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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。
Immunomodulatory and antitumor activities possessed by members of the Fungi immunoregulatory proteins (FIPs) have been extensively studied. In the present study, Lz-8 was expressed recombinantly by using Pichia expression system. The Pichia pastoris strain GS115 was used as the host cell for expression. Both PCR and DNA sequencing showed that Lz-8 cDNA was successfully integrated into the yeast genome. Polyacrylamide gel electrophoresis (SDS-PAGE), laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) and immunological experiments were used for the detection of recombinantly expressed proteins. The experimental results showed that Lz-8 was successfully expressed in Pichia pastoris expression system. A single band of 14000D was observed on SDS-PAGE. The result of MALDI-TOF-MS showed that the molecular weight of rLz-8 was 12722D. In related immunological experiments, rLz-8 can cause sheep red blood cell agglutination, but no agglutination of red blood cells of 4 blood types of human blood, rlz-8 can also induce phagocytosis of macrophages, both with other reported The experimental results match. The above results show that this experiment has successfully used the Pichia expression system for recombinant expression of Lz-8.