The Studies On Trichinella Spp.of Molecular Taxonomy And Expression Of 49ku Structural Protein Gene For Insect Cells

18S rRNA gene, encoded small subunit ribosomal RNA and 1800bp, was 80% in organism and regarded as highly conserved molecules in course of evolution, so 18S rRNA gene was the important marker for organism taxonomy and system evolution. In this study, we cloned and analyzed 18S rRNA gene sequences of five Trichinella isolates. The result is meaningful in identifying and classifying the different Trichinella isolates. The ES protein of Trichinella was the Excretory-Secretory (ES) products of the alive worms. As a detectable antigen of naturally infected animal, ES protein has been proved to have very high levels of sensitivity and specificity. We established baculovirus expression vectors of 49ku ES structural protein gene and obtained recombinant protein. The primers were designed and synthesized according to sequence of the 18S rRNA gene of Trichinella spiralis in GeneBank (U60231). The total RNA of five Trichinella isolates [ISS3 (T. spiralis); ISS10 (T. nativa); HH (Swine isolate); HC (Dog isolate); SW (Cat isolate)] was extracted with Trizol and AGPC improved method according to the manufacture`s instruction. After total RNA was detected with Ultraviolet spectrophotometer, the target gene-18S rRNA gene was amplified by RT-PCR method. We obtained a band about 1800bp. The PCR products were analyzed by agarose gel electrophoresis and cloned by pMD18-T vector and E. coli JM109. The positive recombinant plasmids were sequenced simultaneously by Bioasia Biotechnology co. Ltd (Shanghai, China) and National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences. The sequence analysis result with DNAstar and Genedoc software indicated that the homology of 18S rRNA gene between different isolstes was more than 99%. The homology of Swine isolate and T. spiralis is 99.4% and Dog isolate and T. nativa is 99.3%. And the homology of Cat isolate and T. spiralis is 0.4% more than that of Cat isolate and T. nativa. Accordingly, Swine isolate and T. spiralis, Dog isolate and T. nativa was more closed. This is consistent with the result of traditional identification methods. In this study, we cloned 18S rRNA gene of five Trichinella spiralis isolates successfully and submitted the sequences of T. nativa and Swine isolate to National Center for Biotechnology Information (NCBI). The sequence accession were AY487254 and AY497012 respectively. The study offered a new idea and method for the Trichinella taxonomy.We designed a pair of primer according to the donor plasmid pFastBAC HTa (BAC-TO-BAC Baculovirus Express System). The donor plasmid and the target gene were digested with restriction endonuclease BamHâ… and Hindâ…¢. After the target gene was linked with donor plasmid, the product was cloned by E. coli JM109. The recombinant donor plasmid was transformed into DH10Bac. The bacmid plasmid DNA transfected insect cells through lipid-mediated transfection. We obtained 36ku confluent protein. Through SDS-PAGE and Western blot analysis, it was indicated that the expressed products were specifically detected by positive serum of mouse against Trichinella.Candidate: Li Dongmei Preventive Veterinary MedicineSupervisor: Prof. Song Mingxin...