日本血吸虫脂筏蛋白的重组表达及其生物信息学分析

Recombinant Schistosoma japonicum flotillin 2 expression and its bio-informatic analysis

目的在大肠杆菌中原核表达、纯化日本血吸虫脂筏蛋白(rSjFLOTILLIN2),并对其进行生物信息学分析。方法以日本血吸虫成虫cDNA为模板扩增FLOTILLIN2基因片段,克隆至pET28a(+)质粒,经Xho I和EcoR I双酶切以及测序鉴定;重组质粒再转化入E. coli BL21,含重组质粒的菌株经IPTG诱导表达,采用SDS-聚丙烯酰胺凝胶电泳鉴定目的蛋白分子量大小。同时采用生物信息学分析方法进行了序列比较和进化树分析。结果重组质粒(pET28a-FLOTLLIN2)经双酶切及测序鉴定,证明插入片段序列与预期目的基因序列符合。SDS-聚丙烯酰胺凝胶电泳结果显示,体外原核表达的重组蛋白(rSjFLOTILLIN2)分子量大小为48 kD,其与预测的分子量大小一致。经Blast搜索比对发现,日本三角涡虫、白纹伊蚊、地中海果蝇、苜蓿切叶蜂等FLOTILLIN2序列与日本血吸虫FLOTILLIN2相似性最高;将日本血吸虫和热带爪蟾、原鸡等15种不同物种来源的FLOTILLIN2编码蛋白进行比较,并构建FLOTILLIN2系统进化树,系统发育分析结果显示,日本血吸虫与涡虫同为一支,遗传距离最为接近。结论日本血吸虫脂筏蛋白基因被成功克隆和表达,为进一步研究打下基础。

Objective To clone, express and purify ＆histosoma japonicum flotillin2 （SjFLOTILLIN2） in Escherichia coli, and to analyze the bioinformatics of the recombinants. Methods Flotillin 2 gene was amplified by PCR using cDNA obtained from adult Schistoma japonicum. The amplified product was recombined into pET28a （ ＋ ） plasmid, and subjected to double digestion with Xho Ⅰ and EcoR Ⅰ, sequencing and identification. The recombinant plasmid was then transfected with E. coli BL21 to induce the protein expression with IPTG. SDS - PAGE was performed to identify the molecular weight of the interest protein. Bioinformatics was used to analyze and compare the sequence of FLOTILLIN2 and its phylogenetic tree. Results SjFLOTILLIN2 was successfully amplified by PCR, and identified by Xho I and EcoR I double restriction enzyme digestion and sequence analysis. SDS - PAGE measurement showed that the molecular weight of the recombinant protein was 48 KD, identical with the predicted size. Further match using Basic Local Alignment Search Tool（BLAST） revealed that the flottilin 2 sequence of Dugesia japonica, Aedes albopictus, Mediterranean fruit flies and alfalfa leafcutter bee had the highest similarity with that of Schistosoma japonicum. The flotillin 2 encoding protein was also compared between Schistosoma japonicum and Xenopus tropicalis, gallus and 15 other species of different origin to construct the phylogenetic tree. Phylogenetic analysis indicared that Schistosoma japonicum was closer to the flotillin 2 of Dugesia japonica. Conclusion We successfully cloned and expressed the Flotillin 2 gene from Schistosoma japonicum, and our work may lay a foundation for further research on the function of Schistosoma japonicum recombinant protein.