Eukaryoyic Expression And Biological Function Study Of Insulin-like Peptide Coding Gene From Schistosoma Japonicum

Schistosoma japonicum is a dioecious helminth,which mainly parasitizes the homothermal vertebrate animals.Because of the complexity of its life cycle and the diversity of final hosts,it is very difficult in its prevention and treatment,and leads to serious harm for public health.In the early studies,the endogenous ILP coding genes of S.japonicum was not identified.It was suggested that the growth and metabolism of S.japonicum could be regulated by the host insulin.However,our recently study showed that S.japonicum has a unique ILP coding gene and a unique insulin-like Growth Binding Protein(IGFBP).S.japonicum has endogenous ILP,and can also use the host insulin.So it is important to understand what the functions of its own ILP,and what the interaction relationship between Sj-IGFBP and their own ILP as well as the host insulin It is also meaningful to reveal the mechanism in S.japonicum about how to use insulin signal to control growth.This discovery provides some new measures of prevention and treatment against Schistosomiasis.In this study,we cloned and expressed insulin-like peptide of S.japonicum(Sj-ILP),and identified the interaction relationship between Sj-IGFBP and Sj-ILP.The main results are summarized as follows:1.Cloning and sequence analysis of S.japonicum insulin-like peptide coding gene.The primers were designed according to the genome database annotation information,the c DNA with a length 588 bp from adult worm m RNA was successfully amplified by using RT-PCR.The CDS sequence of Sj-ILP was 393 bp in length.The theoretical molecular mass was 15.53 ku and the isoelectric point was 7.49.The prediction of the tertiary structure was highly similar to the human insulin.2.Sj-ILP eukaryotic yeast expression and the tissue location analysis.Sj-ILP gene codons were optimized based on the codon bias in Pichia pastoris.The recombinant expression vector p PIC9K-Sjilp was constructed and induced in P.pastoris GS115.Sj-ILP recombinant protein was expressed with a molecular weight of about 15 ku,which was evidenced by SDS-PAGE and Western-blot analysis.Polyclonal antibody was prepared by immunization of the New Zealand white rabbits with two synthesized small polypeptides from Sj-ILP which were linked to KLH.The titer of the polyclonal antibodies was determined to be as high as 1: 51200 by indirect ELISA analysis.Furthermore,western-blot analysis showed that the polyclonal antibody had a specific immunogenicity.Immunohistochemical results revealed that Sj-ILP was predominantly expressed in adult worm tegument and ovaries,indicating that Sj-ILP may play an important role in nutrition uptake and development of S.japonicum.3.Interaction analysis between Sj-IGFBP,Sj-ILP and HI protein.The recombinant p CMV-HA-Sj-IGFBP,p CMV-HA-Sj-IGFBP,and p CMV-HA-HI were constructed and cotransfected into CHO cells.Indirect immunofluorescences suggested that Sj-IGFBP and Sj-ILP were successfully expressed in CHO cells.Confocal immunofluorescence test showed that p CMV-HA-Sj-IGFBP expressed with green fluorescent could be were co-expressed with p CMV-myc-Sj-ILP(expressed with red fluorescent)and p CMV-myc-HI protein(expressed with red fluorescent),in CHO cells and merged to be yellow fluorescence.Immune co-precipitation(Co-IP)confirmed that Sj-IGFBP and Sj-ILP/HI proteins had structure interaction.