Studies On Molecular Cloning, Expression And Identification Of Nramp1Gene In Sheep Fetal Fibroblasts And Transgenic Mice

The natural resistance-associated macrophage protein1（Nramp1）gene is alsocalled Bcg/Lsh/Ity gene or solute carrier11a1（Slc11a1）gene. Earlier studies showedthat human tuberculosis and leprosy susceptibility may be associated with a gene fromthe human genome. Recent study shows that the polymorphism of Nramp1gene isnot only related to susceptibility to tuberculosis and leprosy, but also relevant to thepathogenesis of rheumatoid arthritis. Apart from strong disease resistance the Nramp1gene family members are also involved in intestinal iron transport. The Nramp1protein-specific expression in the macrophage lysosomal membrane. Althoughfluorescence analysis showed that the mechanism of Nramp1protein in several otherspecies of mice against intracellular bacteria is not clear, but it may be provided by thedivalent cation concentration in the macrophages within the adjustable restriction ofreplication in bacterial cells.Modern animal husbandry, animal major epidemics is a major limiting factoraffecting the development of modern animal husbandry, such as cattle and sheepbrucellosis, sheep as the most important China’s livestock, It’s meat, wool haspractical value. Cloneing goat Nramp1gene whch with broad-spectrum antimicrobialfeatures, and transfected to80d sheep fetal fibroblasts, cell clones were screened,positive transgenic cell lines would use for the production of disease-resistanttransgenic sheep; Through prokaryotic injection and embryo transplant, the Nramp1transgenic mouse model would use to study the antibacterial mechanism of Nramp1inmice, provide experimental material for further research and lay the foundation forstrong resistance genes for future production of transgenic animals (cattle, sheep).The study includes:(1) extraction goat spleen total RNA by RT-PCR, reversetranscription into cDNA, primers were designed to obtain Nramp1gene sequence, andNramp1gene was inserted to the eukaryotic expression vector pcDNA3.1(+), the recombinant plasmid pcDNA3.1(+)-Nramp1was analysis by double restrictionenzyme digestion and sequencing, the target gene fragmen was completement withexpectations, various regulatory elements of the carrier completely preserved;(2)Small Tail Han sheep fetus primary fibroblast cell lines were transfected witheukaryotic expression vector pcDNA3.1(+)-Nramp1after passage, screeningpositive transgenic cell lines and cell clones were identified by PCR, and two positivecell clones were obtained.(3) injecting the recombinant plasmid PcDNA3.1(+) to0.5dfertilized embryos, genome and RNA levels of transgenic mice were detected andobtained two positive transgenic mice. Distribution on the organization of thetransgenic mice was detected and the result shows that it expressed in heart, spleen,lung, kidney, testis, muscle, and rat tail tissue, There was no expression or littleexpression in the liver.