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目的 构建华支睾吸虫cDNA文库。方法 采集华支睾吸虫阳性鱼 ,分离囊蚴 ,感染实验动物兔 ,解剖兔收集华支睾吸虫成虫虫体。应用“一步法”提取华支睾吸虫总RNA ;经过mRNA纯化、cDNA合成 ,以PcDNA3(Amp +)质粒为载体构建文库。挑取 2 0个克隆进行扩增 ,选择 9个克隆进行DNA序列测定。序列结果与GenBank中相关基因进行比对。结果 获得含有 9 7× 10 5个重组子库容量的华支睾吸虫cDNA文库。其中PC6号克隆基因序列与华支睾吸虫半胱氨酸蛋白酶序列具有 93%的同源性。结论 已构建成华支睾吸虫cDNA文库。
Objective To construct Clonorchis sinensis cDNA library. Methods Clonorchis sinensis positive fish were collected, metacercariae were isolated, rabbits were infected with experimental animals, and adult worms of Clonorchis sinensis were collected. The total RNA was extracted from Clonorchis sinensis by “one-step” method. After purification of mRNA and cDNA synthesis, the library was constructed by using PcDNA3 (Amp +) plasmid as carrier. Twenty clones were picked for amplification, and nine clones were selected for DNA sequencing. The sequence results were aligned with those in GenBank. As a result, a cDNA library of Clonorchis sinensis containing 9 7 × 10 5 recombinants was obtained. Among them, the PC6 clone had 93% homology with the Clonorchis sinensis cysteine protease. Conclusion Clonorchis sinensis cDNA library has been constructed.