Construction Of The Bovine Nrampl Gene Vector For Expression

Natural resistance-associated macrophage protein-1(Nramp1) gene have been shown to impact on susceptibility to intracellular pathogen that survives and replicates in host macrophage. It was demonstrsted that the susceptibility to intracellular pathogen of the host depended on expression of the Nramp1. To explore the function of Nramp1 and seek for the objective genes for resistance breeding against intracellular pathogen via transgenic technology. Qinchuan Cattle's Nramp1 gene and Nramp1 N-terminal fragment gene were cloned and inserted into pET-32a,pGEX-4T-1 vector for prokaryotic expression. The recombinant vector for karyotic expression were gained with promoter of CMV and Nramp1. The results as follow:1 The Nramp1 gene was amplified from total RNA of Qinchuan Cattle's peripheral blood by RT-PCR, inserted into pMD18-T simple vector, and secondly subcloned into pET-32a vector(named pET-N). The recombinant plasmid pET-N was transformed into E.coli BL21(DE3) induced by IPTG..The analysis of SDS-PAGE showed that there was a specific protein expression at 100Ku molecular marker and the protein was further identified by Western-blot using anti-Nramp1 serum. Compare to the gene order of Nramp1 that published at GeneBank, there were two differences in the Qinchuan Cattle's Nramp1 gene order , the first nucleotide mutation changed the threonine into alanine and the second nucleotide mutation didn't change the amino acids order.2 The gene of Nramp1 promoter was amplified from DNA genome of Qinchuan Cattle's peripheral blood by PCR and then was inserted into pIRES2-EGFP instead of CMV gene(named pIRES2-P). pEGFP-PN was got by inserting Nramp1 gene into pIRES2-P. And pEGFP-N and pIRES2-N were made for karyotic expression with the promoter CMV.3 Software BepiPred 1.0b Server was used to analyze the surface accessibility, hydrophilicity and flexibility of amino acid translating from Nramp1, then Qinchuan Cattle's Nramp1 N-terminal fragment gene was amplified from total RNA of Qinchuan Cattle's peripheral blood by RT-PCR, inserted into pMD18-T simple vector, and secondly subcloned into pGEX-4T-1 vector(named pGEX-N2). The recombinant plasmid pGEX-N2 was transformed into E.coli BL21(DE3). The GST-Nramp1-N fusion protein was expressed by induction of IPTG and detected by SDS-PAGE analysis. Eventually, GST-Nramp1-N Cattle's Nramp1 N-terminal fragment gene was 186bp, with a single nucleotide mutation. Recombinant plasmid pGEX-N2 was expressed efficiently in the E.coli BL21(DE3) under the optimized induction condition (0.1 mmol/mL IPTG for 10 h at 35℃) and its expressive product was soluble. The Qinchuan Cattle's Nramp1 Qinchuan Cattle's Nramp1 N-terminal fragment gene had the single nucleotide mutation just as Qinchuan Cattle's Nramp1 gene's first nucleotide.All these conclusions provided a good foundation for further research to determine the biological activity of bovine Nramp1 and laid a pathway for resistance breeding by animal transgenic technology.