【作者】 高波；

【导师】 孙怀昌；

【作者基本信息】 扬州大学 ， 预防兽医学， 2005， 博士

【摘要】 为了构建鸡输卵管特异表达载体,根据已经发表的鸡卵清蛋白基因5′-和3′-调控区序列设计两对引物,用高保真PCR法从中国狼山鸡基因组DNA中分别扩增出长度各为3.0kb的卵清蛋白基因5′-和3′-调控区,将扩增产物克隆入pGEM-T载体,部分序列测定结果证明与国外发表的鸡卵清蛋白基因相应区域同源性为100%。将鸡卵清蛋白基因5′-和3′-调控区插入粘粒载体pHC20,获得的鸡输卵管特异表达载体命名为pOV1。为了证明构建的载体能有效驱动目的基因在鸡输卵管上皮细胞中表达,将绿色荧光蛋白(EGFP)报告基因通过Xho Ⅰ位点克隆入pOV1载体,将获得的重组载体pOV1EGFP与脂质体或多聚阳离子(PEI)包裹后,分别转染产蛋鸡输卵管上皮细胞和成纤维细胞,转染后48 h在荧光显微镜下观察报告基因的表达,结果证明pOV1载体能有效驱动EGFP报告基因在输卵管上皮细胞中表达,而在鸡成纤维细胞中不表达。为了进一步优化pOV1载体的结构,用限制性内切酶将鸡卵清蛋白基因5′-和3′-调控区从pOV1载体中切出,然后克隆入切除CMV启动子和SV40序列的pcDNA3.0载体,获得的鸡输卵管特异表达载体命名为pOV2;将鸡卵清蛋白基因5′-调控区单独克隆入改造后的pcDNA3.0载体,获得的鸡输卵管特异表达载体命名为pOV3。将LacZ报告基因分别克隆入pOV1、pOV2、pOV3的5′-端调控区下游,获得的重组载体pOV1LacZ、pOV2LacZ和pOV3LacZ经基因转染剂PEI包裹后,分别经翅静脉注射产蛋鸡,然后将载体注射鸡扑杀,取其输卵管和内脏组织,分别用RT-PCR和酶活性检测法测定报告基因的表达,结果显示LacZ基因在注射鸡的肝、脾、肾、心等组织不表达,在输卵管膨大部表达,而且表达的重组酶能被分泌到鸡蛋的蛋清中。载体注射前注射雌激素对报告基因的表达具有促进作用,在相同的注射剂量下,pOV3LacZ的表更多还原

【Abstract】 To construct chicken oviduct-specific expression vectors, the 5’- and 3’- regulatory regions of chicken ovalbumin gene were amplified from chicken genome DNA by high fidelity PCR using primers designed according to previously published sequences. The 3.0kb PCR products were subcloned into pGEM-T vector and the correctness of the sequences was confirmed by sequence analysis. The 5’- and 3’- regulatory regions were then subcloned into a cosmid vector pHC20 and the resulted vector was named as pOV1. To evaluate wether the vector can specifically drive expression of gene of interest in hen oviduct epithelial cells, enhanced green fluorescence protein (EGFP) reporter gene was inserted into the downstream of the 5’-regulatory region and the recombinant vector pOVlEGFP was transfected into the primary chicken oviduct epithelial cells and fibroblast cells isolated from egg-laying hens using polyethyleneimine procedure. The results showed that the reporter gene was specificly expressed in epithelial cells, but not in fibroblast cells.To optimize the structure of the chicken oviduct-specific expression vector, the the 5’- and 3’- regulatory regions of chicken ovalbumin gene were excised from the pOVl vector by restriction enzyme digection and subcloned into a modified pcDNA3.0 vector as a Sal I/Not I fragment, resulting in the second oviduct-specific expression vector pOV2. In another strategy, the the 5’- regulatory region of chicken ovalbumin gene was subcloned into the modified pcDNA3.0 vector using bovine growth hormone gene poly (A) as the termination signal and the resultant third oviduct-specific vector was called pOV3.To compare the expression property of the three chicken oviduct-specificexpression vectors, the LacZ reporter gene was subcloned at the downstream of the 5’-regulatory region in pOVl, pOV2 or pOV3 as a Xho I fragment and the resultant recombinant vectors were called pOVlLacZ, pOV2LacZ and pOV3LacZ. Following mixing with polyethyleneimine, the recombinant vectors were injected into egg-dropping hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Similarly, β -galactosidase activity was only detected in the oviduct magnum and the egg white of the injected hens. The expression was enhanced by pre-injection of the hens with additional estrogen. The expression level of pOV3 was highest among the three expression vectors.To optimize the conditions for trascient expression of human tissue kallikrein gene in the oviduct of laying hens, hKLKl cDNA was subcloned into the expression vector pOV1, pOV2 or pOV3, and the resultant recombinant vector pOV1K, pOV2K or pOV3K was injected into laying hens via wing vein after mixing with polyethyleneimine (PEI). The eggs were collected and the enzymatic activity in egg white was detected. The results showed that the expression level of pOV3 was highest among the three vectors tested, which was selected for further experiments. Following mixing with PEI, 2 μg of pOV3K vector was injected once or twice into each laying hen via wing vein and egg white was assayed for enzymatic activity. The results showed that secretion of rhKl into egg white reached a maximal level of 59.1U/ml, which was injection dose-dependent and lasted for more than 7 days. At 10 day after the primary injection, the hens were re-injected with the same dose of the vector and even higher activity was detected in their egg white. Two different breeds of hens were tested with no difference in expression level found.To characterize the recombinant human tissue kallikrein, egg white of the vector-injected laying hens was submitted to Western blotting analysis using an antibody specific for human tissue kallikrein. The results showed that two specificprotein bands of 37kDa and 43kDa were detected, which corresponded to the mature-and pre-enzyme, respectively. Further biochemical studies showed that the recombinant enzyme had a similar thermo-stability, o更多还原