摘要
以乳酸克鲁维酵母(Kluyveromyces lactis,K.lactis)GG799为宿主对人血清白蛋白(HSA)进行分泌表达。以pPIC9k-HSA为模板,采用带有XhoⅠ和NotⅠ酶切位点的引物PCR扩增获得HSA基因,经XhoⅠ和NotⅠ双酶切后插入pKLAC1,构建表达载体pKLAC1-HSA。经SalⅡ线性化后,电击转化K.lactis GG799,用含5 mmol/L乙酰胺的YCB平板筛选阳性转化子。提取基因组DNA,采用PCR方法对转化子鉴定后进行摇瓶发酵。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot分析发酵上清液中的表达产物,并初步分析酵母基础N源(YNB)对HSA在K.lactis GG799中表达的影响。结果表明,HSA成功在K.lactis GG799中分泌表达,表达量为81μg/mL,遗传稳定性好。
The secretory expression of human serum albumin （HSA） in Kluyveromyces lactis GG799 was investigated. The HSA gene was amplified by PCR using pPIC9k-HSA as template and primers with Xho I and Not I restriction sites, respectively, and then cloned into the expression vector pKLAC1 by Xho I and Not. Recombinant plasmid pKLAC 1-HSA was linearized by Sal Ⅱ and transformed into Kluyveromyces lactis strain GG799 by electroporation. Recombinants were screened by YCB plates containing 5 mmol/L acetamide. The genomic DNA of transformants was isolated to identify the positive ones by PCR. The transformants were fermented in shake flask and the expression products were confirmed by SDS-PAGE and Western blot. The effect of YNB in culture medium on expression of HSA in Kluyveromyces lactis were also investigated. The results indicated that HSA was expressed by the Kluyveromyces lactis GG799 expression system and the average yield was 81 μg/mL.
出处
《生物加工过程》
CAS
CSCD
2012年第5期55-60,共6页
Chinese Journal of Bioprocess Engineering
基金
国家自然科学基金资助项目(30970029)
