日本血吸虫SCP/TAPS基因家族的生物信息学分析和克隆表达  

Bioinformatic analysis,cloning and expression of Schistosoma japonicum SCP/TAPS gene family

作　　者：陈静芳[1,2] 胡旭初[2] 王乐旬[2] 余新炳[2] 

机构地区：[1]长沙市疾病预防控制中心,长沙410001 [2]中山大学中山医学院病原微生物学教研室

出　　处：《中国人兽共患病学报》2011年第9期804-807,811,共5页Chinese Journal of Zoonoses

摘　　要：目的以黄蜂Vv Vesv5蛋白为检索源检索日本血吸虫EST数据库,对检索出的序列进行生物信息学分析,并挑选出有代表性的基因进行克隆表达,为日本血吸虫SCP/TAPS家族蛋白的功能研究打下一定的基础。方法利用生物信息学软件对检索出的日本血吸虫SCP/TAPS家族蛋白进行生物信息学分析,经PCR确定后,以AAW24579为代表进行进一步的研究,将其命名为SjVAL2。根据SjVAL2的序列设计引物,通过RT-PCR扩增出全长编码区域,克隆到原核表达载体pET-28a(+)中,在大肠埃希菌中诱导表达并纯化,免疫印迹实验(Western-blotting)鉴定纯化的重组蛋白。结果日本血吸虫SCP/TAPS家族中至少有17个不同的蛋白成员存在,多序列比对分析发现它们初步可以分为3个不同的亚组。以日本血吸虫成虫cDNA为模板RT-PCR扩增出SjVAL2序列,PCR、双酶切及DNA测序均证实pET-28a(+)-SjVAL2重组质粒构建成功。SDS-PAGE结果表明,目的基因在大肠埃希菌中获得高效表达,重组蛋白能识别日本血吸虫感染的小鼠血清,说明其具有免疫反应性。结论日本血吸虫SCP/TAPS家族中至少有17个不同的蛋白成员存在,其中SjVAL2基因可在原核表达系统中高效表达,为进一步研究该家族蛋白的功能打下基础。The Vespula vulgaris Wasp Venom Allergen 5（Vv Vesv5,GenBank accession No.AAA30333） protein sequence was used in a tBLASTn search of S.japonicum ESTs.Detail bioinformatic analysis were conducted and an interesting gene were cloned and expressed to investigate the role in the life cycle of S.japonicum.After PCR confirmation of the sequences from parasite-derived cDNA,AAW24579 was selected for further study and named S.japonicum Venom allergen-like transcript（SjVAL2）.The coding region of SjVAL2 was amplified by RT-PCR using specific primers and cloned into prokaryotic expression vector pET-28a（＋）,then expressed in E.coli BL21（DE3）.The purified recombinant protein was detected by Western Blotting.There were at least 17 different genes among S.japonicum SCP/TAPS gene family.Multiple sequence alignment indicated that they could be divided into three groups.The coding region of SjVAL2 was amplified by RT-PCR using S.japonicum adults cDNA as template.PCR,double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed.The recombinant protein could be highly expressed in E.coli BL21（DE3）.Western blotting indicated that rSjVAL2 could be recognized by serum from S.japonicum naturally infected mouse.SCP/TAPS gene family might play an important role in the life cycle of S.japonicum and its detail biological function need further studies.

关 键 词：日本血吸虫 SCP/TAPS基因家族 基因克隆 原核表达 

分 类 号：R383.2[医药卫生—医学寄生虫学]