旋毛虫河南株TspE1基因克隆及多态性分析

目的：构建旋毛虫河南成囊前期幼虫编码相对分子质量为31000的蛋白原结构基因（TspE1）的重组质粒（pUC18－TspE1)，测定TspE1基因序列，分析旋毛虫河南株的基因多态性。方法：根据TspE1基因已知序设计合成一对引物，采用RT－PCR技术获取旋毛虫成囊前期幼虫目的基因，PCR产物经纯化后用BamHI、HindⅢ进行双酶切，定向克隆入pUC18质粒，转化在肠杆菌JM109；重组质粒用BamHI＋HindⅢ酶切有PCR扩增鉴定。用Sanger双脱氧链终止法进行了DNA序列测定，应用DNASIS软件进行同源性比较。结果：RT－PCR扩增获得成囊前期幼虫TspE1基因（871bp），EcoRI酶鉴定正确；筛选出7个阳性克隆，对目的基因的测序结果显示旋毛虫河南株TspE1基因有5种类型，但都与GenBank中的TspE1基因序列及其由其推测的氨基酸序列不完全相同。结论：应用RT－PCR技术手增出旋毛虫河南株成囊前期成幼虫编码相对分子质量为31000的抗原结构基因，证实TspE1基因在成囊前期幼虫已有表达，结果还提示旋毛虫河南株可能存在有基因多态性。

Molecular cloning and genetic polymorphism analysis of TspE1 gene of Henan isolates of TricHinella spiralis

Aim: To construct a recombinant plasmid containing the structural gene (TspE1) encoding an antigen of 31 000 molecular weight (MW) of Henan isolates of Trichinella spiralis pre-encysted larvae, sequence the TspE1 gene and analyze the genetic polymorphism for Henan isolates of T. spiralis.Methods: One pair of primers was de-signed according to the known sequence of TspE1 gene. The target gene of T. spiralis pre-encysted larvae was ob-tained by using RT-PCR technique. The PCR products were purified and digested by BamHI and HindIII. The generated DNA fragment was cloned into the pUC18, and then transferred into Escherichia coli ( E.coli) strain JM109. The recombinant plasmids were screened and identified by Bam HI and HindIII digestion and PCR amplification.The DNA sequence of TspE1 gene was determined by dideoxy chain termination method.The DANSIS software was used to analyze the TspEl gene sequence, and compare the homology.Results: The TspE1 gene with about 871 bp in length was gained by RT-PCR amplification and was accorded with expected one . Seven positive clones were obtained by screening. The sequencing of target genes showed that there were 5 types of gene sequence in Henan isolates of T.spiralis, and they were different from that of TspEl gene reported in Genebank.Conclusions: The structural gene (TspE1) encoding an antigen of 31 000 molecular weight (MW) of Henan isolates of T. spira-lis pre-encysted larvae has been obtained by RT-PCR and expressed in the stage of pre-encysted larvae. The results also suggest the genetic polymorphism in Henan isolates of T. spiralis.