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(英文)  

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

作　　者：梁重阳[1] 张淑芹[1] 刘志屹[1] 孙非[1] 

机构地区：[1]吉林大学再生医学科学研究所细胞生物研究室,长春130021

出　　处：《生物工程学报》2009年第3期441-447,共7页Chinese Journal of Biotechnology

基　　金：the Major Program of Jilin Provincial Science & Technology Department of Jilin Province (No. 20040401)~~

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14000D的单一条带,MALDI-TOF-MS的实验结果显示rLz-8的分子量为12722D。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。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 μg/mL. However, no aggregation was seen at concentration greater than 50 μg/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.

关 键 词：灵芝 免疫调节蛋白 毕赤酵母 免疫蛋白 

分 类 号：S567.31[农业科学—中草药栽培] Q943.2[农业科学—作物学]