基于cDNA末端快速扩增的刚地弓形虫核仁G蛋白-1的克隆

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。

Cloning of the nucleolar guanosine 5'-triphosphate binding protein 1 of Toxoplasma gondii by rapid amplification of cDNA ends

To clone and characterize the nucleolar guanosine 5'-triphosphate binding protein 1(NOG1) cDNA from Toxoplasma gondii,total RNA of T. gondii was extracted with TRIzol reagent according to the instructions for RNA isolation. Primers used for rapid amplification of cDNA 5'- and 3'- ends(5'-RACE and 3'-RACE) were designed on the basis of the only EST of NOG1 cDNA. 5'-terminal fragment cloned by 5'-RACE, the middle EST and 3'-terminal fragment amplified by 3'-RACE were pieced together integrally and the complete sequence was validated by homologous Blastx and Blastp. The newly identified cDNA fragment was 3?167?bp in length which included the complete ORF or CDS of NOG1 from 650?bp to 2?809?bp coding 719 amino acid(aa). Beside the ORF region 2?160?bp in length, 649?bp upstream from initiation codon ATG and 358bp downstream from stop codon TGA were also amplified by 5'-RACE and 3'-RACE, respectively. Comparing with NOG1s from other species, cDNA and a sequences of T. gondii NOG1 were longest and was some dozens of amino acid more than Plasmodium falciparum and Cryptosporidium parvum of Apicomplexa.This study firstly cloned the cDNA of NOG1 gene from T. gondii. The putative amino acid sequence was scores of amino acid more than congeneric Plasmodium falciparum and Cryptosporidium parvum.