Expression,Purification and Identification of Recombinant Mouse Interleukin 21 Protein in E.coli

Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.

The mRNA–miRNA–lncRNA Regulatory Network and Factors Associated with Prognosis Prediction of Hepatocellular Carcinoma

The aim of this study was to identify novel prognostic mRNA and microRNA(miRNA)biomarkers for hepatocellular carcinoma(HCC)using methods in systems biology.Differentially expressed mRNAs,miRNAs,and long non-coding RNAs(lncRNAs)were compared between HCC tumor tissues and normal liver tissues in The Cancer Genome Atlas(TCGA)database.Subsequently,a prognosis-associated mRNA co-expression network,an mRNA–miRNA reg-ulatory network,and an mRNA–miRNA–lncRNA regulatory network were constructed to identify prognostic biomarkers for HCC through Cox survival analysis.Seven prognosis-associated mRNA co-expression modules were obtained by analyzing these differentially expressed mRNAs.An expression module including 120 mRNAs was significantly corre-lated with HCC patient survival.Combined with patient survival data,several mRNAs and miRNAs,including CHST4,SLC22A8,STC2,hsa-miR-326,and hsa-miR-21 were identified from the network to predict HCC patient prognosis.Clinical significance was investigated using tissue microarray analysis of samples from 258 patients with HCC.Functional annotation of hsa-miR-326 and hsa-miR-21-5p indicated specific associations with several cancer-related pathways.The present study provides a bioinformatics method for biomarker screening,leading to the identification of an integrated mRNA–miRNA–lncRNA regulatory network and their co-expression patterns in relation to predicting HCC patient survival.