灵芝免疫调节蛋白(Lz-8)在毕赤酵母中的表达及其免疫活性鉴定

Ganoderma lucidum immunomodulatory protein(Lz-8) expressed in Pichia pastoris and the identification of immunocompetence

摘要:

真菌免疫调节蛋白家族(Fungi immunoregulatory proteins, FIPs)各成员所具有的免疫调节和抗肿瘤活性已被广泛研究。本研究利用毕赤酵母表达系统对其成员Lz-8进行了重组表达。以毕赤酵母突变株GS115为表达宿主细胞, PCR和DNA测序结果均显示Lz-8的cDNA已被成功地整合入酵母基因组。聚丙烯酰胺凝胶电泳(SDS-PAGE)、激光解析飞行时间质谱(MALDI-TOF-MS)和免疫学实验均被用于重组表达蛋白的检测。实验结果表明Lz-8在毕赤酵母表达系统中得到成功表达, 在SDS-PAGE中可观察到分子量为14 000 D的单一条带, MALDI-TOF-MS的实验结果显示rLz-8的分子量为12 722 D。在相关的免疫学实验中, rLz-8可引起绵羊血红细胞凝集, 但对人血4种血型的红细胞并没有凝集作用, rlz-8还可诱导巨噬细胞吞噬作用, 均与其他报道中的实验结果吻合。以上结果表明, 本实验已成功地利用毕赤酵母表达系统对Lz-8进行重组表达。

Abstract:

Fungi immunoregulatory proteins family is effective in immunological regulation and anti-tumor. We used Pichia pastoris expression system for recombinant expression of Lz-8, an immunomodulatory protein isolated from fruiting body of Ganoderma lucidum. The Gs115 (mut+) strains of P. pastoris was used as host cells. PCR and sequencing of DNA showed that Lz-8 cDNA was successfully integrated into the P. pastoris genome. Electrophoresis(SDS-PAGE), matrix assisted laser desorption ionisation time-of-flight mass spectrometry(MALDI-TOF-MS) and immunological techniques were used to identify recombinant Lz-8 (rLz-8). Lz-8 expressed in Escherichia coli, the Pichia system requires further optimization to obtain more active fungi immunomodulatory protein. Lz-8 was expressed in P. pastoris successfully, and polyacrylamide gel electrophoresis in the presence of SDS-PAGE gave a single band with an apparent Mr=14 000 D. MALDI-TOF-MS also showed that molecular weight of rLz-8 was 12 722 D. Aggregation was observed from sheep red blood cells in the presence of purified rLz-8 within the concentration range of 12.5–50 mg/mL. However, no aggregation was seen at concentration greater than 50 mg/mL for any type of human red blood cell. The dose at 0.5 mg/kg of rLz-8 induced macrophage cytophagocytesis, and set interferon as control at 0.5 mg/kg. These results suggested that active and stable rLz-8 was obtained in P. pastoris expression system.