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目的克隆大劣按蚊(Anopheles dirus)含硫酯键蛋白1(thioester-containing protein 1,TEP1)的cDNA,并分析其序列。方法根据已报道的冈比亚按蚊等多种昆虫的TEP1氨基酸保守序列设计简并引物,以大劣按蚊血淋巴细胞总RNA为模板,进行RT-PCR扩增。对目的片段纯化回收,经TA克隆后测定其核苷酸序列。利用DNASIS程序分析该序列,并推导其氨基酸序列,最后利用BLAST和DNASIS等进行不同昆虫TEP1氨基酸序列的比对。结果RT-PCR扩增出一约770 bp的条带,TA克隆后DNA测序发现,阳性克隆的核苷酸序列长774 bp,推导其氨基酸序列长258 aa。生物信息学分析表明,该序列与冈比亚按蚊、阿拉伯按蚊、斯氏按蚊TEP1基因的氨基酸序列同源性分别达72%、72%和59%,与冈比亚按蚊的TEP4氨基酸序列同源性为59%。结论从大劣按蚊血淋巴细胞中成功克隆出了TEP1的cDNA部分序列。
Objective To clone cDNA of Anopheles dirus thioester-containing protein 1 (TEP1) and analyze its sequence. Methods Degenerate primers were designed according to the reported conserved sequence of TEP1 in many insects, such as Anopheles gambiae. RT-PCR was used to amplify the total RNA of Anopheles dirus. The target fragment was purified and recovered, and its nucleotide sequence was determined by TA cloning. The sequence was analyzed by DNASIS program, and its amino acid sequence was deduced. Finally, the alignment of different insect TEP1 amino acid sequences was carried out by BLAST and DNASIS. Results A 770 bp band was amplified by RT-PCR. The TA cloning DNA sequencing showed that the nucleotide sequence of the positive clone was 774 bp in length and its deduced amino acid sequence was 258 aa. Bioinformatics analysis showed that the homology of this sequence to the TEP1 genes of Anopheles gambiae, Anopheles stephensi and Anopheles stephens was 72%, 72% and 59% respectively, which was homologous to the TEP4 amino acid sequence of Anopheles gambiae Sex is 59%. Conclusion The cDNA sequence of TEP1 was successfully cloned from Anopheles dirus.