重组人血清白蛋白在汉逊酵母中的表达与纯化

优化人血清白蛋白（Human Serum Albumin, HSA）基因的密码子，合成基因连接到用于汉逊酵母（Hansenula polymorpha）表达的表达载体上，构建成重组人血清白蛋白（recombinant Human Serum Albumin, rHSA）表达质粒，转化汉逊酵母细胞，筛选得到的rHSA高表达细胞株HP-rHSA－C，30L发酵罐批式发酵表达量可达1.033g/L，经Streamline SP，Phenyl Bio-Sep 6FF，DEAE Sepharose层析分离获得纯化蛋白，除菌过滤后稀释，进行小鼠免疫原性分析，结果与人血清中提取的人白蛋白具有相同的抗原、抗体反应特性。

Expression and Purification of Recombinant Human Serum Albumin in Hansenula polymorpha

Based on Hansenula polymorpha's genetic code frequency meter, the gene of optimized codon human serum albumin (HSA) was ligated with vector of Hansenula polymorpha by T4 DNA ligase, after EcoR I and Hind III double digestion, to construct recombinant HSA (rHSA) expression vector. Transformed to Hansenula polymorpha strain by electroporation, the transformants were incubated on MD medium to select URA3+ phenotype. The highly expressive strain was selected by the methods of SDS-PAGE and Western-blotting. Fermented in feed-batch 30L-fermenter, about 1.033g/L of rHSA was obtained, according to the result of ELISA. The proteolytic degradation of rHSA turned obviously after 61h of induction and the concentration of rHSA decreased to less in 150h after induction. The rHSA was purified by streamline SP exchange chromatography、phenyl Bio-sep 6FF hydrophohic interaction chromatography and DEAE sepharose exchange chromatography, and electrophoresis-grade HSA could be obtained. Diluting the purified HAS, we immunized several group of mice and got anti-serum. Checking mouse's immunogenity by double agar diffusion test, HSA and rHSA were demonstrated with the similarly reactive character of antibody and antigen.