【作者】 吴丽娟； 蒋建新； 朱佩芳； 康格非；

【Author】 WU Li-juan, JIANG Jian-xin, ZHU Pei-fang, KANG Ge-fei. ( Department of Medical Lab., Doping Hospital, Third Military Medical University, Chongqing 400042, P. R. China; Trauma Branch Lab. National Key Lab of TBC, Research Institute of Surgery. Doping Hospital. Third Military Medical University, Chongqing 400042, P. R. China; Faculty of Laboratory Medicine, Chongqing University of Medical Science, Chongqing 400046, P. R. China.)

【机构】 第三军医大学大坪医院检验科； 第三军医大学大坪医院野战外科研究所第四研究室全军交通医学研究所创伤烧伤与复合伤国家重点实验室； 重庆医科大学医学检验系；

【摘要】 目的巴斯德毕赤酵母是当今生物合成功能性哺乳动物蛋白质及多肽的首选表达体系,挑选整合数恰当的外源基因阳性整合子是实现外源基因稳定高效表达的关键环节之一。本文旨在探索一种巴斯德毕赤酵母表达体系外源基因整合的高通量定量分析方法。方法采用基因工程技术构建pPIC9K-HG质粒作为定量标准品,建立毕赤酵母基因组外源基因整合的高通量定量PCR分析方法,用灵敏度、特异性等对方法进行评价,将之初步用于hA20基因毕赤酵母转化子外源基因整合数的分析。同时对三种酵母基因组制备方法进行了比较。结果方法灵敏度达103拷贝,线性范围在104-109,平均变异系数6.62%,特异性100%。14株hA20基因毕赤酵母转化子外源基因整合数分析显示,成功收获外源基因整合数为单拷贝、3拷贝、4拷贝、5拷贝、15拷贝的阳性转化子。酵母基因组制各经典法、煮沸法和酶法阳性率分别为85.71%、28.57%和85.71%。经X2检验证实,酶法与经典法相比提取酵母基因组的效率一致(P>0.05);煮沸法与经典法相比提取酵母基因组的效率存在显著性差异(P<0.05)。结论成功建立了巴斯德毕赤酵母基因组外源基因整合高通量定量分析方法,方法学评价指标满意,可广泛用于不同外源基因整合之阳性菌株的大批量分析。酶法制备酵母基因组具有可靠、简单、迅速等优点,适合大规模提取酵母基因组。更多还原

【Abstract】 Objective Pichia pastoris expressing system is an important system for producing high levels of functionally active recombinant protein. To pick out integratants with optimal integration number is a key point for resistant expressing target proteins with high levels. Here we establish a high-troughput quantitative method for identification the integration number of exogenous gene in Pichia pastoris genome. Methods pPIC9K-HG was constructed by genetic engineering. Based on pPIC9K-HG sequencing, we took it as the standard for quantitative analysis. A high-throughput method was set up for detecting the integration number of exogenous gene in Pichia pastoris genome. The method was evaluated with methodological indexes such as sensitivity, specificity, etc. Copies of hA20 in integrants were analyzed by it The three methods for isolating the yeast genome were compared, too. Results The sensitivity is 103 and the specificity is 100%. The linearity is between 104 and 109. The average of CV achieved 6.62%. The positive rate of the three methods (classics, boiled and enzymatic) is 85.71%, 28.57% and 85.71% respectively. There was no significant difference between the classics and the enzymatic by X2 test, but there was significant difference between the classics and the boiled . We obtained single copy, 3 copies, 4 copies, 5 copies and 15 copies positive strain with hA20. Conclusions We successfully established a high-throughput quantitative method to determinate the copy number of heterologous gene inserted in yeast genome. Methodological indexes suggested that it meet wholly analysis request. The enzymatic method to prepare yeast genome is effective, simple and rapid, and suit to prepare yeast genome on large scale.更多还原