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目的 克隆弓形虫核仁G蛋白- 1(NOG1)基因的全长cDNA序列。方法 根据NCBIGeneBank中登录的弓形虫NOG1基因的唯一EST序列设计引物,利用cDNA 5’-末端快速扩增法(5’-RACE)和cDNA 3’-末端快速扩增法(3’-RACE)分别对已知序列进行延伸,将扩增获得的3’-端和5’-端未知序列与位于中间位置的已知序列进行拼接,然后经Blast检验全长序列的正确性。结果 5’-RACE扩增获得3个不同长度的片断,最长片断全长12 87bp ,去除引物和夹杂的已知序列后,通过5’-RACE在5’端扩增获得未知序列为1196bp。3’-RACE扩增后得到一条特异性条带,位于大约1.7kb左右,测序全长为16 34bp ,去除引物和已知序列,经3’-RACE扩增获得的3’-端未知序列为12 0 4bp。Blast全长为316 7bp的3段拼接序列,发现其覆盖NOG1基因cDNA的完整ORF或者CDS序列(6 5 0bp 2 80 9bp)。推导翻译出的蛋白质一级结构包含719个氨基酸(aa) ,在所有已发现物种的NOG1中,弓形虫NOG1基因的cDNA和相应的aa序列都是最长的。该序列已登录NCBIGeneBank ,核酸序列登录号AY6 86 734,对应蛋白质的aa序列登录号为AAT94 2 90。结论 本研究首次克隆获得了弓形虫NOG1基因的全长cDNA序列,并对相应的aa序列进行了推导翻译和简单分析。
Objective To clone the full length cDNA of Toxoplasma gondii G protein - 1 (NOG1) gene. Methods According to the unique EST sequence of NOG1 of Toxoplasma gondii registered in NCBIGeneBank, primers were designed and synthesized by rapid amplification of cDNA 5’-RACE and 3’-RACE of cDNA The known sequences were extended, the 3’-end and 5’-end unknown sequences amplified by the amplification were spliced with the known sequences located in the middle position, and the correctness of the full-length sequence was verified by Blast. Results The 3 fragments amplified by 5’-RACE and the longest fragment was 12 87 bp in length. After removing the primers and the known inclusion sequences, the unknown sequence was amplified by 5’-RACE at the 5 ’end to obtain 1196 bp. After 3’-RACE amplification, a specific band was obtained, about 1.7kb, and the full-length sequence was 1634bp. The 3’-RACE amplified 3’-end unknown sequence was 12 0 4bp. The entire length of Blast was 316 7bp 3-segment splicing sequence and was found to cover the complete ORF or CDS sequence of the NOG1 cDNA (650 bp 2 80 9 bp). The deduced protein primary structure contains 719 amino acids (aa), and the NOG1 gene cDNA and corresponding aa sequences are the longest among all the discovered species. This sequence has been registered NCBIGeneBank, nucleic acid sequence accession number AY6 86 734, the corresponding protein aa sequence accession number AAT94 2 90. Conclusion The full-length cDNA sequence of NOG1 gene of Toxoplasma gondii was cloned for the first time in this study. The corresponding aa sequence was deduced and analyzed.