Expression Of Insulin-like Growth Factor Receptor Gene From Taenia Pisiformis And Identification Of Its Interacting Protein

Taenia pisiformis is one of important parasitic worms in the world. Rabbit cysticercosis, causesed by its larvae, has been a severe threat to the rabbit herding. Insulin-like growth factor receptor(IGFR) is an importantly multifunctional cell regulation factor, playing an critical role in cell proliferation, growth and metabolism. Therefore, we predicted that IGFR can be used as a potential drug target and vaccine antigen candidate. Expression of insulin-like growth factor receptor gene from Taenia pisiformis and Identification of its Interacting protein in this study.This results are as follows.1. Cloning and sequence analysis of Tp IGFR gene. The full-length c DNA of Tp IGFR was amplified using RT-PCR and rapid amplification of c DNA end(RACE) methods. The gene structure and phylogenetic evolution were analyzed with bioinformatic software. The results showed that the Tp IGFR c DNA was 5 154 bp in length, including a open reading frame(ORF) of 4 953 bp, encoding a protein of 1 651 amino acids with about 181.92 ku molecular weight. Phylogenetic analysis suggested that Tp IGFR gene was in the same evolutionary branch with the insulin like growth factor receptors gene from Taenia solium. Protein structure prediction indicated that Tp IGFR had the typical characteristics of IGFR structural domain,the extracellular protein ligand binding area(LD), the transmembrane domain(TM) and tyrosine protein kinase domain(TK).2. Preparation of the recombinant Tp IGFR-LD protein and analysis of its tissue localization. The prokaryotic expression vector p ET30a-Tp IGFR-LD was constructed and induced by IPTG. SDS-PAGE analysis showed that the recombinant Tp IGFR-LD protein was expressed in inclusion body, molecular size was 51.48 ku. Western blotting indicated that Tp IGFR-LD could be specifically recognized by the positive serum sample from rabbit infected with Taenia pisiformis metacestode and anti-His antibody. The results showed that the Tp IGFR-LD protein had good reactivity. The Tp IGFR-LD protein was purified by Ni-FF affinity chromatography and used to immune three rabbits. The hyperimmune serum was obtained with the titer of 1:2.56×104 using ELISA test. Immunohistochemical results showed that the Tp IGFR-LD mainly distributed in the adult tegment and reproductive organs, which predicted Tp IGFR might be associated with the nutrition absorbtion and reproductive development of parasite.3. Construction of the eukaryotic expression vector of Tp IGFR-LD and Tp I. The expression vector p CMV-HA-Tp IGFR-LD and p CMV-myc-Tp I were constructed and cotransfected into CHO cells. Indirect immunofluorescence suggested that Tp IGFR-LD and Tp I were successful expressed in CHO cells.4. The interaction analysis of Tp IGFR-LD and Tp I protein. It was identified that Tp IGFR-LD with the red fluorescent and Tp I protein with the green fluorescent could be co-expression in CHO cells merged with yellow fluorescence by confocal laser scanning microscopy. Immune co-precipitation(Co-IP) confirmed that LD and Tp I proteins had interaction with good biological activities.