全文下载：2013030244
刘佳， 李张宇， 金思岑， 邱晓丽
(太极集团有限公司，重庆 401147)
摘要： 将人瘦素成熟蛋白编码序列同义突变成毕赤酵母偏好密码子，人工合成全基因序列。将基因直接连接到pPIC9载体信号肽识别序列之后，构建毕赤酵母真核表达载体pPIC9-hLeptin，电转化毕赤酵母菌株GS115，用PCR法筛选阳性克隆，SDS-PAGE和Western blotting方法筛选能够稳定、高效分泌表达目的蛋白的酵母工程菌。结果表明，成功构建了重组人瘦素毕赤酵母工程菌株，能稳定、高效分泌表达重组人瘦素蛋白，表达量高峰出现在诱导96 h后，摇瓶表达量为200 mg·L-1。
关键词： 人瘦素；毕赤酵母；分泌表达
中图分类号： Ｑ 815文献标志码： A
Research on Secretory Expression of Human Leptin in Pichia Pastoris
LIU Jia， LI Zhang-yu， JIN Si-cen， QIU Xiao-li
(Taiji Group Co., Ltd.，Chongqing401147，China)
Abstract: Optimize the gene of human leptin. The sequence of mature human leptin gene was obtained by synthesis, the expression vector of Pichia Pastoris of pPIC9-hLeptin was constructed. The recombinant plasmid was transformed into Pichia Pastoris GS115 via electroporation. The transformed positive strains were screened by PCR. The supernatant were analyzed by SDS-PAGE and Western blotting to screen Pichia Pastoris engineer strain with highly effective expression of recombinant human leptin. The results showed that genetic engineering strain expressed recombinant human leptin was constructed successfully. Pichia Pastoris can express recombinant human leptin efficiently and stably. The expression peak emerged after 96 h induced with expression level of 200 mg·L-1.
Key words:human leptin; Pichia pastoris; secretory expression
收稿日期：2012-11-19
作者简介： 刘佳（1979—），女，硕士.
