Study Of IFRG15 Gene Expression In Mouse Preimplantation Embryos And Prokaryotic Expression System

Objective: (1) To investigate the expression profile of IFRG15 in mouse preimplantation embryos in vitro, in order to provide theoretical basis and reference for further research of IFRG15 expression in in vitro fertilized embryos and somatic cloned embryos, and to lay the foundation for figuring out the exact role that IFRG15 may plays in the development of mouse early embryos. (2) To isolate and purify proteins of interest from prokaryotic expression system harboring IFRG15 coding sequence, in order to facilitate the future study of biological function of this protein.Methods: (1) Preimplantation embryos were prepared and collected by in vitro culture of in vivo fertilized zygotes of Kunming mouse. We extracted total RNA from embryos at different developmental stages, respectively, and then one-step RT-PCR was used to amplify cDNA of IFRG15. The cDNA was detected by agarose gel electrophoresis. We used image processing software and statistical analysis software for semi-quantitative analysis the expression of IFRG15 in mouse preimplantation embryos at various stages. (2) The genomic DNA was extracted from mouse liver, and which was used as the PCR template to amplify the IFRG15 coding region. The amplified product and pGEM-T cloning vector were ligated after being digested with EcoR I and Xho I restriction enzyme. The recombinant plasmid was then transformed into E.coli DH5αcompetent cells. Plasmid DNA was extracted from bacterium lysate and confirmed by double digestion and DNA sequencing. The correct sequencing of pGEM-T-Mifrg15 recombinant plasmid was digested with EcoR I and Xho I to gain IFRG15 fragments. The fragments were connected to prokaryotic expression vetor pET-41c which aslo by double enzyme digestion. pET-41c-Mifrg15 recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and recombinant plasmid DNA was detected by double enzyme digestion. We used IPTG inducting the correct connection recombinant plasmids for fusion protein expreesion. The expressed products were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and used two kinds of affinity chromatography for recombinant protein purification.Results: (1) Preimplantation embryos were collected by in vitro culture and extracted the total RNA successfully. We used one-step RT-PCR reaction to amplify the IFRG15 cDNA and amplified products were analysised by 1.2% agarose gel electrophoresis. The result showed that IFRG15 express in all developmental stages of mouse preimplantation embryos and expression of IFRG15 in the zygote, 2-cell stage, 4-cell stage embryos were no significant differences. The expression of IFRG15 in the 8-cell stage, morula and blastocyst stage embryos were significantly different with other stage (P<0.01). The IFRG15 expressed in blastocyst stage embryos show the highest expression level.(2)We used genomic DNA as the PCR template to amplify the IFRG15 coding region. The amplified products were analysised by 1.2% agarose gel electrophoresis. The band was clear and bright and the size is about 411 bp. pGEM-T-Mifrg15 recombinant plasmid DNA was digested with EcoR I and Xho I and the products were analysised by 1.2% agarose gel electrophoresis. It showed two clear electrophoresis bands, one was the target gene fragment of 411 bp, another was the fragment of pGEM-T vector fragment. Plasmid DNA sequencing was consistented with the mouse IFRG15 coding sequence from NCBI. pET-41c-Mifrg15 recombinant plasmid DNA was digested with EcoR I and Xho I and the products were analysised by 1.2% agarose gel electrophoresis. It showed two clear bands, one was the target gene fragment of 411 bp, another was the fragment of pET-41c vector fragment. SDS-PAGE result showed that, in the positions of relative molecule about 47 kD showed a specific protein band after IPTG induction. The size of recombinant protein was consistented with the theoretical value. SDS-PAGE showed that two kinds of affinity chromatography purified proteins were obtained a single, clear and the size is about 47 kD, it consistented with the theoretical value.In conclusion, the expression of IFRG15 in mouse preimplantation embryos was changed with the developmental time change. IFRG15 on embryonic development maybe had very important. IFRG15 could be highly expressed in prokaryotic cells. Using HisLink resin could be efficiently purified MIFRG15 protein in denatured condition.