【作者】 程祥；

【导师】 王华岩；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2012， 博士

【摘要】 巨噬细胞天然抗性蛋白1（natural resistance-associated macrophage protein I，Nramp1）是含有12个跨膜域的膜蛋白，定位于溶酶体膜表面，能够抑制布鲁氏菌、结核杆菌、沙门氏菌等胞内寄生菌的增殖。本研究从秦川牛中克隆了Nramp1基因，对Nramp1的亚细胞定位、抑制胞内菌生长的功能和转Nramp1基因克隆牛的研制等进行了研究，主要得到以下结果：1.本研究通过RT-PCR的方法从秦川牛脾脏中扩增了牛Nramp1基因的编码区序列，测序表明秦川牛Nramp1基因的两个核苷酸位点存在单核苷酸多态性；同时克隆了Nramp1基因的同源异构体NRAMP1-ISO，NRAMP1-ISO基因比Nramp1基因多了第三内含子，NRAMP1-ISO终止密码子位于第三内含子内，NRAMP1-ISO共编码200个氨基酸。真核表达载体pEGFP-NRAMP1在细胞质的溶酶体表达，而pEGFP-NRAMP1-ISO在细胞核和细胞质中都有表达。2.本文构建了EGFP标记的NRAMP1不同编码区域的融合表达载体，与定位于溶酶体的特异性载体Lamp1-RFP共转染CHO细胞中，筛选NRAMP1蛋白中与溶酶体定位有关的氨基酸序列。研究结果表明，氨基端的包含第二个跨膜域的AA.73-140区域、含有³²⁷YAPI³³⁰结构域的AA.263-334区域、含有MORN结构域的AA.372-430区域和两个内部信号肽AA.430-451与AA.489-522等都能够将NRAMP1定位到溶酶体上。3.本研究构建了Nramp1的表达载体pCMV-NRAMP1-HA（简称pCMV-N）和巨噬细胞特异性表达载体pSP-NRAMP1-HA（简称pSP-N）。将两个载体分别稳定转染入NRAMP1蛋白功能缺陷的小鼠巨噬细胞系RAW264.7，经RT-PCR和免疫荧光检测表明pCMV-N和pSP-N都能在RAW264.7细胞中表达。分别用牛布鲁氏菌强毒株2308和羊沙门氏菌感染转染牛Nramp1基因的和未转染的RAW264.7细胞，验证牛Nramp1基因的抗菌功能；结果表明，与未转染的细胞相比，牛Nramp1基因能够显著地抑制胞内菌的增殖与生长。4.通过组织块培养法分离培养秦川犊牛的耳皮肤成纤维细胞（BEF），用pSP-N转染BEF，加入G418进行筛选，并经PCR鉴定，获得了整合有外源Nramp1基因的单克隆细胞。进一步研究表明2号转基因单克隆细胞（SP-BEF2）的生长活力与未转染的BEF类似，且SP-BEF2的染色体核型正常（2n=60，XX）。用SP-BEF2和BEF作为供体细胞制备克隆胚胎，两者胚胎的卵裂率和囊胚率差异不显著（P>0.05）。5.用转基因细胞株SP-BEF2作为供体细胞，通过核移植制备克隆胚胎，移植到43头秦川受体牛，其中14头受体牛怀孕，1头受体牛妊娠200天时产下胎儿，经PCR鉴定表明，该流产胎儿基因组中整合有外源Nramp1基因。更多还原

【Abstract】 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.更多还原