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目的:构建旋毛虫河南株成囊前期幼虫编码相对分子质量为31 000的蛋白抗原结构基因(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,定向克隆人 pUC18质粒,转化大肠杆菌JM109;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。结果:RT－PCR扩增获得成囊前期幼虫TspE1基因(871 bp),EcoR I 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基因序列及由其推测的氨基酸序列不完全相同。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相对分子质量为31 000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。
Objective: To construct a recombinant plasmid (pUC18-TspE1) encoding the TspE1 gene of pre-larvae of Trichinella spiralis strain of Henan Strain and to determine the TspE1 gene sequence and analyze the genetic polymorphism of Trichinella spiralis Henan strain . Methods: According to the known sequence of TspE1 gene, a pair of primers was designed and synthesized. RT-PCR was used to obtain the target gene of pre-larvae of Trichinella spiralis. The PCR products were purified by double enzyme digestion with BamHI and HindⅢ, and cloned into pUC18 plasmid. Escherichia coli JM109; recombinant plasmid was digested with BamHI + Hind Ⅲ and identified by PCR amplification. DNA sequencing was performed using the Sanger dideoxy chain termination method and DNASIS software was used for homology comparison. Results: The TspE1 gene (871 bp) was amplified by RT-PCR and identified by EcoR I restriction endonuclease digestion. Seven positive clones were screened. Sequencing results showed that TspE1 gene of Trichinella spiralis strain had 5 types , But both of them are not exactly the same as the TspE1 gene in GenBank and the deduced amino acid sequence. Conclusion: RT-PCR technique was used to amplify the antigenic gene with a relative molecular mass of 31 000 encoded by the larvae of the adult Trichinella spiralis strain. Confirmed TspE1 gene in pre-sac larvae have been expressed, the results also prompted Trichinella Henan strains may have genetic polymorphisms.