生物技术减毒沙门氏菌介导apo-t-PA-GFP融合基因在鸡体内表达和在卵黄中沉积的研究

 目的 探讨以减毒沙门氏菌为载体携带目的基因在家禽体内表达,并实现表达产物在卵黄中定向沉积的可行性。方法 将人t-PA基因与鸡VLDLy组装前体apo基因融合,构建pApo-tPA-GFP表达质粒,电转化至减毒鼠伤寒沙门氏菌△crp SL1344中,阳性重组菌静脉注射高产蛋鸡,收集不同时期鸡蛋,涂片荧光镜检和ELISA检测表达产物在卵黄中沉积情况和表达水平,平板溶圈法检测卵黄中表达产物的活性。结果 注射表达质粒第8天开始卵黄中有荧光颗粒出现,第21天表达量最高,为10.4 μg·mL-1,活性相当于50.2 μg·mL-1标准品t-PA。结论 研究表明,减毒沙门氏菌能够介导apo-t-PA-GFP融合基因在鸡体内表达,且apo载脂蛋白能够引导t-PA透过卵黄膜实现在卵黄中沉积,这一途径简捷、方便,为外源基因在动物体内的表达研究提供了理论和技术基础。

Expression of apo-t-PA-GFP Fusion Gene Delivered by Attenuated Salmonella typhimurium in Vivo and Deposition of Expression Product in Yolk

OBJECTIVE To evaluate the expression of apo-t-PA fusion gene in vivo delivered by attenuated Salmonella and to detect the deposition of expression product in the yolk. METHODS Recombinant plasmid apo-tPA-GFP combined human tissue-type plasminogen activator (t-PA with apo and green fluorescent fusion protein (GFP fusion gene was constructed. The resultant construct expression plasmid apo-tPA-GFP was transformed into attenuated Salmonella typhimurium strain using electroporation method. Hens were transfected the attenuated Salmonella typhimurium strain carrying pApo-tPA-GFP by wing intravenous injection. The expression of fusion gene was detected by smear fluorescence microscopy, ELISA and fibrinolysis test. RESULTS The results of smear fluorescence microscopy and ELISA showed that t-PA accumulated in yolk on the 8th day after the injection of recombinant plasmid, and the expression yield was 10.4 μg·mL-1 on the 21 days. Fibrinolysis test showed that the activity of apo-t-PA-GFP contained in yolk per milliliter corresponded to that of 50.2 μg standard t-PA. CONCLUSION Attenuated Salmonella typhimurium can medicate the expression of apo-t-PA-GFP in vivo, and the expression product depos in yolk. This technique is simple and convenient, which provides the theoretical and technical basis for the expression of extrinsic genes in animals.