Research On Molecular Taxonomy And Expression Of 28KU Glutathione S-Transferse Gene From Clonorchis Sinensis

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.