日本血吸虫硫氧还蛋白编码基因的克隆和表达

目的克隆和表达日本血吸虫大陆株硫氧还蛋白(SjcTrx)的编码基因。方法根据日本血吸虫菲律宾株硫氧还蛋白基因序列设计一对引物,上游引物引入BamHI酶切位点和起始密码子ATG,下游引物引入SalI酶切位点和终止密码子TAA。以日本血吸虫大陆株成虫总RNA为模板,经反转录-聚合酶链反应(RT-PCR)扩增SjcTrx基因。经双酶切并纯化的PCR产物与同样双酶切并纯化的pET28a质粒DNA片段用T4 DNA连接酶连接,构建重组质粒pET28a-SjcTrx,转化BL21感受态菌并大量扩增。重组质粒DNA经限制性内切酶双酶切、PCR、琼脂糖凝胶电泳和核苷酸序列测定进行鉴定。pET28a-SjcTrx/BL21用IPTG诱导表达。结果SjcTrx编码基因RT-PCR产物约334 bp,构建的重组pET28a-SjcTrx表达质粒DNA经限制性内切酶双酶切和PCR扩增产物于琼脂糖凝胶电泳均观察到相同大小基因片段。根据核苷酸序列测序结果推导的氨基酸序列与日本血吸虫菲律宾株和曼氏血吸虫Trx分别有97%和43%的同源性。表达的重组蛋白经SDS-PAGE分析,约14kDa。Western blotting显示该重组蛋白可被日本血吸虫感染兔血清和重组抗原免疫小鼠血清所识别。结论SjcTrx基因的表达获得成功,并获得纯化蛋白,为开展动物保护性免疫试验创造了条件。

Cloning and expression of the gene encoding thioredoxin of Schistosoma japonicum

To clone and express the gene encoding thioredoxin of Schistosoma japonicum(SjcTrx),according to cDNA sequence of thioredoxin in S.japanicum(Phillippine strain),a couple of primers was designed with BamHI restriction endonuclease site introduced in forward primer with ATG as the start codon and SalI in reverse primer with TAA as the termination codon.Total RNA was extracted from adult worms of S.japonicum and the SjcTrx gene was amplified by RT-PCR.Meanwhile,the PCR products and the pET28a plasmid were digested with both BamHI and SalI,and the target DNA fragments were purified and cloned properly into the prokaryotic expression vector pET-28a. After identification by endonuclease digestion,PCR analysis and sequencing.The recombinant plasmid pET28a-SjcTrx was transformed into competent E.coli BL21 and expressed in the presence of IPTG.The experimental results showed that size of RT-PCR product as judged by agarose gel electrophoresis was around 334 bp,and the same fragments were obtained by restriction enzyme digestion from the recombinant plasmid pET28a-SjcTrx and PCR with the plasmid as a template.DNA of the recombinant plasmid pET28a-SjcTrx was sequenced and shown to be 97% and 43% identical to the deduced amino acid sequence to those of thioredoxin in S.japonicum Phillippine strain and S.mansoni respectively.Induced expression in E.coli BL21 could result in a constant level of the production of recombinant protein.The results of SDS-PAGE and Western blot analysis revealed that the expressed protein,which was around 14 kDa,could be recognized by sera from rabbit infected with S.japonicum and from mice immunized with recombinant reSjcTrx.It is concluded that the expression of gene encoding thioredoxin of S.japonicum was successful and the immunogenicity of the recombinant gene product to different animal models should be further investigated.