论文部分内容阅读
目的克隆一个新的旋毛虫基因即亲肌肉抗原(myophilin)并分析其序列同源性,为旋毛虫病疫苗的研制奠定基础。方法检索GenBank旋毛虫基因组数据库,获得旋毛虫亲肌肉抗原的cDNA序列,利用Primer5.0软件设计引物(上、下游引物分别含NdeⅠ和XhoⅠ酶切位点)。收集云南株旋毛虫肌幼虫,提取总RNA,并以此为模板进行RTPCR。将PCR产物进行2%琼脂糖凝胶电泳,目的基因切胶回收后与克隆载体pMD-19T分别用NdeⅠ、XhoⅠ双酶切,酶切产物以3︰1的比例与pMD-19T连接,然后转化到DH5a大肠埃希菌感受态细胞中,用Amp抗性培养基培养,阳性克隆提质粒后做PCR及双酶切鉴定,阳性菌液进行测序,测序结果与检索基因核苷酸序列通过DNAMAN软件比较,分析其同源性。结果 RT-PCR扩增出旋毛虫亲肌肉抗原全长基因序列,大小为579bp;亲肌肉抗原基因重组质粒经双酶切得到的片段大小分别为579、2 300和300bp,与预期值相符;该基因序列与检索基因核苷酸序列相比,同源性为100%。结论成功克隆出旋毛虫亲肌肉抗原基因,为旋毛虫病疫苗的研制奠定了基础。
Objective To clone a new Trichinella spiralis gene, myophilin, and analyze its sequence homology, and lay a foundation for the development of a trichinosis vaccine. Methods The GenBank Trichinella spiralis genome database was searched and the cDNA sequence of Trichinella spiralis muscle antigen was obtained. Primer5.0 software was used to design the primers (Nde Ⅰ and Xho Ⅰ restriction sites). Collecting Trichinella spiralis larvae of Yunnan strain, extracting total RNA, and using it as a template for RTPCR. The PCR product was subjected to 2% agarose gel electrophoresis. The target gene was recovered by gel filtration and cloned into pMD-19T with NdeI and XhoI digestion respectively. The digested product was ligated to pMD-19T in a ratio of 3: 1, then transformed To E. coli DH5a competent cells, with Amp resistant culture medium, the positive cloning plasmid and PCR and double restriction enzyme digestion, positive bacteria were sequenced, sequencing and nucleotide sequence of the search gene DNAMAN software Compare and analyze their homology. Results The full-length gene sequence of Trichinella spiralis progenitor muscle antigen was amplified by RT-PCR and its size was 579bp. The size of the recombinant plasmid of pro-muscle antigen gene was 579,2300 and 300bp respectively, which was consistent with the expected value Compared with the nucleotide sequence of the retrieved gene, the homology was 100%. Conclusion The cloned muscle antigen of Trichinella spiralis was successfully cloned, which lays the foundation for the development of trichinosis vaccine.