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(英文)

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

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 Sm16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 c DNA文库中扩增 Sj16基因 ;将 Sj16基因定向克隆入 pc DNA3,转化感受态 DH5α菌 ;用酶切、PCR扩增鉴定筛选得到的重组阳性克隆。以重组 pc DNA3- 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 c DNA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 pc DNA3- Sj16 ;Sj16基因开放读码框有 35 4碱基 ,编码 117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。

Objective To further study the immunodulatory function of Schistosoma japonicum Sj16 and more obtain the knowledge about the immune evasion of schistosome. Methods A couple of primers were designed according to the known sequence of Schistosoma mansoni Sm16 and the Sj16 gene obtained by amplification from cDNA library of S. japonicum Chinese strain by using PCR technique. By cloning Sj16 gene into a eukaryotic expression vector, pcDNA3, a recombinant pcDNA3 Sj16 was constructed and transferred into DH5α. The positive recombinant pcDNA3 Sj16 was screened and identified by agarose gel electrophoresis, endonuclease digestion and PCR technique. The pcDNA3 Sj16 recombinant plasmid was used as template and nucleotide sequence of Sj16 gene was determined by the dideoxy chain termination method. Using software to analyze the structure and sequence homology of Sj16 gene and Sm16 gene of S. mansoni . Results The specific fragment of Sj16 was amplified and the recombination eukaryotic expression plasmid pcDNA3 Sj16 was successfully constructed. The Sj16 gene is 354 base pairs in open reading frame, encording 117 amino acids and the Sj16 gene and Sm16 gene of S. mansoni shared quite high homology with only a different amino acid. Conclusion The recombination eukaryotic expression plasmid pcDNA3 Sj16 was successfully constructed and the Sj16 gene was sequenced and analyzed. It paved the way for studying further the immunodulatory function of S. japanicum Sj16 and obtaining the knowledge about the immune evasion of schistosome.