安徽省石台县日本血吸虫rDNA-ITS2和mtDNA-COX1遗传多态性研究

Genetic polymorphisms of rDNA-ITS2 and mtDNA-COX1 of schistosomajaponicum in Shitai County, Anhui Province

目的:研究安徽石台县日本血吸虫的核糖体DNA第二内转录间隔区(rDNA-ITS2)基因和线粒体细胞色素C氧化亚基1基因(mtDNA-COX1)的遗传变异。方法:收集两种不同来源的阳性钉螺(石台和南京),逸出尾蚴后感染小鼠,每只小鼠感染(20±2)尾蚴,35天后解剖小鼠,收集血吸虫成虫,PCR-RFLP扩增成虫的rDNA-ITS2和mtDNA-COX1基因,限制性核酸内切酶Taq I水解,观察结果;同时收集小鼠的肝脏和血液,HE染色观察肝脏肉芽肿的大小,ELISA检测小鼠血清中的谷丙转氨酶(ALT)。结果:石台组和南京组均获得500 bp的ITS2序列和1400 bp的mtDNA-COX1序列;两组的ITS2序列经Taq I酶切后,得到一样的片段;而mtDNA-COX1序列经Taq I酶切后,两组结果不同,石台组在1000 bp检测到1条清晰的条带,而南京组在此处无任何条带;两组小鼠的肝脏纤维化病理结果与谷氨酸转氨酶测定结果均无明显差异。结论:石台组和南京组的日本血吸虫虽然在致病性方面具有相似性,但在mtDNA-COX1基因序列存在差异,本研究为今后日本血吸虫的种群遗传结构研究提供科学依据。

Objective:To analysis the genetic variation of rDNA-ITS2 and mtDNA-COX1 of Schistosoma japonicum in Shitai County,Anhui province.Methods:Oncomelania hupensis were collected from the Shitai County and the Nanjing City.Cercariae escaped from the infected snails obtained in diverse locality were used to infect laboratory-raised female C57BL/6 mice by dose of(20±2)cercariae in individual mouse.Then the mice were dissected 35 days after infection to collect the adult worms via portal vein perfusion technique.DNA was extracted from the adults to undergo PCR-RFLP amplification ITS2 and mtDNA-COX1 gene fragments.The gene was digested with restriction endonuclease Taq I,and the results were observed.At the same time,the liver and blood of mice were collected,the size of liver granuloma was observed by HE staining,and the serum glutamic transaminase(ALT)was detected by ELISA.Results:A 500 bp ITS2 sequence and A 1400 bp COX1 sequence were obtained from Shitai group and Nanjing group.The ITS2 sequences of the two groups were digested by Taq I,and the results were the same.However,the results of Taq I digestion of mtDNA-COX1 sequences of the two groups were different.A clear band can be seen in the Shitai group with a size of 1000 bp.No bands can be seen in the Nanjing group.There was no significant difference in liver fibrosis and ALT between the two groups.Conclusion:The Schistosoma japonicum in the two groups were similar in pathogenicity,but there were differences in the COX1 genetic sequence.The results of the study provide data for elucidating the genetic structure of Schistosoma japonicum from China in the future.