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Optimization of Prokaryotic Expression of Xinjiang Bovine Theileria Annulata Tams1 Gene and the Research of Immunological Reactivity

Author: MiaoZhongQiu
Tutor: JianZiJian
School: Xinjiang Agricultural University
Course: Basic Veterinary Science
Keywords: Cattle Theileria annulata Tams1 gene Optimizing expression conditions Immune response
CLC: S852.723
Type: Master's thesis
Year: 2009
Downloads: 62
Quote: 2
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Abstract


Ring theileriosis make the world 250,000,000 cattle threatened to cause significant economic losses, distributed in North Africa, southern Europe, the Indian subcontinent, the Middle East and Central Asia. The case of cattle ring theileriosis by 泰勒科泰勒, ring Theileria caused by parasitic on cattle body as the main clinical symptoms of fever, anemia, bleeding, swollen lymph nodes and body surface a hematologic protozoosis the. The disease is widely popular in Xinjiang, seriously endangering the health of the cattle industry. This research mainly includes the following three parts: 1. Xinjiang Niu of Theileria annulata Tams1 gene expression conditions optimization in order to improve the amount of of Xinjiang cattle ring of Theileria Tams1 gene expression in E. coli to study the induction temperature, induction time and inducer The Fusion (IPTG) concentration of different conditions on Tams1 amount of protein. The test results show that the E. coli strain BL21 (DE3) and cultured at 37 ℃ 2.5h after, using a final concentration of 2mmol / L of IPTG at 25 ° C oscillation induced culture 8h, GST-Tams1 fusion protein expression, reaching the highest 2.5mg / mL. GST-Sepharose 4B affinity chromatography on the GST-Tams1 fusion protein purified by SDS-PAGE analysis showed that the fusion protein GST-Tams1 size of approximately 56KDa with the expected molecular weight of the same size. Construction and immune response studies. Xinjiang cattle ring-shaped of Theileria Tams1 gene eukaryotic expression vector by PCR amplification to get the 846bp of Tams1 gene fragment, and cloned into the eukaryotic genome from of giardiasis patients cattle blood expression vector pcDNA3.1 () to build Tams1 gene eukaryotic expression plasmid. After sequencing, the Tams1 eukaryotic expression plasmid genes tail vein injection of mice gene transient expression Tams1. Take inoculated mice liver extraction of total RNA by reverse transcription polymerase chain reaction (RT-PCR) amplified bands; recombinant plasmid pcDNA3.1 ()-Tams1 subcutaneous injection of mice immunized 4 times, The immune antibody detection. The test results showed that the recombinant plasmid pcDNA3.1 ()-Tams1 to contain complete Tmas1 gene fragment. Western-blot test available antigen - antibody complexes produced specific bands, indicating that the recombinant plasmid pcDNA3.1 ()-Tams1 immunogenicity. 3. Xinjiang cattle Theileria annulata GST-Tams1 fusion protein of the immune response by ELISA method, the results show the best package of antigen concentration of 2.5μg/ml serum dilution of 1:20. Next are the fusion protein GST-Tams1 antiserum titer assessment results show that the four protein serum titer gradually increased With the increase in the number of immunization, which ginsenoside adjuvant immunohistochemistry highest titer; Freund's adjuvant adjuvant immunohistochemistry; injection of recombinant eukaryotic plasmid pcDNA3.1 () the-Tams1 immunohistochemistry and injection of GST-Tams1 fusion protein immunohistochemistry titer similar injection of saline and no-load Plasmid pcDNA3.1 () of the control group is very low almost no antibody production.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Livestock parasitology > Veterinary protozoan school > Cryptosporidium
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