Objective To clone the human Islet neogenesis associated protein(rhINGAP)gene,express the gene extraeellulary in Pichia yeast for.further study on biological function and animal test on INGAP.Methods INGAP gene Was amplified with PCR and inserted into the recombinant plasmidα/pUC18.Then,the fusion gene of α and INGAP was digested and inserted into the expression plasmid pPIC9K.The positive recombinant plasmid which integrated INGAP Was confirmed by restriction enzyme digestion and sequencing,and it Was linearized with Sal Ⅰ digestion and transfered into the yeast host strain GS115 through electroporation.The yeast transformants that harbor the desired gene INGAP with high copy were selected by the auxotroph mediam G418,and verified by PCR.The condition of hake-flask culture was optimized,and the recombinant human INGAP Was induced expression with methanol as the only Carbone source.The antigen activity of the desired protein Was detected by Western blotting and ELISA method.Results Recombinant plasmid αINGAP/pPIC9K were successfully constructed and three positive Pichia yeast transformants were obtained.The expressed protein had satisfactory antigen activity,which Was confirmed by the Western blotting and ELISA method.Conclusions Pichia yeast expressing human Islet neogenesis associated protein (rhINGAP)gene was successfully constructed.

Objective:To explore the key genes and its biological functions of aldosterone producing adenoma (APA) using bioinformatics analysis.Methods:Differentially expressed genes of APA were identified from two training datasets GSE60042 and GSE64957 in GEO database. Function and pathway enrichment analyses for differentially expressed genes were performed and transcriptional regulation network among these genes were determined. Hub genes were extracted by node analysis from the protein-protein interaction (PPI) network. The expression of key genes was verified by a testing dataset GSE8514. Receiver operating characteristic(ROC) curve analysis was applied to assess the diagnostic efficiency of key genes in APA. The biofunction of each key gene were determined by gene set enrichment analysis (GSEA).Results:A total of 68 differentially expressed genes, including 33 up-regulated genes and 35 down-regulated genes, were detected from the training datasets. These genes were mainly enriched in aldosterone biosynthetic process, calcium signaling pathway, serotonin receptor signaling pathway, transcriptional activator activity, and regulation of transcription. JUN and VDR were at the center of the transcriptional factor-gene network. Furthermore, we identified nine Hub genes from the PPI network. In testing dataset, CYP11B2 and VDR showed the higher expression, while JUN, NFKBIZ, EGR3, and KLF6 showed lower expression in APA (all P<0.05), and the value of area under ROC curve analysis was 0.936, 0.833, 0.953, 0.854, 0.868, and 0.929, respectively. GSEA indicated the alter of key genes in APA led to up-regulation of the steroid biosynthesis, cell adhesion molecules, immune cells signaling pathway, and complement and coagulation cascades [all normalized enrichment score (NES)>1.5, P<0.05], but down-regulation of the DNA replication, ribosome, and autophagy (all NES<-1.5, P<0.05). Conclusion:Results of bioinformatics indicate that JUN and VDR are key transcriptional factors, and CYP11B2, NFKBIZ, EGR3, and KLF6 are the key genes for APA, which are involved in the steroid biosynthesis, cell adhesion molecules, immune cells signaling pathway in APA.