旋毛虫成囊前期幼虫编码31kDa抗原基因的分子克隆及序列分析

目的　克隆旋毛虫河南株成囊前期幼虫编码 31kDa抗原基因 (TspE1)片段 ,并测定其基因序列 ,以了解该基因序列与已报道虫株的差异。方法　根据TspE1基因已知序列设计合成一对引物 ,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pC18质粒 ,转化大肠杆菌JM10 9;重组质粒用BamHⅠ +HindⅢ酶切及PCR扩增鉴定。用Sanger双脱氧链终止法进行DNA序列测定 ,应用DNASIS软件进行同源性比较及预测抗原表位。结果　RT -PCR扩增获得成囊前期幼虫TspE1基因 (约 870bp) ,EcoRⅠ酶切鉴定正确 ;筛选出 7个阳性克隆 ,对目的基因的测序结果显示有 5种类型 ,但都与GenBank中的TspE1基因序列及由其推测的氨基酸序列有较高同源性 ,尤其是Ts HN3核苷酸及氨基酸序列同源性分别达 99 6 %和 98 9% ;TspE1基因中可能存在 7个抗原表位。 结论　应用RT -PCR技术扩增出旋毛虫河南株成囊前期幼虫编码 31kDa抗原结构基因 ,证实TspE1基因在成囊前期幼虫已有表达 ;结果还提示在旋毛虫河南株之间可能存在有遗传多态性

Cloning and sequencing of the gene encoding a 31 kDa antigen of Trichinella spiralis pre-encysted larvae

Aim To clone the gene encoding a 31 kDa antigen of Trichinella spiralis (Henan isolates) pre encysted larvae(TspE1),sequence the TspE1 gene and find out the differences of the TspE1 gene sequence between Henan isolates and other isolates previously reported Methods One pair of primers was designed according to the known sequence of TspE1 gene The target gene of T spiralis pre encysted larvae was obtained 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 BamHⅠ 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 TspE1 gene sequence,compare the homology and predict the antigenic epitopes Results The TspE1 gene with about 870 base pairs was gained by RT PCR amplification and 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 T spiralis (Henan isolates),and little difference from that of TspE1 gene reported in Genbank Ts HN3 isolate and other isolates exhibited 99 6% and 98 9% of homology in DNA and amino acid sequences,respectively There may be seven antigenic epitopes in the encoding region of TspE1 gene Conclusion The structural gene encoding a 31 kDa antigen of T spiralis(Henan isolates)pre encysted larvae(TspE1)was obtained by RT PCR and expressed in the stage of pre encysted larvae The results also suggested the intraspecies polymorphism among Henan isolates of T spiralis