重组人血清白蛋白在酵母中的表达及分析鉴定

目的：实现重组人白蛋白（rHSA)在巴斯德毕赤酵母(Pichia pastoris)中的高效表达，方法：将本室构建的rHSA表达载体转化毕赤酵母，用G418筛选抗性克隆，SDS－PAGE与Western印迹检测并鉴定表达产物；采用生化法、电泳扫描及Brodford法、竞争ELISA3种方法测定摇瓶培养条件下rHSA的表达量。结果：Western印迹分析发现转移至膜上的酵母表达产物能与抗人血清白蛋白单克隆抗体结合，相对分子质量与人血白蛋白一致，证明在酵母中成功地表达了rHSA。在甲醇诱导下摇瓶培养，rHSA表达量随诱导表达时间的延长而增加，第8天产量约为3．5g/L。结论：人血清白蛋白表达载体与毕赤酵母基因组整合，获得酵母表达株，摇瓶培养，该株表达量为3．5g/L。

High expression and analysis of recombinant human serum albumin in Pichia pastoris

Objective: To develop a highlevel human serum al bumin (HSA) expression system using Pichia pastoris~{!<~}WT~{!=~}. Metho dsThe expression vector for HSA which was constructed in this lab was t ransformed to P.pastoris~{!<~}WT~{!=~} by electroporation. The transformants wer e selected by growth on G418 and screened for HSA production by SDSPAGE and We stern blot. The HSA secretion levels were estimated by biochemical method, elect rophoresis scan and Brodford~{!d~}s method, and competitive ELISA method. Re sults The Western blot analysis showed tha t the recombinant products as a 67?000(M~{*-~}r~{!<~}WT~{!=~}) secretion protein reac ted with an antiHSA monoclonal antibody. By induced expression in shake flask culture for eight days, about 3.5?g/L of rHSA was obtained from Z~{*-~}162 strain supernatant of P.pastoris. Conclusions:A yeast strai n was constructed introducing an expression vector for HSA into the P.pastoris genome. The strain prod uced as much as 3.5?g HSA per liter of shake flask culture medium.