表达乙型肝炎表面抗原重组汉逊酵母细胞破碎方法的比较

Comparison of various methods for breakage of recombinant Hansenula polymorpha cells for expression of hepatitis B surface antigen

目的比较球磨法、低温超高压破碎法和Yeast Buster酵母细胞裂解液法破碎表达乙型肝炎表面抗原(HBsAg)的重组汉逊酵母(Hansenula polymorpha,HP)细胞的效果。方法分别采用球磨法、低温超高压破碎法和细胞裂解液法破碎表达HBsAg的重组HP细胞和重组酿酒酵母(Saccharomyces cerevisiea,SC)细胞,测定其细胞破碎率、细胞破碎后蛋白和HBsAg释出量,比较3种方法破碎细胞的效果。结果球磨法使球磨罐和磨球中少量杂质渗入样品,其中氧化锆罐-氧化锆珠组合破碎重组HP细胞形成的杂质较少,破碎率约为60%,氧化锆罐-玻璃珠组合破碎重组HP细胞后蛋白浓度最高达2.71 mg/ml,HBsAg释出量最高为180.04μg/ml;低温超高压破碎法破碎重组HP细胞破碎率最高为71.18%,细胞破碎后蛋白浓度最高可达6.72 mg/ml,HBsAg释出量最高为350.63μg/ml;细胞裂解液法破碎细胞平均破碎率为60.30%,破碎不同次数的蛋白浓度及HBsAg释出量差异较大。结论低温超高压破碎法更适合破碎表达HBsAg的重组HP细胞。

Objective To compare the breakage effects of recombinant Hansenula polymorpha （HP） cells for expression of hepatitis B surface antigen （HBsAg） by bead-milling, low-temperature high-pressure homogenization and Yeast Buster cell lysate treatment. Methods Recombinant HP cells for expression of HBsAg were broken by bead-milling, low-temperature high-pressure homogenization and Yeast Buster cell lysate treatment respectively, using recombinant Saccharomyces cerevisiae cells as control. The breakage rates of cells and the releases of protein and HBsAg were determined, based on which the effects of three methods were compared. Results The components in either jars or beads were abraded into the cells broken by bead-milling. Agate jars and zirconia beads produced fewer impurities, by which the breakage rate of HP cells was only about 60%. The highest protein content of HP cells broken by bead-milling with agate jars and glass beads was 2. 71 mg/ml, while the highest release of HBsAg was 180. 04 ug/ml. The breakage rate of recombinant HP cells by low-temperature high-pressure homogenization was 71. 18% at most, while the highest protein content was 6. 72 mg/ ml, and the highest release of HBsAg was 350. 63 ug/ml. However, the breakage rate of HP cells by Yeast Buster cell lysate was 60. 30%, while significant differences were observed in protein contents and HBsAg releases in the cells broken for various times. Conclusion Compared with bead-milling and Yeast Buster cell lysate treatment, low-temperature high pressure homogenization is more suitable for breakage of recombinant HP cells for expression of HBsAg.