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拟获得adw2亚型乙型肝炎(乙肝)病毒表面抗原preS2+S基因在PichiaPastoris酵母分泌型表达系统(pPIC9K)的高效表达。实验首先将adw2亚型乙肝病毒表面抗原preS2+S基因重组到分泌型酵母表达载体(pPIC9K)形成表达质粒,电转化酵母细胞KM71,G418筛选多拷贝整合克隆,经甲醇诱导表达并用SDS-PAGE电泳及酶免疫法检测表达产物。经100个克隆筛选获得了表达量较高的表达菌株WC4。该菌株甲醇诱导后细胞上清10倍浓缩SDS-PAGE电泳检测显示,细胞上清中有特异蛋白条带,且第6天表达量最高,表达产物单体分子量为31kD左右。用美国雅培公司AUZYMEMONOCLONAL试剂盒估算表达量为2μg/100OD600细胞。上述结果表明,乙肝病毒表面抗原preS2+S基因在本系统中获得了分泌表达。同时检测了酵母细胞裂解液中特异蛋白质的表达,结果发现,自甲醇诱导后第一天即可检测到表达产物,而且除了第6天细胞外表达量高于细胞内外,其余各天的表达水平均表现为细胞内高于细胞外。以上结果提示,利用分泌型酵母表达系统表达乙肝病毒表面抗原在技术上可行,但表达产量偏低,一些蛋白滞留在细胞内未能分泌到培养基中。
The preS2 + S gene of hepatitis B virus (hepatitis B) surface antigen of adw2 is highly expressed in Pichia pastoris yeast secretory expression system (pPIC9K). First of all, the preS2 + S gene of adw2 subtype hepatitis B virus surface antigen was recombined into the secretory yeast expression vector (pPIC9K) to form the expression plasmid. The multi-copy integrated clones were electroporated into yeast cells KM71 and G418, induced by methanol and analyzed by SDS-PAGE And enzyme immunoassay to detect the expression product. After 100 clones were screened, a higher expression strain WC4 was obtained. After the strain was induced by methanol, 10-fold concentration of the supernatant of the strain was detected by SDS-PAGE electrophoresis. The specific protein bands were observed in the supernatant of the strain and the highest expression was obtained on the 6th day. The molecular weight of the expressed product was about 31 kD. The expression level was estimated to be 2 μg / 100 OD600 cells using the Abbott AUZYMEMONOCLONAL kit. The above results showed that the preS2 + S gene of hepatitis B virus surface antigen was secreted in this system. At the same time, we detected the expression of specific protein in yeast cell lysis solution. The results showed that the expression product was detected on the first day after methanol induction, and the expression level was higher than that on the 6th day Both showed intracellular than extracellular. The above results suggest that the expression of hepatitis B virus surface antigen by the secretory yeast expression system is technically feasible, but the expression yield is low, and some proteins remain in the cells and can not be secreted into the culture medium.