavy chain variable region [IGHV] gene mutated [n=55] and unmutated [n=29] and also indolent [n=42] and progressive [n=39] subtypes. ROR1 expression was identified in 94% of our CLL patients, but none of the AML patients expressed ROR1. No significant differences were observed between different CLL subtypes for ROR1 expression. Taken together the present data and our previous results on ROR1 expression in ALL, our findings propose ROR1 as a tumor-associated antigen overexpressed in a large proportion of lymphoid [CLL and ALL], but not myeloid [AML] leukemias. Expression of ROR1 seems to be associated to lineage and differentiation stages of leukemic cells with a potential implication for immunotherapy

Optimization of gene transfection in murine myeloma cell lines using different transfection reagents

Purification and isolation of cellular target proteins for monoclonal antibody [MAb] production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines [SP[2]/O, NSO, NS1, Ag8, and P3U1] were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein [EGFP], were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP[2]/O, 55.7%, 21.1% and 9.3% for NSO, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NSO and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NSO cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia: association between CD38 expression and disease progression

Patients with B-cell chronic lymphocytic leukemia [B-CLL] have heterogeneous clinical courses, thus several biological parameters need to be added to the current clinical staging systems to predict disease outcome. Recent immunophenotypic studies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. To investigate the expression pattern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between indolent and progressive groups. In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. In all cases, the neoplastic cells displayed B-CLL phenotype [CD5[+]/CD19[+]/sIg[+]]. The vast majority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 [84/87, 95.4%] and CD45RO [74/87, 83.9%] molecules, suggesting a memory B-cell phenotype. Comparison between the indolent [n=42] and progressive [n=37] patients revealed significantly higher frequency and intensity of CD38 expression in progressive group [40.5%] compared to indolent [11.9%] patients [p<0.05]. None of the other membrane antigens were differentially expressed in these two groups of patients. Our results obtained in an Asian ethnic population confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL

Over expression of Wilm's tumor gene 1[WT1] in Iranian patients with acute myeloblastic leukemia

The Wilm's tumor gene 1 [WT1] encodes a zinc finger transcription factor that is inactivated in a subset of Wilm's tumors. It plays a crucial role in growth, proliferation and development of some embryonic and adult organs. WT1 is expressed as a tumor associated antigen [TAA] in various types of solid and hematopoietic malignancies and can be employed as a useful marker for targeted immunotherapy and monitoring of minimal residual disease [MRD]. To investigate the profile of WT1 gene expression in Iranian patients with acute myeloblastic leukemia. RT-PCR method was used to determine the WT1 gene expression in bone marrow [BM] and/or peripheral blood [PB] samples from 11 patients with AML and PB samples of 36 normal subjects. Isolated cells from all patients were immunophenotyped by flow cytometry. The leukemic cells from 10 patients [91%] were found moderately or strongly positive for WT1 expression whereas only 3 out of 36 normal subjects expressed WT1 at very low levels. A highly significant correlation was observed for WT1 expression between paired BM and PB samples of the AML patients. Our results indicate that WT1 is expressed in the majority of Iranian AML patients and may be employed for screening and monitoring of minimal residual disease in these patients