ABSTRACT
Background: Purified mouse IgG2a [a product that could be used in medical research] subclass could be used for animal immunization to production of polyclonal Antibody and to obtain hybridomas in Monoclonal Antibody Production Procedures. The goal of this study was to purify the mouse IgG2a
Methods: In one step, Ion exchange chromatography was carried out for purification of mouse IgG and then in second step, ProA affinity chromatography was used for IgG2a purification .The chosen method for determination of purity was reduced and non-reduced SDS-PAGE. ELISA method was used for titer and isotype determination
Results: Mouse Igs with a protein concentration of 27mg/ml [volume: 3CC] was applied on Ion exchange column. Purification by Ion exchange chromatography yielded about 28mg of mouse IgG. Eight mg mouse IgG2a was obtained by ProA affinity chromatography. In reduced SDS-PAGE analysis of purified antibody, two bands were seen in 25and 50 KDa MW positions. Isotype determination of purified mouse IgG2a with mouse isotyping Kit showed the presence of mouse IgG2a isotype with a kappa light chain in related fraction
Conclusion: Purified mouse IgG2a subclass was obtained with purity more than 95%. Due to the obtained high purity we concluded that Ion exchange chromatography following by ProA affinity chromatography could be a suitable method for purification of mouse IgG subclasses with high quality. Our product is an economical and suitable product that takes a step towards self-sufficiency of the country
ABSTRACT
CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5 alpha competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using Kpnl and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells [29 and 93%, respectively]. Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera
Subject(s)
NIH 3T3 Cells , Cell Line , B-Lymphocytes , Cloning, Organism , Gene Expression , Immunogenetics , MiceABSTRACT
Background: Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 [TIM-1] glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases
Objectives: In this study, we aimed to express TIM-1 protein on Human Embryonic kidney [HEK] 293T cell line in order to have an available source of the TIM-1 antigen
Materials and Methods: The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells [PBMC] and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA[TM]3.1/Hygro [+] and transformed in Escherichia coli TOP 10 F'. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR
Results: The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells
Conclusions: Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1
ABSTRACT
In addition to Human Leukocyte Antigens [HLA] compatibility, gene polymorphisms in cytokines might also be important in the quality of allogeneic immune response. To evaluate the influence of HLA-DR matching and a number of cytokine gene polymorphisms on acute rejection after living-unrelated donor [LURD] kidney transplantation. A total of 42 renal transplants performed at Hashemi Nejad Kidney Hospital [Tehran/Iran] and followed up for 3 months post-transplantation were included. Using PCR-SSP, HLA-DR alleles [DR1-18] of recipients and donors and gene polymorphisms in TNF-a, TGF-b1, IL-10, IL- 6, and IFN-g of recipients were determined. Acute rejection was observed in 11[26.2%] of renal recipients. The frequency of one and two HLA-DR mismatches in rejector group was 2[18.2%] and 9[81.8%] and in non-rejector group was 13[41.9%] and 17[54.8%], respectively. HLA-DR incompatibility was not significantly higher in rejector [1.82 +/- 0.40] compared with non-rejector [1.52 +/- 0.57] recipients [P=0.069] and more than half of non-rejectors had ompletely mismatched HLA-DR antigens with donors. Polymorphisms associated with the mentioned cytokines had no correlation with acute rejection. The predictive value of HLA-DR mismatching for acute rejection is not as prominent in LURD kidney transplantation as in the cadaveric one. In addition, we failed to demonstrate an association between combined cytokine genotypes and HLA-DR matching with acute rejection. Further and more detailed immunogenetic investigations are required in order to have a better prediction of the transplant outcome