Theileria Sergenti P33 Gene And Cattle Interleukin-18 Tandem Expression

Theileria sergenti is blood hemozoon of theileriidae and theileria.which parasitize the bovine erythrocyte, Infected bovine mainly for chronic anemia, hyperpyrexia, emaciation, lymphadenectasis and other symptoms. Due to infected bovine decreased immunity can lead to secondary infection Which led to death, caused huge economic losses to cattle industry. Recent years, with the booming of the farming cattle in China, Bovine Theileria sergenti prevalence in China showed an increasing trend, which poses a great threat to the cattle industry. Therefore, effective control of bovine Theileria sergenti incidence become a top priority. This test will conduct recombinant bovine Theileria sergenti major surface antigen gene p33 and bovine interleukin-18 (IL-18) gene and express in E.coli expression system, the fusion protein has good reactiongenicity.Theileria sergenti p33 gene is one of the major surface antigen cancause specific immune response of the host. Designed two pairs of specific primers based on p33 gene sequence published in GenBank [GenBank:DQ078264.1] Interleukin-18 gene sequence (IL-18) [GenBank:EU574909.1], Extraction bovine Theileria sergenti positive blood DNA genome as a template to amplify 821bp fragment by PCR technology. Extract pMD-18-T-IL-18 plasmid DNA as template to obtain a size for the 627bp fragment by PCR technology. To obtain the tandem gene IL-18-p33 in the size of 1416bp apply SOE-PCR technique to recombine two genes. The tandem gene IL-18-p33 was cloned into pMD-19-T simple vector and transformed into E.coli DH5α. Extract recombinant plasmid DNA and employ PCR, BamH Ⅰ and Xho Ⅰ double enzyme digestion and DNA sequencing to be sure that the tandem gene IL-18-p33 was successfully cloned into pMD-19-T simple vector, then named the recombinant plasmid DNA as pMD-19T-IL18- p33. Employ BamH Ⅰ and Xho Ⅰ double enzyme to digest the recombinant plasmid pMD-19T-IL18- p33 and prokaryotic expression vector PGEX-4T1, then gather the target band and connect 16℃ overnight. Transformed ligation products into prokaryotic expression strain E.coli BL21. Extracted plasmid DNA was identified with PCR and BamH Ⅰ、 Xho Ⅰ double enzyme digestion, then named the recombinant transforming bacteria as BL21-pGEX4T1-IL18-p33. Induced BL21-pGEX4T1-IL18-p33 with IPTG at Thousandth of the concentration, the inducing product was analyzed with SDS-PAGE gel electrophoresis, the result showed that about 79 kDa bands appear between 66.4 kDa and 97.2 kDa, which consistent with the expected size, then the inducing product was analyzed with Western blot, The results show that the recombinant protein has good reactiongenicity...