鸡输卵管特异表达载体的优化及体内表达

Optimization and in vivo Expression of Chicken Oviduct- Specific Expression Vector

摘要:

为了实现鸡输卵管特异表达载体在相应组织特异高产表达, 并简化质粒DNA的制备过程, 本研究在已经构建的鸡输卵管特异表达载体pOV1基础上进行了优化改造, 为进一步进行重组药物蛋白的暂态表达及转基因鸡研究奠定基础。首先用限制性内切酶将克隆在pOV1载体的鸡卵清蛋白基因5￠-和3￠-调控区切出, 同时克隆到切除neo基因及CMV启动子的pcDNA3.0载体, 构建成另一输卵管特异表达载体pOV2; 将鸡卵清蛋白基因5￠-调控区单独克隆入同样载体, 获得第三个鸡输卵管特异表达载体pOV3。为了检验三个输卵管表达载体驱动外源基因在鸡体内输卵管细胞中表达的有效性和特异性, 将LacZ报告基因分别克隆入pOV1、pOV2、pOV3中5'-调控区的下游, 获得的重组载体pOV1LacZ、pOV2LacZ和pOV3LacZ经聚乙烯亚胺包裹后, 经翅静脉注射产蛋鸡。用RT-PCR和酶活性检测法对LacZ基因在载体注射鸡体内的表达进行检测, 结果显示肝、脾、肾、心等组织中无LacZ基因的表达, 而输卵管膨大部不仅有LacZ基因的表达, 而且表达的重组酶能分泌到蛋清中, 雌激素注射对报告基因的表达具有促进作用, 其中pOV3LacZ的表达水平较高。这些试验结果表明, 鸡输卵管特异表达载体pOV3具有结构相对简单、表达水平较高、组织特异性较好等优点, 能用于鸡输卵管生物反应器的研制。

Abstract:

We modified a previously constructed vector pOV1 and compared expression difference among modified constructs. First. 5￠-and 3￠-regulatory regions of chicken ovalbumin gene were excised from the previously constructed vector pOV1 by endonuclease digestion and subcloned into modified pcDNA3.0, named as pOV2. Then only the 5′-regulatory region was subcloned into the same vector and this resulted in the third oviduct-specific expression vector pOV3. To compare expression property of the three constructs in hen oviduct, the LacZ reporter gene was subcloned at the down-stream of the 5′-regulatory region in vectors pOV1, pOV2 and pOV3, respectively. The resultant recombinant constructs pOV1LacZ, pOV2LacZ and pOV3LacZ were injected into laying hens via wing vein rout. RT-PCR of the vector-injected hen tissues showed that the LacZ gene was transcribed only in the oviduct, but not in the heart, liver, kidney and spleen tested. Similarly, β-galactosidase activity was detected only in the oviduct magnum, which was secreted into egg white of the injected hens and enhanced by injecting estrogen into the hens. Among the three vectors tested, expression of LacZ gene in pOV3LacZ-injected hen oviduct magnum was at a relatively higher level and thus pOV3 vector was selected for further studies.