Using COⅠand Cyt B Sequences To Analysis Echinococcus Isolates And Preliminary Analysis On Protein Expression By Western-blot Of Qinghai Plateau

Objective 1. To analysis the gene polymorphism of Echinococcus in the intermediate host by mitochondrial cytochrome C oxidase subunit Ⅰ(COⅠ) gene and mitochondrial cytochrome B(Cyt B) gene in Qinghai province. 2. To provide theoretical basis for selecting diagnostic antigens and vaccine antigens, and the separation and identification of drug target molecule antigen by analysing the protein expression between cystic echinococcosis and alveolar hydatid.Methods 1. The analysis for nucleotide homology and composition and genetic distance calculation in all sequences were based on Cyt B gene and COⅠ gene of Echinococcus.spp by appropriate DNA softwares. Echinococcus haplotype network diagrams were drawn. Bayesian tree and NJ tree were constructed. 2. SDS-PAGE was used to analyse the protein expression extracted from wall, cyst fluid, protoscolex and alveolar hydatid organization in different human isolates. Specific proteins from cystic echinococcosis and alveolar hydatid were identified by Western-blot when the serum from patient were treated as first antibody.Results 1. Echinococcus haplotype network diagram and phylogenetic tree were successfully constructed based on COⅠand Cyt B gene. 2. There are 241 variable sites and 149 parsimony informative sites in 816 nucleotide sites of COⅠgene. A, T, C, G nucleotide frequencies were 0.163, 0.491, 0.103, 0.242 respectively. All sequences average genetic distance was 0.068, whereas the genetic distance between outgroup Taenia solium(AB086256) and CH3 was 0.204 at largest. 3. 541 nucleotide sequences of Cyt B gene were identified with 241 variable sites and 149 parsimony informative sites. of which the A, T, C, G nucleotide frequencies were 0.191,0.101,0.24,0.468, respectively. All sequences genetic distance was 0.068 on average, in which the distance outgroup Taenia solium(AB086256) and CH14,ZH22 was 0.311. 4. Patients infected by cystic echinococcosis were with rich protoscolex protein strip tested by SDS-PAGE. The results of Western-blot indicated the expression of protoscolex protein were rich with specific strips at 117 KD, 49 KD, 34 KD and no significant differences between cyst wall and cyst fluid. 5. SDS-PAGE showed alveolar hydatid protein expressed affluent at 72 KD, 55 KD and 26 KD and specific strips were appeared at 55 KDand 26KD by Western-blot. Conclusion 1. Echinococcus granulosus and Echinococcus multilocularis were identified from the intermediate host basd on COⅠgene and Cyt B gene. 2. Echinococcus granulosus is G1 genotype. Haplotype treated for analysis presented a comb-shaped kind in Bayesian tree and NJ tree with intraspecific mutations. 3. CH14 was the advantage haplotype in Qinghai province. 4.Expression of Echinococcus granulosus protein in different human body was insignificant, whereas the expression were significant between cyst wall, cyst fluid and protoscolex protein in the same worm. 5. Echinococcus multilocularis protein expression were different from distinctive regions and inhabitants.