【作者】 胡林勇；

【导师】 靳亚平；

【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】 制备“乳腺生物反应器”生产药用蛋白或生产“人源化”羊奶或牛奶是近年来转基因研究的热点。基因打靶技术不仅可以实现对基因组中特定基因的敲除，从根本上阻断目的基因的表达；而且可以将外源目的基因定位整合到基因组的特定位点，借助细胞内源性调控序列指导外源目的基因高效表达，从而有效降低随机整合带来的位置效应的影响。α-乳白蛋白（α-lactalbumin，ALA）是人乳中主要的乳清蛋白，富含色氨酸、赖氨酸及半胱氨酸等必需氨基酸，近年的研究发现其在预防新生儿胃肠道感染等方面有重要作用。而作为山羊乳中主要的乳清蛋白，β-乳球蛋白（β-lactoglobulin，BLG）在人乳中并不存在，因而可能和牛乳中BLG一样是引起婴幼儿乳过敏症的一种重要的过敏原。因此，本研究通过基因打靶的手段，将BLG基因敲除或用hALA基因置换BLG基因CDS区，以期为生产“人源化”山羊乳奠定基础。1.以人血液基因组DNA为模板，通过PCR成功克隆获得人乳白蛋白（hALA）基因；构建了hALA基因的真核表达载体，将其转染到293T细胞中作为人乳腺上皮细胞的替代细胞，通过RT-PCR的方法成功克隆得到hALA cDNA。这一方法为克隆组织材料难以采集的cDNA提供了一种有效的备选方案。2.克隆了山羊BLG基因3个不同长度的5’端调控序列片段，并分别插入到荧光报告载体pAdTrack-RFP中，构建了重组腺病毒载体：pAd-B51RFP、pAd-B52RFP和pAd-B53RFP，以这3个片段为调控序列来启动mRFP基因的表达。将包装得到的重组腺病毒分别感染山羊乳腺上皮细胞，用倒置荧光显微镜检测mRFP表达。结果表明3个片段均能够启动mRFP的表达，其中B51和B52启动mRFP的表达水平高于B53。3.构建了4个靶向山羊BLG基因的打靶载体pBAT、pBATGFP、pBATM和pBAcT。其中pBATGFP含有neo和GFP两个阳性筛选标记基因，其他3个均只含有neo一个阳性筛选标记基因；pBATM的两个同源臂长度较pBAT短； pBAcT与pBATM载体的差异在于前者的目的基因为hALA cDNA。经酶切和测序鉴定，4个载体序列组装正确。以pBATM为代表，用其中的表达元件BLG基因5’调控序列、目的基因hALA和3’端调控序列置换pAdTrack-RFP中的mRFP和polyA，构建重组腺病毒载体。将包装得到的重组腺病毒感染山羊乳腺上皮细胞，用促乳素、EGF、胰岛素等进行诱导，取培养上清进行Western杂交检测，结果表明hALA能够在BLG基因调控序列指导下表达。4.将构建的4个打靶载体及本实验室保存的旨在敲除山羊BLG基因的打靶载体pBLG2T线性化后分别转染山羊胎儿成纤维细胞，用G418和GCV进行药物筛选。结果发现从pBATGFP组筛选到的胎儿成纤维细胞中GFP表达量非常微弱，不能实现剔除野生型细胞污染的目的。在pBAT和pBLG2T两组中分别筛选鉴定得到2株（B412F1和B7732F6）和3株（T229F2，T1041F1，T7963F6）打靶细胞。5.以5株打靶阳性细胞作为核供体进行体细胞核移植，将早期卵裂基因打靶克隆胚胎移植到同期发情的受体山羊输卵管，45d左右妊娠率达到了44.4%。妊娠足月顺产获得3只克隆山羊胎儿。经PCR及Southern杂交检测，其中有一只为BLG基因敲除山羊胎儿，另有一只为hALA定位整合的基因打靶阳性山羊。更多还原

【Abstract】 Production of transgenic domestic animals which can be used to produce pharmaceuticalproteins or humanized milk is the popular research topics in recent years. Target genes can beknockout to disrupt its expression and the interested gene can be inserted into the specificlocus of the genome and expressed under the control of the internal regulatory elements withgene targeting techniques. As the main whey proteins in human milk, α-lactalbumin (ALA)was thought as the rich sources of essential amino acids, such as tryptophan, lysine andcysteine, especially for infants. It has been found that ALA may take part in the prevention ofgastrointestinal infections among neonates. In addition, as the main whey protein of theruminant milk, BLG was not contained in the milk of the human, and was considered as themain allergen of the cattle or goat milk. With the aim of producing transgenic goat which cansecrete humanized milk, we want to knockout the BLG gene in goat and/or to replace it withhALA using the gene targeting combined with somatic cell nuclear transfer techniques.1. Firstly, we cloned the human ALA gene by PCR with serum genomic DNA as template.Meanwhile, in order to clone the cDNA of hALA, we integrated the hALA into the eukaryoticexpression vector pIRES2-EGFP. Then, the vector was transfected into the293T cells toconstruct the surrogate cells from which the total RNAs were extracted to clone the cDNA byRT-PCR. This protocol can be served as an alternative method to clone cDNA which onlyexpressed in the tissues not so conveniently to collect.2. Three fragments of BLG in goat were cloned by PCR, and inserted into the fluorescentreporter vector pAdTrack-RFP. After recombination with pAdEasy-1in BJ5183, theadenoviral vector pAd-B51RFP, pAd-B52RFP and pAd-B53RFP were constructed in whichthe expression of mRFP was directed by the regulatory element, respectively. Then theadenovirus were packaged and used to infect the goat mammary epithelial cells. Wheninduced with corresponding hormones the expression of mRFP can be dtectected in the goatmammary epithelial cells, which showed that the BLG fragments were cloned successfully.3. Four targeting vectors pBAT, pBATGFP, pBATM and pBAcT were constructedsuccessfully identified by enzymic digestion and sequencing. In order to assay the expressional elements in pBATM, the hALA combined with5’ and3’ flanking regions of BLGin pBATM was sub-cloned to replace the RFPPA in pAdTrack-RFP to construct theadenovirus. Induction the lactation of goat mammary epithelial cells infected with theadenovirus. The hALA was detected by western bloting in the culture medium, which furtherdemonstrated that the pBATM were constructed correctly.4. Transfecting the goat fibroblast cells with the linearized four targeting vector andknockout vector pBLG2T, respectively. After selection with G418combined with ganciclovir(GCV),2(B412F1and B7732F6） and3(T229F2, T1041F1and T7963F6） strains of usabletargeted cells with normal karyotype were got in the group transfected with pBAT or pBLG2T,respectively. Unfortunately, GFP almostly cannot be detected in the clones transfected withpBATGFP, which suggested that it cannot be used to reject the contaminant wild cells mixedin the selected positive cell strains.5. With the cells from the five selected targeted strains as donors, the recombinantembryos were transferred into the oviducts of the recipient goats after cultured24h in vitro.Totally about18goats were transferred and8pregnant goats were detected about45dayslater and3full term development fetuses were got finally. After detection with PCR andsouthern blot, one fetus was BLG knockout and one was hALA targeted integrated.更多还原