华支睾吸虫分子分类及其28kuGSTs原核表达的研究
|
摘要
华支睾吸虫病是由于华支睾吸虫(Clonorchis sinensis)寄生于人、猫、犬等动物的肝脏和胆囊内而引起的一种人兽共患寄生虫病。近年来,华支睾吸虫的研究主要集中在其形态特征的描述、生活史的研究及华支睾吸虫病的流行病学调查、诊断、药物防治和预防措施等方面,关于其分子分类和重组抗原的研究少见报道。核糖体DNA(rDNA)是细胞核内编码核糖体RNA的基因,由内部转录间隔区(ITS)和3种核糖体基因编码区(18S、5.8S、28S)等组成,不同区域其进化速率不同。线粒体DNA(mtDNA)为母系遗传,其基因间很少重组,所以可以反映出母系的进化历史,这样线粒体一个基因就可以代表整个线粒体基因组的变异情况。因此,本实验以华支睾吸虫核糖体内转录间隔区1(ITS1)基因、线粒体细胞色素c氧化酶亚基2(COX2)基因和烟酰胺腺嘌呤二核苷酸脱氢酶亚基3(NAD3)基因为研究对象,对目的片段进行克隆和序列测定,构建分子系统发生树,来探讨华支睾吸虫在后睾科中的系统发生位置。谷胱甘肽S转移酶(GSTs)是广泛存在于各种生物体内的一组抗氧化反应同工酶,通过催化内源性和外源性毒素与谷胱甘肽的结合,从而解除毒性复合物的毒性,对寄生虫在宿主体内存活发挥着重要作用。同时,GSTs具有较强的免疫原性,可用于免疫诊断或作为候选疫苗分子用于免疫预防。
本研究以采自宾县、大庆、海伦、双城、泰来、同江和长春7个地区的华支睾吸虫为研究对象,经PCR扩增ITS1、COX2和NAD3基因,采用序列分析方法研究不同地区华支睾吸虫ITS1、COX2和NAD3基因的多态性,并与GenBank登录的麝猫后睾吸虫、猫后睾属吸虫、东方次睾吸虫、广西株、沈阳株、韩国株和美国株华支睾吸虫等进行比对分析。使用Phylip3.66软件采用邻接法(NJ)绘制系统发生树。序列分析结果表明,华支睾吸虫核糖体ITS1、COX2和NAD3编码基因大小分别为661 bp,636 bp和357 bp;不同地区华支睾吸虫ITS1、COX2和NAD3基因序列存不同程度的差异,同源性分别在99.4 %~100 %、98.9 %~100 %和99.2 %~100 %之间。以肝片形吸虫作外群,基于ITS1、COX2和NAD3基因序列采用NJ法构建的系统发生树,其结果一致,均显示华支睾吸虫的分类地位处于后睾科内,同时证明各地区存在差异。以上结果显示:ITS1、COX2和NAD3基因均可作为遗传标记用以鉴定华支睾吸虫在后睾科的位置,科内属间的遗传关系,还可以用来区分属下种间的遗传关系。
提取人源华支睾吸虫总RNA,应用RT-PCR技术扩增出华支睾吸虫28ku谷胱甘肽S-转移酶(GSTs)基因。对此基因片段进行克隆,经鉴定正确后,亚克隆到表达载体pET-32a(+)上,构建重组质粒pET-32a(+)-GSTs,转化大肠埃希菌BL21中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达;表达产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot)分析和鉴定。本研究结果显示人源华支睾吸虫GSTs编码区含有639个碱基,编码212个氨基酸残基;表达的蛋白存在于上清中,大小约为42.68ku,可被感染华支睾吸虫人的阳性血清识别,证明GSTs基因可在原核表达系统中高效表达并具有反应原性。
Adult worms of Clonorchis sinensis live in the liver or gallbladder of human, cat ,dog and other animals. In recent years, researches are mainly focused on the morphology, life cycle of this pathogen, and epidemiology, diagnosis, treatment, prevention. However, little has been done for the classification and recombination protein of C.sinensis. Ribosomal DNA (rDNA) was generally existed in the living nature, it has numerous characters for using as the DNA molecular maker, and for example it has a lot of multicopy and different revolutionary rates in different regions. The main compositions in the ribosomal DNA are internal transcribed spacer region (ITS), three ribosomal gene coding regions (18S, 5.8S and 28S), which be applied in molecular phylogeny. Since mitochondrial DNA (mtDNA) is maternal inheritance, seldom recombination between them, and one of the mitochondrial genomes may represent the whole variation, they can be used to determine the molecular phylogeny. The ITS1, the COX2 and NAD3 genes were amplified by PCR and cloned into pMD18-T easy vector and then sequenced and analyzed, and compared with other sequences of schistosome, counstucted phylogeny tree then determined the molecular phylogeny. Glutathione S-transferases (GSTs) is a superenzyne encoded by several genes and possessing multifunctions. It is also a major detoxification system in a number of organisms.
Clonorchis sinensis isolates were collected from Binxian, Daqing, Hailun, Shuangcheng, Tailai, Tongjiang and Changchun, respectively. The complete ITS1, COX2 and NAD3 gene of these isolates were sequenced by PCR. The objective was to examine the DNA polymorphism in ITS1, COX2 and NAD3 of C.sinensis from different geographical locations in China. To explore the sequence divergences a neighbor-joining tree was established by comparing with the ITS1, COX2 and NAD3 sequences of Opisthorchis viverrini, Opisthorchis felineus, Metorchis.orientalis, C.sinensis (Guangxi, Shenyang, Korea and America isolate) deposed in GenBank, respectively. Sequencing results showed that ITS1, COX2 and NAD3 were 661 bp, 636 bp and 357 bp, respectively. Identity of the ITS1, COX2 and NAD3 sequences was 99.4% to 100%, 98.9 % to 100 % and 99.2 % to 100 %, respectively. This study showed that ITS1, COX2 and NAD3 could be applied as genetic marker in identifying intergeneric genetic relation and classifying interspecific genetic relation of C.sinensis.
The gene of 28 ku Glutathione S-transferse (GSTs) was amplified by RT-PCR from total RNA of Clonorchis sinensis was extracted from human. The PCR product was cloned into pET-32a(+)vector, and the recombinant plasmids were transformed into E.coli BL21.The expressed protein induced by IPTG was purified and identified by SDS-PAGE and western blot.The results showed that the GSTs contained 639 base pairs encoding 212 amino acids. The SDS-PAGE electrophoresis analysis illustrated that the fusion protein mainly existed in the supernatant, indicating a soluble expression. The protein had a MW about 42.68 ku. The recombinant protein could react with positive serum from human naturally infected with C.sinensis, indicating that the GSTs expressed in prokaryotic expression system retained antigenicity of the native protein.
本研究以采自宾县、大庆、海伦、双城、泰来、同江和长春7个地区的华支睾吸虫为研究对象,经PCR扩增ITS1、COX2和NAD3基因,采用序列分析方法研究不同地区华支睾吸虫ITS1、COX2和NAD3基因的多态性,并与GenBank登录的麝猫后睾吸虫、猫后睾属吸虫、东方次睾吸虫、广西株、沈阳株、韩国株和美国株华支睾吸虫等进行比对分析。使用Phylip3.66软件采用邻接法(NJ)绘制系统发生树。序列分析结果表明,华支睾吸虫核糖体ITS1、COX2和NAD3编码基因大小分别为661 bp,636 bp和357 bp;不同地区华支睾吸虫ITS1、COX2和NAD3基因序列存不同程度的差异,同源性分别在99.4 %~100 %、98.9 %~100 %和99.2 %~100 %之间。以肝片形吸虫作外群,基于ITS1、COX2和NAD3基因序列采用NJ法构建的系统发生树,其结果一致,均显示华支睾吸虫的分类地位处于后睾科内,同时证明各地区存在差异。以上结果显示:ITS1、COX2和NAD3基因均可作为遗传标记用以鉴定华支睾吸虫在后睾科的位置,科内属间的遗传关系,还可以用来区分属下种间的遗传关系。
提取人源华支睾吸虫总RNA,应用RT-PCR技术扩增出华支睾吸虫28ku谷胱甘肽S-转移酶(GSTs)基因。对此基因片段进行克隆,经鉴定正确后,亚克隆到表达载体pET-32a(+)上,构建重组质粒pET-32a(+)-GSTs,转化大肠埃希菌BL21中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达;表达产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot)分析和鉴定。本研究结果显示人源华支睾吸虫GSTs编码区含有639个碱基,编码212个氨基酸残基;表达的蛋白存在于上清中,大小约为42.68ku,可被感染华支睾吸虫人的阳性血清识别,证明GSTs基因可在原核表达系统中高效表达并具有反应原性。
Adult worms of Clonorchis sinensis live in the liver or gallbladder of human, cat ,dog and other animals. In recent years, researches are mainly focused on the morphology, life cycle of this pathogen, and epidemiology, diagnosis, treatment, prevention. However, little has been done for the classification and recombination protein of C.sinensis. Ribosomal DNA (rDNA) was generally existed in the living nature, it has numerous characters for using as the DNA molecular maker, and for example it has a lot of multicopy and different revolutionary rates in different regions. The main compositions in the ribosomal DNA are internal transcribed spacer region (ITS), three ribosomal gene coding regions (18S, 5.8S and 28S), which be applied in molecular phylogeny. Since mitochondrial DNA (mtDNA) is maternal inheritance, seldom recombination between them, and one of the mitochondrial genomes may represent the whole variation, they can be used to determine the molecular phylogeny. The ITS1, the COX2 and NAD3 genes were amplified by PCR and cloned into pMD18-T easy vector and then sequenced and analyzed, and compared with other sequences of schistosome, counstucted phylogeny tree then determined the molecular phylogeny. Glutathione S-transferases (GSTs) is a superenzyne encoded by several genes and possessing multifunctions. It is also a major detoxification system in a number of organisms.
Clonorchis sinensis isolates were collected from Binxian, Daqing, Hailun, Shuangcheng, Tailai, Tongjiang and Changchun, respectively. The complete ITS1, COX2 and NAD3 gene of these isolates were sequenced by PCR. The objective was to examine the DNA polymorphism in ITS1, COX2 and NAD3 of C.sinensis from different geographical locations in China. To explore the sequence divergences a neighbor-joining tree was established by comparing with the ITS1, COX2 and NAD3 sequences of Opisthorchis viverrini, Opisthorchis felineus, Metorchis.orientalis, C.sinensis (Guangxi, Shenyang, Korea and America isolate) deposed in GenBank, respectively. Sequencing results showed that ITS1, COX2 and NAD3 were 661 bp, 636 bp and 357 bp, respectively. Identity of the ITS1, COX2 and NAD3 sequences was 99.4% to 100%, 98.9 % to 100 % and 99.2 % to 100 %, respectively. This study showed that ITS1, COX2 and NAD3 could be applied as genetic marker in identifying intergeneric genetic relation and classifying interspecific genetic relation of C.sinensis.
The gene of 28 ku Glutathione S-transferse (GSTs) was amplified by RT-PCR from total RNA of Clonorchis sinensis was extracted from human. The PCR product was cloned into pET-32a(+)vector, and the recombinant plasmids were transformed into E.coli BL21.The expressed protein induced by IPTG was purified and identified by SDS-PAGE and western blot.The results showed that the GSTs contained 639 base pairs encoding 212 amino acids. The SDS-PAGE electrophoresis analysis illustrated that the fusion protein mainly existed in the supernatant, indicating a soluble expression. The protein had a MW about 42.68 ku. The recombinant protein could react with positive serum from human naturally infected with C.sinensis, indicating that the GSTs expressed in prokaryotic expression system retained antigenicity of the native protein.
引文
仇建华,李利,王春仁等.2008.牛羊源土耳其斯坦东毕吸虫ITS和28SrNDA-LSU序列分析[J].中国寄生虫学与寄生虫病杂志.26(3):183-186
崔惠儿,裴福全,长野功等.2004.华支睾吸虫重组蛋白应用于ABC-ELISA检测特异性循环抗体的研究[J].热带医学杂志4(2):136-138
董晓波,李冬梅,花丽茹等.2006.旋毛虫ITS II区基因的克隆及其在分类学上的应用[J].畜牧兽医学报.37(2):173-175
郭凯文,牛安欧.2004.PCR-SSCP研究中国日本血吸虫线粒体DNA的遗传变异[J].中国人兽共患病杂志.20(6):529-532
何芳,屈仁建,吴绍强等.2005.用PCR-RFLP技术鉴别鲁道夫对盲囊线虫姊妹种的研究[J].华南农业大学学报.26(2):115-117
何丽洁,邹学华,罗金萍.2005.重组华支睾吸虫26 ku GST蛋白用于检测特异抗体的研究[J].热带医学杂志.5(2):197-199
胡旭初,徐劲,陈守义等.2003.华支睾吸虫病金标诊断试剂盒的研制和现场初步实验[J].中国寄生虫病防治杂志.16(3):152-154
李利,邢继兰,王春仁等.2008.4种终末宿主土耳其斯坦东毕吸虫COX1和NAD1基因的序列分析[J].中国兽医科学.38(04):303-307
林瑞庆,董世娟,胥楚雄等.2004.片形吸虫第一内转录间隔区DNA多态性的研究[J].中国兽医科技.34(7):8-12
牛安欧,冯友仁,方正明等.2003中国日本血吸虫DNA遗传变异及聚类分析[J].寄生虫与医学昆虫学报.10(2):70-73
全国人体重要寄生虫病现状调查办公室.2005.全国人体重要寄生虫病现状调查报告[J].中国寄生虫学与寄生虫病杂志.23(5)增刊:332-340
吴德.2002.华支睾吸虫病的流行概况[J].热带医学杂志.2(3):289-294
吴太葆,夏斌,邹志文等.2007.椭圆食粉螨线粒体DNA COI基因片段序列分析[J].蛛形学报.16(2):79-82
伍忠銮,余新炳,吴德等.2004.华支睾吸虫的一个μ型GST基因的表达、纯化与免疫反应性鉴定[J].中山大学学报.25(2):105-109
谢觅,何毅勋,裘丽妹等.1997.我国与日本山梨不同地域品系日本血吸虫DNA的限制性片段长度多态性分析中国寄生虫学与寄生虫病杂志.15(4):216-219
许隆祺,余森海,徐淑惠等.2000.中国人体寄生虫分布与危害[M].北京:人民卫生出版社.695-697
薛亚娟,惠清法.2006.食源性寄生虫病的研究进展[J].延安大学学报.4(2):3-4
杨毅梅,何作顺.2004.我国食源性寄生虫病的流行与致病现状[J].中国自然医学杂志.6(3);206-208
杨毅梅.2004.常见食源性寄生虫病的感染与防治[J].大理学院学报.3(3):49-51
易冰,李卓雅,孟锦绣等.2003.微卫星DNA在寄生虫学研究中的应用[J].中国人兽共患病杂志.19(2):85-87.
张朝云,杨明,张科等.2006.云贵两省三地牛带绦虫核糖体rDNA-ITS1序列分析[J].贵阳医学院学报.31(4):291-295
左胜利,桂爱芳,杨连第等.1998.华枝墨吸虫病流行现状与防治对策的研究[J].疾病控制杂志.2(2):124-126
Anou N, Yanwen X, Youren F. 2002. Schistosoma japonicum strains: differentiation by RAPD and SSR-PCR [J]. Southeast Asian J Trop Med Public Health. 33(4): 720-724
Choi B I, Han J K, Hang S T, et al. 2004. Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis[J]. Clin Microbiol Rev.17(3):540-542
Choi M H, Ryu J S, Lee M J, et al. 2008. Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda) [J]. Korean J Parasitol. 41(3): 155-163
Crampton A, Mckay I, Barker SC. 1996. Phylogeny of ticks (Ixodida) inferred from nuclear ribosomal DNA [J]. Int J Parasitol. 26(5): 511-517
Dietrich CH, Rakitov RA, Holmes JL, et al. 2001. Phylogeny of the major lineages of Membracoidea (Insecta: Hemiptera: Cicadomorpha) based on 28S rDNA sequences [J]. Molecular Phylogenetics and Evolution. 18(2): 293-305
Gasser GB, Chilton NB, Hoste H, et al. 1993. Raqid sequencing of rDNA from single worms and eggs of parasitic helminthes [J]. Nucleic Acids Res.21(10): 2525-2526
Gasser RB, Qian BZ, Nansen P, et al. 1996. Use of RAPD for the detection of genetic variation in the human blood fiuke, Schistosoma japonicum, from mainland China [J]. Mol Cell Probes. 10(5): 353-358
Hong S J, Kang S Y, Chung Y B, et al. 2000 Paragonimus westermani: A cytosolic glutathione S-transferase of a sigma class in adultstage[J]. Exp Parasitol. 94(3):180-189
Hong S T, Hong S J. 2005. Clonorchis sinensis in Korea[J]. AsianParasitology Series Monograph. 1:35-49
Hoste H, Chilton NB, Beveridge I, et al. 1998. A comparison of the frist internal transcribed spacer of ribosomal DNA in seven species of Trichost rongylus (Nematoda: Trichostrongylidag) [J]. Int J Parasitol. 28(8): 1251-1260.
Huang W Y, He B, Wang C R, et al. 2004. Characterisation of Fasciolaspecies from Mainland China by ITS-2 ribosomal DNA sequence[J]. Vet Parasitol. 120(1-2):75-83
Humbert JF, Cabaret J. 1995. Use of random amplified polymorphic DNA of identification of ruminant trichostrongylid nematodes [J]. Parasitol Res. 81(1): l-5
Jennifer K, Xiao Shu-Hua, Jacques C, et al. 2007. Evaluation of the in vivo activity of Tribendimidine against Schistosoma mansoni, Fasciola hepatica, Clonorchis sinensis, and Opisthorchis viverrini [J]. Antimicrob Agents Chemother. 51(3): 1096-1098
Kang S Y, Ahn I Y, Park C Y, et al. 2001. Clonorchis sinensis: Molecular cloning and characterization of 28 ku glutathione S-transferase[J]. Exp Parasitol. 97(4):186-195
Kim E M, Kim J L, Sung Y C, et al. 2008. Infection status of freshwater fish with metacercariae of Clonorchis sinensis in Korea [J]. Korean J Parasitol. 46(4): 247-251
Lee G S, Cho I S, Lee Y H, et al. 2002.Epidemiological study of clonorchiasis and metagonimiasis along the Geum-gang(River) in Okcheon-gun(county)[J].Korean J Parasito1.40(1):9-16
Lee S U, Sun H. 2006. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis [J]. Korean J Parasitol. 42(3): 145-148
Liebau E,Bergmann B,Campbell A M,et al. 2002. The glutathioneS-transferase from plasmodium falciparum[J].Mol Biochem Parasitol. 124(1):85-90
Maxam AM, Gilbert W. 1977. A new method for sequencing DNA [J]. Proc Natl Acad Sci. 74(2): 560-564
Nadler SA. 1992. Phylogeny of some ascaridoid nematodes, inferred from comparison of 18S and 28S rRNA sequences [J]. Mol Biol Evol. 9(5): 932-944
Neil D, Young B E, Campbell D, et al. 2010. Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini [J]. PLoS Negl Trop Dis, 2010. 4(6): e719
Oliveira RP, Macedo AM, Chiari E, et al. 1997. An alternative approach to evaluating the intraspecific genetic variability of parasites [J]. Parasitology Today. 13(5): 196-200
Roberts LJ, Baldwin TM, Speed TP, et a1. 1999. Chromosomes X, 9, and the H2 locus interact epistatically to control Leishmania major infection [J]. Eur J Immunol. 29(9): 3047
Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with chain-terminating inhibitors [J]. Proc Natl Acad Sci. 74(12): 5463-5467
Sohnoon W M, Zhang Hong-Man, Choi M H, et al. 2006. Susceptibility of experimental animals to reinfection with Clonorchis sinensis [J]. Korean J Parasitol. 44(2): 163-166
Srivatanakul P,Sriplung H,Deerasamee S. 2004.Epidemiology of livercancer an overview[J].Asian Pac J Cancer Prev. 5(2):118
Stevens J, Wall R. 1995. The use of random amplified polymorphic DNA (RAPD) analysis for studies of genetic variation in populations of the blowfly Lucilia sericata (Diptera: Calliphoridae) in southern England [J]. Bull Entomol Res. 85: 549-555
Traub R J, Macaranas J, Mungthin M, et al. 2009. A new PCR-based approach indicates the range of Clonorchis sinensis now extends to central Thailand [J]. PLoS Negl Trop Dis. 3(1): e367
Tsai M H,Marx K A,Ismail M M,et al.2000. Randomly amplifiedpolymorphic DNA (RAPD) polymerase chain reaction assayfor identification ofSchistosoma mansonistrains sensitive ortolerant to anti.schistosomal drugs[J]. J Parasitol. 86(1): 146-149
Van Herwerden L, Blair D, Agatsuma T. 1999. Intra and interindividual variation in ITS-1 of Paragonimus westermani (Trematoda: Digenea) and related species: implications for phylogenetic studies [J]. Mol Phylogenet Evol. 12(1): 67-73
Vaughan HE, Heslop-Harrison JS, Hewitt GM. 1999. The localization of mitochondrial sequences to chromosomal DNA in orthopterans [J]. Genome. 42(5): 874-880
Vermeulen A.N. 1998. Progress in recombinant vaccine development against coccidiosis A review and prospects into the next millennium[J]. International Journal for Parasitology. 28: 1121～1130. .
崔惠儿,裴福全,长野功等.2004.华支睾吸虫重组蛋白应用于ABC-ELISA检测特异性循环抗体的研究[J].热带医学杂志4(2):136-138
董晓波,李冬梅,花丽茹等.2006.旋毛虫ITS II区基因的克隆及其在分类学上的应用[J].畜牧兽医学报.37(2):173-175
郭凯文,牛安欧.2004.PCR-SSCP研究中国日本血吸虫线粒体DNA的遗传变异[J].中国人兽共患病杂志.20(6):529-532
何芳,屈仁建,吴绍强等.2005.用PCR-RFLP技术鉴别鲁道夫对盲囊线虫姊妹种的研究[J].华南农业大学学报.26(2):115-117
何丽洁,邹学华,罗金萍.2005.重组华支睾吸虫26 ku GST蛋白用于检测特异抗体的研究[J].热带医学杂志.5(2):197-199
胡旭初,徐劲,陈守义等.2003.华支睾吸虫病金标诊断试剂盒的研制和现场初步实验[J].中国寄生虫病防治杂志.16(3):152-154
李利,邢继兰,王春仁等.2008.4种终末宿主土耳其斯坦东毕吸虫COX1和NAD1基因的序列分析[J].中国兽医科学.38(04):303-307
林瑞庆,董世娟,胥楚雄等.2004.片形吸虫第一内转录间隔区DNA多态性的研究[J].中国兽医科技.34(7):8-12
牛安欧,冯友仁,方正明等.2003中国日本血吸虫DNA遗传变异及聚类分析[J].寄生虫与医学昆虫学报.10(2):70-73
全国人体重要寄生虫病现状调查办公室.2005.全国人体重要寄生虫病现状调查报告[J].中国寄生虫学与寄生虫病杂志.23(5)增刊:332-340
吴德.2002.华支睾吸虫病的流行概况[J].热带医学杂志.2(3):289-294
吴太葆,夏斌,邹志文等.2007.椭圆食粉螨线粒体DNA COI基因片段序列分析[J].蛛形学报.16(2):79-82
伍忠銮,余新炳,吴德等.2004.华支睾吸虫的一个μ型GST基因的表达、纯化与免疫反应性鉴定[J].中山大学学报.25(2):105-109
谢觅,何毅勋,裘丽妹等.1997.我国与日本山梨不同地域品系日本血吸虫DNA的限制性片段长度多态性分析中国寄生虫学与寄生虫病杂志.15(4):216-219
许隆祺,余森海,徐淑惠等.2000.中国人体寄生虫分布与危害[M].北京:人民卫生出版社.695-697
薛亚娟,惠清法.2006.食源性寄生虫病的研究进展[J].延安大学学报.4(2):3-4
杨毅梅,何作顺.2004.我国食源性寄生虫病的流行与致病现状[J].中国自然医学杂志.6(3);206-208
杨毅梅.2004.常见食源性寄生虫病的感染与防治[J].大理学院学报.3(3):49-51
易冰,李卓雅,孟锦绣等.2003.微卫星DNA在寄生虫学研究中的应用[J].中国人兽共患病杂志.19(2):85-87.
张朝云,杨明,张科等.2006.云贵两省三地牛带绦虫核糖体rDNA-ITS1序列分析[J].贵阳医学院学报.31(4):291-295
左胜利,桂爱芳,杨连第等.1998.华枝墨吸虫病流行现状与防治对策的研究[J].疾病控制杂志.2(2):124-126
Anou N, Yanwen X, Youren F. 2002. Schistosoma japonicum strains: differentiation by RAPD and SSR-PCR [J]. Southeast Asian J Trop Med Public Health. 33(4): 720-724
Choi B I, Han J K, Hang S T, et al. 2004. Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis[J]. Clin Microbiol Rev.17(3):540-542
Choi M H, Ryu J S, Lee M J, et al. 2008. Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda) [J]. Korean J Parasitol. 41(3): 155-163
Crampton A, Mckay I, Barker SC. 1996. Phylogeny of ticks (Ixodida) inferred from nuclear ribosomal DNA [J]. Int J Parasitol. 26(5): 511-517
Dietrich CH, Rakitov RA, Holmes JL, et al. 2001. Phylogeny of the major lineages of Membracoidea (Insecta: Hemiptera: Cicadomorpha) based on 28S rDNA sequences [J]. Molecular Phylogenetics and Evolution. 18(2): 293-305
Gasser GB, Chilton NB, Hoste H, et al. 1993. Raqid sequencing of rDNA from single worms and eggs of parasitic helminthes [J]. Nucleic Acids Res.21(10): 2525-2526
Gasser RB, Qian BZ, Nansen P, et al. 1996. Use of RAPD for the detection of genetic variation in the human blood fiuke, Schistosoma japonicum, from mainland China [J]. Mol Cell Probes. 10(5): 353-358
Hong S J, Kang S Y, Chung Y B, et al. 2000 Paragonimus westermani: A cytosolic glutathione S-transferase of a sigma class in adultstage[J]. Exp Parasitol. 94(3):180-189
Hong S T, Hong S J. 2005. Clonorchis sinensis in Korea[J]. AsianParasitology Series Monograph. 1:35-49
Hoste H, Chilton NB, Beveridge I, et al. 1998. A comparison of the frist internal transcribed spacer of ribosomal DNA in seven species of Trichost rongylus (Nematoda: Trichostrongylidag) [J]. Int J Parasitol. 28(8): 1251-1260.
Huang W Y, He B, Wang C R, et al. 2004. Characterisation of Fasciolaspecies from Mainland China by ITS-2 ribosomal DNA sequence[J]. Vet Parasitol. 120(1-2):75-83
Humbert JF, Cabaret J. 1995. Use of random amplified polymorphic DNA of identification of ruminant trichostrongylid nematodes [J]. Parasitol Res. 81(1): l-5
Jennifer K, Xiao Shu-Hua, Jacques C, et al. 2007. Evaluation of the in vivo activity of Tribendimidine against Schistosoma mansoni, Fasciola hepatica, Clonorchis sinensis, and Opisthorchis viverrini [J]. Antimicrob Agents Chemother. 51(3): 1096-1098
Kang S Y, Ahn I Y, Park C Y, et al. 2001. Clonorchis sinensis: Molecular cloning and characterization of 28 ku glutathione S-transferase[J]. Exp Parasitol. 97(4):186-195
Kim E M, Kim J L, Sung Y C, et al. 2008. Infection status of freshwater fish with metacercariae of Clonorchis sinensis in Korea [J]. Korean J Parasitol. 46(4): 247-251
Lee G S, Cho I S, Lee Y H, et al. 2002.Epidemiological study of clonorchiasis and metagonimiasis along the Geum-gang(River) in Okcheon-gun(county)[J].Korean J Parasito1.40(1):9-16
Lee S U, Sun H. 2006. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis [J]. Korean J Parasitol. 42(3): 145-148
Liebau E,Bergmann B,Campbell A M,et al. 2002. The glutathioneS-transferase from plasmodium falciparum[J].Mol Biochem Parasitol. 124(1):85-90
Maxam AM, Gilbert W. 1977. A new method for sequencing DNA [J]. Proc Natl Acad Sci. 74(2): 560-564
Nadler SA. 1992. Phylogeny of some ascaridoid nematodes, inferred from comparison of 18S and 28S rRNA sequences [J]. Mol Biol Evol. 9(5): 932-944
Neil D, Young B E, Campbell D, et al. 2010. Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini [J]. PLoS Negl Trop Dis, 2010. 4(6): e719
Oliveira RP, Macedo AM, Chiari E, et al. 1997. An alternative approach to evaluating the intraspecific genetic variability of parasites [J]. Parasitology Today. 13(5): 196-200
Roberts LJ, Baldwin TM, Speed TP, et a1. 1999. Chromosomes X, 9, and the H2 locus interact epistatically to control Leishmania major infection [J]. Eur J Immunol. 29(9): 3047
Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with chain-terminating inhibitors [J]. Proc Natl Acad Sci. 74(12): 5463-5467
Sohnoon W M, Zhang Hong-Man, Choi M H, et al. 2006. Susceptibility of experimental animals to reinfection with Clonorchis sinensis [J]. Korean J Parasitol. 44(2): 163-166
Srivatanakul P,Sriplung H,Deerasamee S. 2004.Epidemiology of livercancer an overview[J].Asian Pac J Cancer Prev. 5(2):118
Stevens J, Wall R. 1995. The use of random amplified polymorphic DNA (RAPD) analysis for studies of genetic variation in populations of the blowfly Lucilia sericata (Diptera: Calliphoridae) in southern England [J]. Bull Entomol Res. 85: 549-555
Traub R J, Macaranas J, Mungthin M, et al. 2009. A new PCR-based approach indicates the range of Clonorchis sinensis now extends to central Thailand [J]. PLoS Negl Trop Dis. 3(1): e367
Tsai M H,Marx K A,Ismail M M,et al.2000. Randomly amplifiedpolymorphic DNA (RAPD) polymerase chain reaction assayfor identification ofSchistosoma mansonistrains sensitive ortolerant to anti.schistosomal drugs[J]. J Parasitol. 86(1): 146-149
Van Herwerden L, Blair D, Agatsuma T. 1999. Intra and interindividual variation in ITS-1 of Paragonimus westermani (Trematoda: Digenea) and related species: implications for phylogenetic studies [J]. Mol Phylogenet Evol. 12(1): 67-73
Vaughan HE, Heslop-Harrison JS, Hewitt GM. 1999. The localization of mitochondrial sequences to chromosomal DNA in orthopterans [J]. Genome. 42(5): 874-880
Vermeulen A.N. 1998. Progress in recombinant vaccine development against coccidiosis A review and prospects into the next millennium[J]. International Journal for Parasitology. 28: 1121～1130. .