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目的构建刺毛虫毒刺的cDNA文库,克隆其毒素编码的基因。方法提取刺毛虫毒刺中的总RNA;使用clontech公司的cDNA文库构建试剂盒进行反转录,长距离PCR方法合成第二条链,酶切纯化后与载体连接,电转化入感受态细胞XL1-Blue,通过抗性筛选后进行菌落PCR鉴定,挑选插入子在300bp以上的单克隆送至测序公司测序。结果成功构建了刺毛虫毒刺cDNA文库,初步鉴定其插入子长度为250～1 000bp。结论刺毛虫毒刺cDNA文库构建成功,并获得大量的编码刺毛虫毒素的全长基因,为研究其致病机制奠定了基础。
Objective To construct a cDNA library of thorn cercarillas and clone the gene encoding the toxin. Methods The total RNA was extracted from the thorn of the cercariae. The clontech cDNA library was used for reverse transcription. The second strand was synthesized by the long-range PCR method. After digestion and purification, the vector was ligated with the vector and transformed into competent cells XL1 -Blue, identified by colony PCR after resistance screening, and the clones with the insertion of more than 300bp were selected and sent to the sequencing company for sequencing. Results The cDNA library of thorn crab stinger was successfully constructed. The length of the insert was 250-1 000 bp. Conclusion The cDNA library of thorn crab stings was successfully constructed and a large number of full-length genes encoding toxin were obtained, which laid the foundation for the study of its pathogenesis.