摘要
应用PCR技术从含有罗曼鸡白介素18全长基因质粒pMD18-T-ChIL-18中扩增出鸡IL-18成熟肽基因,亚克隆于毕赤酵母表达载体pPICZαA上,构建了重组质粒pPICZαA-ChIL-18。经酶切、PCR和测序鉴定正确后,电转化入毕赤酵母菌X-33,筛选多拷贝单克隆进行诱导表达,并进一步分析了培养液pH值、诱导剂浓度、诱导时间和诱导温度对重组菌表达水平的影响,从而获得最优化的表达条件。结果表明,重组毕赤酵母菌能够表达鸡IL-18,且在培养液pH6.0,甲醇诱导浓度1.5%,诱导温度26℃的条件下诱导96 h,目的蛋白的表达量最高,可占菌体总蛋白的60.2%。本文为鸡IL-18的规模化发酵生产奠定了基础。
Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR, and subcloned into Pichia pastoris expression vector pPICZαA, thus constructing the recombinant plasmid pPICZαA-ChlL-18. After identified by restriction enzymes digestion, PCR and DNA sequencing, the recombinant plasmid was transformed into P. pastoris X-33. Then the multi-copy recombinant strains were induced for expression. The expression conditions were further optimized by analyzing the influences of pH value, inducer concentration, inducing time, and inducing temperature on the yields of target protein. The results showed that chicken IL-18 could be secreted by the recombinant P. pastoris and the expression level of recombinant protein was highest under pH 6.0, 1.5% of methanol, temperature 26℃, and 96 hours of inducing time. This study laid a foundation for the large-scale fermentation of chicken IL-18.
出处
《畜牧与兽医》
北大核心
2009年第4期28-32,共5页
Animal Husbandry & Veterinary Medicine
基金
河南省自然基金项目(0511032300)
关键词
鸡IL-18
毕赤酵母
表达
优化
chicken IL-18
Pichia pastoris
expression
optimization
