Molecular Cloning And Functional Characterization Of Bovine Nramp1Gene

Natural resistance-associated macrophage protein I (Nramp1) which contains12transmembrane domains is located in the lysosomal membrane. Nramp1could inhibit thegrowth of intracellular pathogens including Brucella, Mycobacterium and Salmonella. In thisstudy, the bovine Nramp1gene was cloned from Qinchuan bovine. Several studies includingthe subcellular localization, the function of inhibiting the growth of intracellular pathogensand the production of transgenic cloned cattle were investigated.1. The NRAMP1coding region was amplified by RT-PCR from the spleen of Qinchuancattle. The sequence analysis showed that there were two nucleotides associated with singlenucleotide polymorphism (SNP) within Qinchuan bovine Nramp1gene. At the same time, anNramp1isoform (NRAMP1-ISO) was cloned and the sequence analysis showed thatNRAMP1-ISO had a third intron in comparison with Nramp1, which resulted in the proteincontaining only200amino acids because the translation stopped in the third intron. Theeukaryotic expression vector pEGFP-NRAMP1expressed around the lysosomal membrane inthe cytoplasm, while pEGFP-NRAMP1-ISO could express both in the nucleus and in thecytoplasm.2. The lysosomal targeting motifs of NRAMP1were investigated by expressingEGFP-tagged full-length and truncated NRAMP1proteins and overlapping with thelysosomal marker Lamp1-RFP in Chinese hamster ovary (CHO) cells. The colocalizationshowed that the NH2-terminal amino acids (AA)73-140region including transmembranedomain2(TMD2), the AA.263-334region containing the tyrosine-based motif³²⁷YAPI³³⁰,and two internal signal peptides AA.451-483and AA.489-522were identified as lysosomaltargeting motifs.3. The expression plasmid pCMV-NRAMP1-HA (pCMV-N) driven by cytomegalovirus(CMV) promoter and macrophage-specific expression plasmid pSP-NRAMP1-HA (pSP-N)driven by macrophage-specific synthetic promoters (SP) were constructed, respectively. Afterstably transfecting these plasmids into mouse macrophage cell line RAW264.7which containsa defective NRAMP1, RT-PCR and the immunofluorescence assay showed that pCMV-N andpSP-N could express in the cells. The Nramp1transfected and untransfected RAW264.7cellswere infected with Brucella abortus and Salmonella abortusovis in order to evaluate the function of bovine Nramp1gene in vitro. Overexpression of NRAMP1in macrophagessignificantly inhibited the replication and growth of Brucella abortus and Salmonellaabortusovis.4. Bovine ear fibroblast (BEF) were isolated and cultured with the tissue-culture methodfrom Qinchuan calf. The pSP-N was stably transfected into BEF, and the cell clones wereobtained with G418selection. The integration of Nramp1transgene was confirmed by PCRamplification. The2^nd transgenic cell clone (SP-BEF2) maintained the normal karyotype(2n=60, XX), and the growth curve of the SP-BEF2was similar with the untransfected BEF.The SP-BEF2and untransfected BEF were used as donor cells to produce cloned embryos bySCNT, of which the cleavage rate difference and the blastocyst rate difference were notsignificant (P>0.05).5. The embryos were produced from transgenic SP-BEF2by SCNT, and were thentransferred to43Qinchuan recipient cattle. Fourteen out of43recipients were pregnant butthe pregnancy was not maintained to term. One cloned fetus was aborted at Day200andcontained the Nramp1transgene by confirming with PCR using specific primers.