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目的通过分析18SrRNA基因序列同源性,对河南猪株旋毛虫进行分子鉴定及分类。方法收集河南猪株旋毛虫成虫,提取总RNA,反转录合成cDNA,经特异引物扩增获得18SrRNA基因片段。将此目的基因与pMD18-T载体连接,转化大肠埃希菌感受态细胞,阳性克隆经PCR及酶切鉴定后进行序列测定及分析,构建系统发育树。结果构建的重组质粒酶切片段大小分别为2 700和1 800bp,与预期值相符。根据18SrRNA碱基序列构建系统发生树,河南猪株旋毛虫与虫株Trichinella nativa(AY487254.1)的亲缘关系较近,同源性为99.1%。结论河南猪株旋毛虫归属于T2。
Objective To identify and classify the Trichinella spiralis strain of Henan pig by analyzing the sequence homology of 18S rRNA gene. Methods Adult Trichinella spiralis strains were collected from Henan pigs. Total RNA was extracted and cDNA was reverse transcribed to obtain 18S rRNA gene fragments amplified by specific primers. The target gene was ligated with pMD18-T vector and transformed into E.coli competent cells. The positive clones were identified by PCR and restriction enzyme digestion, then sequenced and analyzed to construct phylogenetic tree. Results The size of constructed recombinant plasmid fragments was 2 700 and 1 800 bp, respectively, which was consistent with the expected value. According to the 18S rRNA sequence, phylogenetic tree was constructed. The genetic relationship between Trichinella nativa (AY487254.1) and Trichinella nativa (AY487254.1) in Henan pig was close and the homology was 99.1%. Conclusion Trichinella in Henan pig belongs to T2.