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目的构建日本血吸虫(中国大陆株)信号蛋白14-3-3编码基因原核表达质粒pET28a-Sj14-3-3,测定Sj14-3-3基因序列,并为中国大陆株Sj14-3-3的体外表达奠定基础。方法采用RT-PCR技术从日本血吸虫中国大陆株成虫总RNA中扩增出Sj14-3-3基因,扩增产物经纯化后。用BamH I+Xho I双酶切,定向克隆入pET28a质粒,转化大肠杆菌DH5a,再用BamH I+Xho I双酶切及PCR扩增对重组子进行鉴定。用Sanger双脱氧链终止法进行DNA序列测定,并应用在线软件进行同源性比较及预测抗原表位。结果 PCR扩增产物约为765bp,符合预计大小,并成功地定向克隆到原核表达质粒pET28a,对插入片段的测序结果表明,该序列和推测的氨基酸序列与GenBank收录的日本血吸虫14-3-3基因分别有99.5％和99.2％的同源性。序列分析显示,Sj14-3-3基因序列中可能有抗原表位存在。结论 pET28a-Sj14-3-3重组质粒构建成功,为日本血吸虫5j14-3-3分子疫苗及信号转导研究奠定基础。
Objective To construct the prokaryotic expression plasmid pET28a-Sj14-3-3 of the gene encoding 14-4-3-3 of Schistosoma japonicum (Chinese mainland strain) and determine the sequence of Sj14-3-3 gene. Lay the foundation for expression. Methods Sj14-3-3 gene was amplified from the total RNA of Schistosoma japonicum adult strains by RT-PCR. The amplified product was purified. The recombinant plasmid was digested with BamH I + Xho I and cloned into pET28a plasmid. The recombinant plasmid was transformed into E. coli DH5a. The recombinant plasmid was identified by double digestion with BamH I + Xho I and PCR amplification. Sanger dideoxy chain termination method for DNA sequence determination, and application of online software homology comparison and prediction of epitopes. Results The PCR product was approximately 765 bp in size and was successfully cloned into the prokaryotic expression plasmid pET28a. The sequencing results of the inserted fragment showed that the sequence and deduced amino acid sequence were identical with those of GenBank Genes have 99.5% and 99.2% homology, respectively. Sequence analysis showed that the Sj14-3-3 gene sequence may have antigenic epitopes. Conclusion The recombinant plasmid pET28a-Sj14-3-3 was successfully constructed, which laid the foundation for the study of molecular vaccine and signal transduction of Schistosoma japonicum 5j14-3-3.