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Neospora NcSRS2 and NcSAG1 gene and recombinant protein immunogenicity

Author: YuJinShu
Tutor: LiuQun
School: China Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Neospora NcSRS2 NcSAG1 Gene fusion Immunogenicity
CLC: S852.723
Type: Master's thesis
Year: 2005
Downloads: 106
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Abstract


Neosporosis animals were suffering from a variety of protozoal, the most serious hazards for cattle. Neospora surface protein NcSRS2 (Nc-p43), NcSAG1 (Nc-p36) is mediated by Neospora adhesion and invasion of host cells is the main surface protein is the most promising candidate antigen Neospora vaccine and diagnostic antigens. This study NcSRS2 gene was cloned and expressed, and the genes and gene fusion to achieve co-expression NcSAG1 while preliminary study NcSRS2 protein and NcSRS2-NcSAG1 protein immunogenicity. Analyzed using molecular biology software NcSRS2, NcSAG1 genes, gene truncated NcSRS2 hydrophobic N-terminal part after the gene (dNcSRS2) was cloned into the prokaryotic expression vector pGEX-6P-1 recombinant plasmid pGEX-dNcSRS2, induced in 1mMIPTG , expressed in E. coli BL21 and a large number of protein (GST-dNcSRS2). Inclusion body protein is both soluble and exists in two forms, a molecular weight of 62.6KD, expression was 32.3%. With GST antibody Western blot analysis, successfully detected specific bands. Glutathione Sepharose 4B with the fusion protein was purified. NcSRS2 and NcSAG1 the strong antigen gene fragment was cloned into the same series prokaryotic expression vector pGEX-6P-1, the recombinant plasmid pGEX-tNcP43-P36, to obtain a strong antigenic fusion protein of 1mM IPTG induction in , the target protein (GST-tNcP43-P36) in the form of inclusion bodies in E. coli, a molecular weight of 64.5KD, expression was 14.9%. Serum with anti-GST Western blot analysis, successfully detected specific bands. At low protein concentration (0.042mg/ml) under denaturing gradient dialysis make recombinant protein refolding get better. The purified GST-dNcSRS2 protein and GST-tNcP43-P36 protein three immunized Balb / C mice in order to detect the immunogenicity of two proteins. GST-dNcSRS2 protein and GST-tNcP43-P36 protein were set high (100μg / only), medium (50μg / only), low (25μg / only) and three doses of immunization of mice with Freund's adjuvant, a separate GST -dNcSRS2 protein and GST-tNcP43-P36 protein in two middle dose group (without adjuvant) and the control group. Before the first immunization, respectively, 10 days after each immunization blood serum was separated, ELISA serum antibody titers; three free after each immunization group have gained a good antibody levels, especially GST-dNcSRS2 middle dose (adjuvant) Immunohistochemical best. 10 days after each immunization spleen lymphocytes isolated MTT assay was used to do T lymphocyte proliferation test showed GST-tNcP43-P36 immunohistochemistry each dose of each dose than the GST-dNcSRS2 immunohistochemistry with strong cellular immune response induced functional .

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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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