Construction and stable expression of a truncated human receptor tyrosine kinase ror1 [Ror1-ECD]

Expression of receptor tyrosine kinase Ror1 in a wide variety of cancers has emerged as a new era focusing on targeting this receptor in cancer therapy. Our preliminary results indicate the presence of a truncated transcript of Ror1 in tumor cells. The truncated Ror1 encompasses extracellular and transmembrane domains, lacking catalytic kinase domain [Ror1-ECD]. As enzyme activity is highly dependent on the catalytic domain, we were wondering how this transcript and its encoded protein could play a possible role in tumorigenesis. To understand the function of this truncated transcript and whether or not the encoded protein translocates to the cell surface, we construct-ed a mammalian expression vector containing exon 1 to exon 8 of human Ror1 gene as a model system. The encoded protein by this construct covers the entire extracellular and transmembrane domains of Ror1. The Chinese Hamster Ovary Cell line [CHO] was used for transfection. Our results showed that this construct could express Ror1-ECD at protein level and also the protein could effectively translocate to the surface of transfected cells. Such model may suggest that a proportion of Ror1 molecules ex-pressed by tumor cells are not full-length Ror1. This notion may be considered when applying flow cytometry using antibodies against Ror1 for screening of tumor cells in order to avoid any miscalculation in the number of Ror1 molecules expressed by tumor cells. Furthermore, such expression may bring about assumptions on functional roles of Ror1-ECD in tumorigenesis, which requires extensive functional studies

Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium

Mycoplasma genitalium [M.genitalium] is a sexually transmitted pathogen. Detection of this microorganism in clinical specimens by culture is rather difficult and time consuming. The aim of this study was to produce polyclonal antibody against a synthetic peptide from P110 protein of M.genitalium in order to develop a diagnostic tool for detection of this microorganism in clinical specimens. A synthetic peptide from P110 protein was conjugated to Keyhole Limpet Hemocyanin]KLH[and used for immunization of a white New Zealand rabbit. The produced antibody was purified by affinity chromatography and its specific interaction with immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by Western blotting in bacterial cell lysate prepared from M.genitalium G-37. To confirm its application as a diagnostic tool, indirect immunofluorescent staining method was performed on M.genitalium-infected PBMC using anti-P110 as the primary antibody. The results showed that produced antibody has excellent reactivity with immunizing peptide and also detected a single band of 110 kDa corresponding to P110 protein. M.genitalium-infected PBMC showed a bright fluorescent signal in IF staining. This antibody might be used as a tool in diagnostic applications