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Prokaryotic Expression of tNcSRS2 Gene from N.caninum Xinjiang Strain and Development of an Indirect ELISA

Author: YangFan
Tutor: BaYinChaHan
School: Xinjiang Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Xinjiang strain Neospora tNcSRS2 gene Prokaryotic expression Indirect ELISA
CLC: S852.7
Type: Master's thesis
Year: 2010
Downloads: 47
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Abstract

The Neospora (Neosporosis) is a variety of livestock parasites in the host body caused by N. caninum (N. caninum) were suffering from a protozoal disease is particularly serious harm to the cattle, mainly caused by cow abortion stillbirth, and neonatal motor nerve system diseases, caused huge economic losses to the livestock industry. To date, no effective drugs and vaccines for prevention and treatment of cryptosporidiosis, did not establish a comprehensive prevention and control measures, so the establishment of the diagnosis method is particularly important. The constructed Neospora prokaryotic expression vector explore the inclusion body protein extraction and refolding, and Neospora indirect ELISA. Sis for our cattle spores epidemiological surveillance, to provide an effective means of diagnosis. The test published in GenBank. Neospora NcSRS2 of gene sequences, biological Oligo 6.0 software to design a pair of primers, the introduction of restriction sites and to remove NcSRS2 protein signal peptide region. IDEXX kit for detection of Neospora positive blood samples, the extraction of total DNA. About the 975bp tNcSRS2 gene fragment was amplified by PCR and cloned into PMD-18-T vector, restriction enzyme digestion and sequencing. Then further target fragment was cloned into the pGEX-4T-2 expression vector, and transformed into BL21 expression bacteria. And optimize the expression condition was 37 ° C conditions, the final concentration of 0.5 mmol / L IPTG induction the 4h, obtained recombinant proteins the GST-tNcSRS2 expressed as inclusion bodies, and a molecular weight of about 62.2kDa expression levels can be accounted for 35% of the total protein. Western blotting showed that the recombinant protein has a good reactogenicity, and laid the foundation for the establishment of new cryptosporidiosis diagnostic kit developed. In order to explore the GST-tNcSRS2 inclusion body protein extraction conditions and Neospora diagnostic methods. The test will induce the obtained of GST-tNcSRS2, inclusion body protein was washed to a purity of 87%. Then denatured, purified, refolded GST affinity chromatography purification. To obtain a concentration of 275μg/mL, purity of 95% the GST-tNcSRS2 recombinant protein. And then the protein as coating antigen, the establishment of an indirect ELISA method used to detect Neospora antibody. The establishment of the indirect ELISA results: antigen concentration 7μg/mL, package 4 ° C overnight, blocking solution of 0.5% skim milk, and the closure time 37 ° C role 1h; optimum packet optimal serum dilution 1:100 The reaction time was 1 hour; HRP optimal dilution of 1:4000, the role of time at 37 ℃ for 1h. Criteria (threshold OD450/630 ≥ 0.35 who ruled antibodies OD450/630 lt; 0.25 sentenced antibody negative, between the two as suspicious. Specificity failed to detect cross-reactivity. Intra repeat the test and Repeat the test batch coefficient of variation less than 10%, indicating good repeatability for canine neosporosis detection, diagnosis and provide technical support.

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