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目的获取弓形虫RH株速殖子苹果酸脱氢酶(MDH)的cDNA全长序列,并对推导翻译出的氨基酸(aa)序列进行分析。方法将NCBIGenBank中登录的弓形虫MDHEST序列进行比对、拼接和电子延伸,发现近5’末端的部分核酸序列缺失。基于分析,设计的上游、下游引物分别包含起始密码子ATG和终止密码子TGA(TGA→UGA),使经RTPCR扩增出的片段对应完整的CDS或者ORF。之后对MDHCDS推导翻译出的aa序列进行了保守功能域和同源性分析。结果本研究克隆的MDH的cDNA全长951bp,推导翻译出的蛋白质有316个aa组成,约35kDa,与预期大小接近。保守功能域分析表明,弓形虫MDH最接近类乳酸脱氢酶型(lactatedehydrogenaselike)MDH,其准确定名缩写为LDHlikeMDH,同时弓形虫MDH还具有其它脱氢酶类的特征。弓形虫MDH除与隐孢子虫和恶性疟原虫高度同源外,还与很多细菌等物种同源,但与人的同源性最低(22%)。弓形虫MDHcDNA序列在NCBIGenBank中登录号为AY650028,对应aa序列的登录号为AAT67462。结论本研究首次获得了弓形虫MDH基因的全长cDNA序列和对应的aa序列。MDH是三羧酸循环中的重要酶类,该酶活性的改变,将直接影响弓形虫的存活。弓形虫MDHaa序列与人MDH蛋白间的同源性很低,提示基于MDH蛋白作为药靶,经高通量技术筛选抗弓形虫药具有可行性。
Objective To obtain the full-length cDNA sequence of tachyzoite malate dehydrogenase (MDH) from RH strain Toxoplasma gondii and analyze the deduced deduced amino acid (aa) sequence. Methods Toxoplasma gondii MDHEST sequences registered in NCBIGenBank were aligned, spliced and electronically extended to find that the partial nucleotide sequence near the 5 ’end was deleted. Based on the analysis, the designed upstream and downstream primers contain the initiation codon ATG and the stop codon TGA (TGA → UGA), respectively, so that the fragment amplified by RTPCR corresponds to the complete CDS or ORF. Afterwards, the conservative domain and homology analysis of deduced aa sequences from MDHCDS were carried out. Results The cloned MDH cDNA was 951 bp in length. The deduced protein had 316 aa, about 35 kDa, which was close to the expected size. Conserved domain analysis showed that Toxoplasma MDH was closest to lactate dehydrogenase-like MDH and its accurate name was LDHlikeMDH. Meanwhile, Toxoplasma MDH also has other dehydrogenase enzymes. Toxoplasma MDH is highly homologous to Cryptosporidium and Plasmodium falciparum and is homologous to many species such as bacteria but has the lowest homology (22%) to humans. Toxoplasma MDH cDNA sequence in NCBIGenBank accession number AY650028, corresponding to the aa sequence accession number AAT67462. Conclusion The full-length cDNA sequence and corresponding aa sequence of MDH gene of Toxoplasma gondii were obtained for the first time in this study. MDH is an important enzyme in the TCA cycle. The change of the enzyme activity will directly affect the survival of Toxoplasma gondii. Toxoplasma MDHaa sequence and human MDH protein homology between the low, suggesting that based on MDH protein as a drug target, high-throughput screening of anti-Toxoplasma gondii drug feasibility.