日本血吸虫(中国大陆株)Sj16基因真核表达载体的构建及序列分析

目的为了进一步研究日本血吸虫(中国大陆株)Sj16的免疫调节功能,丰富有关血吸虫免疫逃避知识.方法根据曼氏血吸虫Sm1 6基因已知序列设计合成一对引物,用PCR技术从日本血吸虫(中国大陆株)成虫cDNA文库中扩增Sj16基因;将Sj16基因定向克隆入pcDNA3,转化感受态DH5a菌;用酶切、PCR扩增鉴定筛选得到的重组阳性克隆.以重组pcDNA3-Sj16质粒为模板,用双脱氧链末端终止法测定Sj16基因序列,应用软件辅助分析Sj16序列及进行Sj16与曼氏血吸虫的Sm16同源性比较.结果从日本血吸虫(中国大陆株)成虫cDNA文库中获取Sj16基因,重组质粒中含有Sj16基因,成功构建日本血吸虫(中国大陆株)Sj16基因真核表达重组质粒pcDNA3-Sj16;Sj16基因开放读码框有354碱基,编码¨7氨基酸,N端18个氨基酸可能为信号肽序列;Sj16与Sm16有高度同源性,只存在一个氨基酸的差异.结论成功克隆了Sj16基因的真核表达载体,并测定、分析了Sj16基因序列,为深入研究Sj16的免疫调节功能,丰富有关血吸虫免疫逃避知识打下基础.

CONSTRUCTION OF EUKARYOTIC EXPRESSION PLASMID AND SEQUENCE ANALYSIS OF Sj16 GENE OF SCHISTOSOMA JAPONICUM CHINESE STRAIN

Objective To further study the immunodulatory function of Schistosoma japonicum Sj16 and more obtain theknowledge about the immune evasion of schistosome. Methods A couple of primers were designed according to theknown sequence of Schistosoma mansoni Sm16 and the Sj16 gene obtained by amplification from cDNA library of S. japon-icum Chinese strain by using PCR technique. By cloning Sj16 gene into a eukaryotic expression vector, pcDNA3, a recombi-nant pcDNA3-Sj16 was constructed and transferred into DH5α. The positive recombinant pcDNA3-Sj16 was screened and i-dentified by agarose gel electrophoresis, endonuclease digestion and PCR technique. The pcDNA3-Sj1 6 recombinant plasmidwas used as template and nucleotide sequence of Sj16 gene was determined by the dideoxy chain termination method. Usingsoftware to analyze the structure and sequence homology of Sj16 gene and Sm16 gene of S. mansoni. Results The specif-ic fragment of Sj16 was amplified and the recombination eukaryotic expression plasmid pcDNA3-Sj16 was successfully con-structed. The Sj16 gene is 354 base pairs in open reading frame, encording 117 amino acids and the Sj16 gene and Sm16gene of S. mansoni shared quite high homology with only a different amino acid. Conclusion The recombination eukary-otic expression plasmid pcDNA3-Sj16 was successfully constructed and the Sj16 gene was sequenced and analyzed. It pavedthe way for studying further the immunodulatory function of S. japanicum Sj16 and obtaining the knowledge about the im-mune evasion of schistosome.