【作者】 罗艳；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2013， 博士

【摘要】 人血清白蛋白（HSA）是血液中最丰富的循环蛋白，它是一种单体多结构域大分子，是一种非糖基化的蛋白，在医学治疗和生物药剂学等领域中已经得到了广泛的应用。目前临床应用的HSA主要是来源于人血浆的人血清白蛋白（pdHSA），由于pdHSA的来源有限，价格昂贵，而且有传染病原微生物污染的风险（例如，病毒和朊病毒），因此重组人血清白蛋白（rHSA）成为pdHSA最理想的替代品。HSA非糖基化的特点使得它可以在许多的宿主器官中进行高效的表达和正确的折叠。目前，rHSA的生产已经在大肠杆菌、酵母和植物种子（水稻）等表达系统中得到了一定研究。尽管如此，对于生产具有天然活性的真核生物蛋白，动物乳腺反应器更具有优势。本研究的目的在于通过优化载体的构建，筛选高效表达HSA的乳腺特异性表达载体，制备高效表达HSA转基因牛。通过phiC31整合酶系统和cHS4绝缘子等调控元件的综合应用构建了HSA牛乳腺特异性表达载体，经体外和体内验证实验，筛选出高效表达HSA的牛乳腺特异性表达载体，将其转染牛胎儿成纤维细胞，通过体细胞核移植技术制备生产转HSA基因克隆牛。1.通过PCR和RT-PCR扩增HSA基因组序列和cDNA序列，通过HSA第1外显子中的BstEⅡ和第7外显子中的NcoⅠ位点将这两个序列片段进行整合构建HSA微基因，其包含前14个外显子、前6个内含子和部分的第15个外显子。通过对比分析得出，HSA微基因与模板DNA序列的同源性为99.5%，其编码区氨基酸序列与模板氨基酸序列同源性为100%，其二级结构和三级结构与其模板相比无明显差异。利用构建的HSA基因与EGFP融合表达载体pN-H16将HSA微基因导入293细胞，荧光显微镜下可见EGFP的表达，RT-PCR可检测到HSA mRNA正常表达，结果证实了HSA微基因具有被正确转录表达的功能。2.通过序列分析得出PCR扩增得到的牛β-酪蛋白启动子和cHS4绝缘子核心区基因序列与其模板序列的同源性都大于99%，LoxP序列、attB序列和BGHpolyA序列与模板同源性为100%。通过镜下观察质粒p-C（含有β-酪蛋白启动子-EGFP表达盒子）转染的HC11细胞和293细胞的EGFP表达情况，检测牛β-酪蛋白启动子是否具有指导基因乳腺特异性表达的活性；通过比较转染质粒pC1-E（neo基因表达启动子与增强子间无绝缘子插入）和转染质粒pC1-EP（neo基因表达启动子与增强子间插入绝缘子）后所产生的G418细胞克隆数，分析绝缘子的活性；通过检测转染质粒pC1-A（包含attB序列）的成纤维细胞中的外源基因整合位点，分析attB序列是否能够被phiC31整合酶介导整合；通过对Cre酶处理的G418克隆细胞中的neo基因的PCR扩增，检测loxP序列在Cre酶介导下是否能将其所包括的基因删除；通过流式细胞仪检测转染包含和不包含BGHpolyA质粒的293细胞的EGFP表达情况，分析BGHpolyA是否具有增强基因表达的活性。功能验证试验表明，所有调控元件都具有相应的功能。3.利用上述原件构建了4种HSA乳腺特异性表达载体pACH（含attB序列）、pIACH（含attB和3个正向绝缘子序列）、pIACH（-）（含attB和一对反向绝缘子序列）和pIACH（+）（含attB和一对正向绝缘子序列），分别与整合酶表达载体共转染牛乳腺上皮细胞，筛选阳性转基因细胞，并以不加整合酶表达载体组作对照，利用反向巢式PCR，RT-PCR和western-blotting分别检测各种载体的整合效率、整合位点和HSA表达水平。结果显示在整合酶介导下pACH整合率显著高于其它各组，而pIACH（-）组表达HSA的水平显著高于其它各组。结果表明，整合酶系统可以提高外源基因整合效率，与绝缘子配合可以提高HSA的表达量。4.通过电转染将HSA乳腺特异性表达载体转染泌乳期小鼠乳腺，利用RT-PCR、western-blotting和ELISA检测了体内环境中牛β-酪蛋白启动子-HSA表达盒子的表达效率以及phiC31整合酶对HSA表达效率的影响。结果显示，HSA乳腺特异性表达载体在小鼠乳腺中具有表达HSA的功能，在phiC31整合酶的介导下小鼠乳汁中的HSA水平较无整合酶对照组显著提高。5.将质粒pIACH（-）和pCMVint共转牛胎儿成纤维细胞，应用G418筛选阳性细胞克隆，PCR检测外源基因的整合，反向PCR鉴定转基因细胞整合位点，选择整合良好的细胞作为核供体细胞，通过体细胞核移植技术制备转基因牛2头，经HSA基因PCR和southern-blotting鉴定为转HSA基因牛，通过反向巢式PCR和实时定量PCR进行的基因整合位点和整合基因拷贝数的分析，得出HSA基因在这两头牛基因组中的整合位点为BF19，基因拷贝数为单拷贝。更多还原

【Abstract】 Human serum albumin (HSA), the most abundant protein in plasma, is a monomericmulti-domain macromolecule, and is also a non-glycosylated protein. HSA is widely used inmedical treatment and biopharmacy. HSA for clinical applications is mainly derived fromhuman plasma. Plasma-derived HSA (pdHSA) sources are limited, expensive, and have therisk of infecting with pathogenic microorganisms (e.g. viruses and prions). So, recombinanthuman serum albumin (rHSA) is the ideal substitute of pdHSA. Non-glycosylated feature ofHSA makes it can be efficient expressed and correct folded in many hosts. Currently, rHSAhas been produced in E. coli, yeast, and plant seeds (e.g. rice) expression systems.Nevertheless, for the production of natural active eukaryotic proteins, animal mammary glandbioreactor has more advantages. The purpose of this study is to optimize the vectorconstruction, screen more efficient HSA mammary-specific expression systems, and produceHSA transgenic cattle. We constructed HSA bovine mammary specific expression vector byusing phiC31integrase enzyme systems, cHS4insulators and other regulatory elements,screened out efficiently HSA-expressing vector by verification experiments in vitro and invivo. Then we transfected bovine fetal fibroblast cells with the vector, used the transfecedclonal cells as donor cells and produced HSA transgenic cattle by somatic cell nucleartransfer.1. Using PCR and RT-PCR to amplify HSA genomic and cDNA sequences, through HSAfirst exon of BstE Ⅱ and7th exon of Nco Ⅰ sites to integrate these two sequences fragmentsto construct HSA minigene including the first14exons, introns and before6, Section15exons. The results of comparative analysis showed that the homology between HSA minigeneand template DNA sequence is99.5%, the homology of amino acid sequences is99.8%, andthat the secondary and tertiary structure are not significant different from template. Weconstructed a HSA gene and EGFP fusion expression vector pN-H16, and transfected thisvector into293cells. Observing EGFP expression under a fluorescence microscope anddetecting HSA mRNA expression by RT-PCR to confirm the HSA gene has been correctlytranscripted.2. Sequence analysis of bovine β-casein promoter and cHS4insulator core genesequence showed their homology compared with template sequence was higher than99%. The homologies of LoxP sequence, attB sequence and BGHpolyA with their templates were100%. Transfected plasmid p-C (containing the β-casein promoter-EGFP expression cassette)into HC11cells and293cells to detect the tissue-specific activity of bovine β-casein promoterby observation of EGFP expression; Comparing the number of G418-cell clones fromtransfected pC1-E (insulator inserted between neo gene expression promoter and enhancer)and transfected pC1-EP (there is no insulator between neo gene expression promoter andenhancer) cells to analyze insulator activity; Detecting the integration site of pC1-A(including attB sequence) in fibroblasts to test whether attB sequences is able to be mediatedby phiC31integrase; Detecting whether loxP sequences are able to be Cre-mediated to deletegene flanked by them by PCR amplification of neo gene in G418neo gene cloned cellstreated by Cre enzyme; Comparing the efficiency of the293cells transfected by plasmidscontaining or uncontaining by flow cytometry to test whether BGHpolyA has the activity ofenhancing gene expression. Functional verification tests showed that all regulatory elementshave their corresponding functions.3. Using the above-mentioned regulatory elements, we constructed four tissue-specificHSA expression vectors pACH (including attB sequences), pIACH (including attB sequenceand three forward insulators), pIACH (-)(containing attB sequence and a pair of reverseinsulators) and pIACH (+)(containing attB and a pair of forward insulators), used them withintegrase expression vector to co-transfect bovine mammary epithelial cells (BMECs) andscreened positive transgenic cells. As the same time, we also transfected the cells withoutintegrase as a nagetive control. We detected the effects of phiC31and insulator on transfectionefficiency and gene expression by reverse nested PCR, RT-PCR and western-blotting. Theresults show the transfection efficiency of group transfected by pACH and pCMVint wassignificantly higher than other groups; HSA expression level of group transfected by pIACH(-) and pCMVint was obviously higher than other groups. The results showed that theintegrase system can improve the efficiency of gene transfection efficiency and expressionlevel of foreign genes, and the combination of phiC31integrase and insulator can furtherenhance the level of HSA expression.4. We transfected mammary glands of lactating mice by electroporation, detected theactivity of β-casine promoter-HSA expression cassette and effects of phiC31integrase onHSA expression level in vivo by RT-PCR, western-blotting and ELISA. The results showedthat mammary-specific HSA expression vector was capable of expressing HSA in mousemammary glands and phiC31integrase significantly improved the level of HSA expressionthan the control group without integase in vivo.5. We used plasmids pIACH (-) and pCMVint co-transfected bovine fetal fibroblasts, screened positive clones by G418, identified integration of exgious gene by PCR, tested theintegration sites of transgenic cells by neverse PCR, and chose well-integrated cells as nucleardonor cells for somatic cell nuclear transfer. Then we had2transgenic cattle by somatic cellnuclear transfer technology and confirmed the cattle were transfected with HSA gene by PCRand southern-blotting. The results of detection of integration site, which were performed byreal-time quantity PCR and nested-reverse PCR, showed the copy number of inserted-genewas one and the integration site was BF19.更多还原