高温和红光诱导的鱼腥藻7120短藻丝体中TNF-α基因表达效率的提高

Elevating Expression of Human Tumor Necrosis Factor Alpha Gene in Anabaena sp. PCC7120 Short Filaments Induced by High Temperature and Red Light

目前解决外源基因在蓝藻中低效表达的主要策略是改进供体DNA元件。这项工作尝试改变受体系统生理状态,研究对外源基因表达效率的影响。以前已经报道用高温(45℃)和红光处理鱼腥藻7120(Anabaenasp.PCC7120)可以诱导其形成短藻丝体,这里报道以此作为受体细胞,将构建的含重组人肿瘤坏死因子α(TNF-α)基因的穿梭表达载体pKT-TNF通过三亲接合转移法进行转化。红光和高温诱导形成的短藻丝体中,TNF-α基因接合转移效率为在正常营养藻丝中的5～6倍,Southern杂交结果证明pKT-TNF已在受体细胞中复制。Western印迹表明TNF-α基因已在受体系统中表达,放射免疫测定结果显示在短藻丝体中的表达效率提高到在正常营养藻丝中的4～5倍。这可能为提高外源基因在丝状蓝藻中的表达效率提供了一条新途径。此外,研究还分析了人TNF-α基因密码子使用偏向性对在鱼腥藻7120中表达效率的影响。

The strategies for improving the expression efficiency of foreign genes in cyanobacterial hosts are,so far,focusing on modifying donor DNA elements.This work described here was an attempt to physiologically change host systems and see how they affected the expression efficiency of foreign genes in cyanobacteria.Our previous work presented that filamentous cyanobacterium Anabaena sp.PCC7120 cells induced by high temperature and red light can generate short filaments (SHT and SRL,respectively) with photosynthetic activities that has the ability to normally differentiate into vegetable filaments.We transferred here a foreign gene (human tumor necrosis factorα,hTNFα) into these short filaments.A shuttle expression vector pKTTNF harboring human TNFα gene was constructed and transconjugated into the cells of SHT,SRL and the normal vegetative filaments (control) by triparental conjugal mating,respectively.The data showed that the transconjugative efficiencies of pKTTNFs in the SHT and SRL were 500%-600% of those in the control cells.The Southern blotting confirmed TNFα gene was replicated in transconjugative cells of SHT,SRT and control.The Western blotting suggested that pKTTNFs were expressed in these cells.The radioimmunoassay (RIA) showed expression efficiency of pKTTNFs in the SHT and SRL cells were about 400%-500% of those in the control cells.This might provide a novel potential avenue to improve the expression efficiency of foreign genes in filamentous cynobacteria.In addition,we also analyzed the effect of genetic code bias of human TNFα gene on its expression in Anabaena sp.PCC7120.