弓形虫乳酸脱氢酶LDH2基因启动子的克隆及鉴定

摘要

To clone promoter fragment of Toxoplasma gondii LDH2 gene and to evaluate the promoting activity, a series of fragments of 5 UTR of Toxoplasma gondii LDH2 gene were amplified by PCR, and then inserted into pTX3-basic via Kpn I and Xhol I to construct luciferase reporter plasmids. Forty eight hours after tachyzoites being co-transfected by luciferase reporter plasmids and reference plasmid pRL-Tg, dual-lucif erase assays were carried out to determinate activities of the fragments to initiate luciferase transcription. Results showed that the sequence of the 5 UTR of Toxoplasma gondii LDH2 gene fragments was proved to be correct by sequencing, and a series of luciferase reporter plasmids were verified by PCR, restrictive endonuclease digest, and sequencing. The luciferase activities of all the luciferase reporter plasmids with 5 UTR of Toxoplasma gondii LDH2 in tachyzoites were similar with the negative control pTX3-basic. While in bradyzoites, all the plasmids yielded 40 to 50 folds of luciferase activity to the negative control, and 1. 2 to 1. 5 folds to the positive control of pTX3-basic, excepting for the plasmids pTX3-293 and pTX3-193. In conclusion, the promoter region of Toxoplasma gondii LDH2 was located and cloned successfully. Our results provide evidence for investigation of the mechanism on regulation of the LDH2 gene.%目的 克隆弓形虫乳酸脱氢酶LDH2基因启动子并进行初步鉴定.方法 PCR扩增出LDH2基因5′侧翼序列系列截短突变体,定向插入载体pTX3–basic的Kpn I / Xhol I酶切位点之间,转化大肠埃希菌Top 10,氨苄西林筛选,建立重组LDH2基因5′侧翼序列系列截短突变体荧光素酶表达载体.转染刚地弓形虫速殖子,利用荧光素酶检测试剂盒测定所克隆片段的启动转录活性.结果 成功扩增出LDH2基因5′侧翼序列系列突变体;系列重组荧光素酶表达质粒经PCR、酶切及测序鉴定正确无误;系列重组荧光素酶表达质粒在弓形虫速殖子中的相对活性与阴性对照pTX3-basic基本相同,而在弓形虫缓殖子中,除pTX3-293和pTX3-193外,其他重组荧光素酶活性均是阴性对照pTX3-basic的40~50倍之多,是阳性对照pTX3-control的1.2~1.5倍,说明插入片段具有较强的启动子活性.结论 成功定位并克隆弓形虫LDH2基因启动子,为今后研究LDH2基因上游调控序列提供试验和理论依据.