摘要
在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。
In order to express Edwardsiella tarda Eta1-L-Gapdh fusion protein in blue algae（ Anabaena sp.PCC7120）,genome DNA of E. tarda was purified and was used as a PCR template for the amplification of eta1 and gapdh gene,two immunogenic genes that can be developed into vaccine against E. tarda. Then overlap extension PCR was employed to link eta1 and gapdh gene into the target fusion gene eta1-L-gapdh. The target gene was inserted into the two Bam H I sites of an expression vector pRL489 in order to construct an expression vector that can express the target gene in cyanobacteria. The accuracy of the expression vector was confirmed by such methods as plasmid extraction,PCR,restriction enzyme digestion and sequencing. The confirmed vector was transformed into wide-type fishy alga Anabaena sp. PCC7120 using the method of triparental conjugal transferand neomycin sulfate to screen the transgenic cyanobacteria. The selected positive cyanobacterium was confirmed by plasmid extraction and PCR. Finally the expression of the fusion gene was detected at the transcription and translation level using RT-PCR and Western-blot respectively. The results showed and concluded that the expression vector was successfully constructed,and the target gene was transcribed in Anabaena sp. PCC7120 and a fusion protein was also detected. In addition,the relative expression level of the fusion protein was approximately 2. 46%.
出处
《微生物学杂志》
CAS
CSCD
2016年第5期78-84,共7页
Journal of Microbiology
基金
北京中医药大学自主选题课题(2013-JYBZZ-JS-139)
