Cao J P, Liu S X
Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, WHO Collaborating Center of Malaria, Schistosomiasis, and Filariasis, Shanghai 200025.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2001;19(1):22-5.
To clone and express the cDNA encoding Schistosoma japonicum tropomyosin.
The cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were ligated with pGEM-T vectors and then for transformations. After characterization of white clones by agarose gel electrophoresis, endonucleases digestion and PCR, some recombinant plasmids with inserts were used for sequencing. Then the gene was subcloned into prokaryotic expression vector pQE30 and expression was induced by IPTG.
The PCR products was 823 bp judged by agarose gel electrophoresis and sequencing. A cDNA encoding S. japonicum tropomyosin, except for 14 amino acids at the amino terminus and 2 at the carboxyl terminus, has been constructed and cloned successfully. The colony, designated pGSjcTM12, was sequenced and shown to be 91.1% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The gene was subcloned into pQE30 and an expressed protein of about 32 kDa was obtained.
The cloning and expression of the gene encoding S. japonicum tropomyosin had been successfully made.
克隆并表达日本血吸虫原肌球蛋白的编码cDNA。
通过逆转录-聚合酶链反应（RT-PCR）扩增cDNA。将PCR产物与pGEM-T载体连接，然后进行转化。通过琼脂糖凝胶电泳、核酸内切酶消化和PCR对白色菌落进行鉴定后，一些带有插入片段的重组质粒用于测序。然后将该基因亚克隆到原核表达载体pQE30中，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。
经琼脂糖凝胶电泳和测序判断，PCR产物为823 bp。已成功构建并克隆了一个编码日本血吸虫原肌球蛋白的cDNA，其氨基末端除14个氨基酸和羧基末端除2个氨基酸外。命名为pGSjcTM12的菌落经测序，在核酸水平上与曼氏血吸虫原肌球蛋白的同源性为91.1%，推导的氨基酸序列同源性为98.1%。该基因亚克隆到pQE30中，获得了一个约32 kDa的表达蛋白。
已成功实现日本血吸虫原肌球蛋白编码基因的克隆与表达。
