Cloning And Prokaryotic Expression Of 49 Ku Structural Protein Gene For Trichinella Nativa

The ES protein of Trichinella nativa(T. nativa, T2) is the Excretory-Secretory products of their living worms. As a detectable antigen of naturally infected animal, ES protein has been proved to have very high levels of sensitivity and specificity. While as an immunogen, it can induce a strong immunologoca! protection to animals. At the same time, the encoding gene of the ES protein has the important application in classification of Trichinella. However, the extensive study and large-scale application of ES protein are restricted because of its difficult preparation. Therefore, it is possible to prepare for the specific recombinant antigen if only we can acquire the specific genes encoding for T. nativa, and we expect to clone and express the gene encoding for the 49 ku antigen in this report.We cultured T. nativa in vitro and analyzed the antigenicity of ES protein. We verified that the ES antigen can induce specific immune reaction.The primers were designed and synthesized based on the gene sequence of the 49 ku protein of Trichinella spiralis(T. spiralis, T1), which was published by Su XZ. T. nativa RNA was extracted with Trizol, which was used according to the manufacture's instruction.After detected with Ultrospec 3000 The target gene-TNPG was amplified by RT-PCR method. The PCR products were analyzed by agarose gel electrophoresis and cloned by pMD-18 T vector and E. coli. The sequence analysis indicated that TNPG consist of 951bp and are closely related with T. spiralis with 98.63% nucleotide identity and 99.05% derived amino acid identity respectively.The TNPG gene was digested from cloned T vector and cloned into the BamH I site of the prokaryotic expression vector pET-30a, resulting in the construct pET-30a-TNPG. The recombinant expression plasmid was transformed into the prokaryotic cell BL-21. The TNPG gene was expressed by induction with IPTG. After ultrasonic disruption, the expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that TNPG was expressed efficiently in prokaryotic cell BL-21. The expression products were 40.8 ku confluent protein and account for 22.8% of the whole proteins.It was also demonstrated from western blot that the expression products can be specificallydetected by positive serum of mouse against T. nativa.In summary, T. nativa recombinant protein expressed in E.coli is a very good candidate for development of T. nativa recombinant antigen and has intriguing potential application for the detection and immunization of T. nativa.Candidate: Zheng Baoliang Preventive Veterinary ScienceSupervisor: Song Mingxin...