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Cloning and Expression of Three Low-molecularweight Antigen Genes of Cysticercus Cellilosae and Establishment of ELISA Using Recombinant Protain

Author: WangQiuXia
Tutor: NingChangShen；ZhangGaiPing
School: Henan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Cysticercosis 8kDa antigen family TsRS1 gene M13h gene Ts8B2 gene Clone Expression Protein Purification Indirect ELISA
CLC: S852.7
Type: Master's thesis
Year: 2009
Downloads: 21
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Abstract


Cysticercosis is an important zoonotic parasitic diseases, not only caused the decline in pork quality, and cause significant harm to human health. At present, the role of the low molecular weight antigens in the diagnosis of cysticercosis immune and immune prevention is increasingly subject to the attention of scholars at home and abroad. This study through genetic engineering methods cloned pig the cysticerci 8kDa antigen family of three genes were constructed recombinant expression vector, efficient expression; three recombinant proteins were purified using GST affinity chromatography methods; and compare three recombinant protein antigen , to elect the the antigenic best establish an indirect ELISA method to detect cysticercosis antibody. Cysticercosis detection test strip kit for reference. 1, total RNA extraction technology, reverse transcription and PCR techniques pig the cysticerci 8KDa family excretion secretion antigen M13h gene, Ts8B2 gene, the the lentil lectin the antigen TsRS1 gene length 258bp, 270bp and 321bp respectively. cloned into pMD19-T and sequenced. 2, sequenced three genes mature peptide portion were subcloned into the prokaryotic expression vector pGEX-6p-1, the recombinant expression vector pGEX-6p-1/TsRS1 pGEX-6p-1/Ts8B2 pGEX-6p- 1/M13h; After PCR amplification and double restriction enzyme digestion and sequencing analysis proved that the gene is properly inserted into an expression vector and the correct reading frame. Three recombinant plasmids were transformed prokaryotic expression host strain BL21 (DE3), expression products by SDS-PAGE and Western blot analysis. The results show that: successfully constructed three expression vectors and expressed in E. coli three proteins; expression by Western blot prove three kinds of rabbit anti-porcine Cysticercus cyst fluid protein may be antiserum specific recognition may as a diagnostic antigen. Induced expression of bacterial ultrasonic lysis, SDS-PAGE showed that the three proteins are soluble expression; purified through the the GST column of the three proteins, the tagged protein purification empty as a control. Respectively with purified expressed protein and GST as a coating antigen coated microtiter plates, eliminate the GST protein reactive, and comparison of three of the recombinant protein antigen, to elect the best antigenicity of the recombinant protein for establishing a rapid detection of swine the cysticercosis indirect ELISA method. 4, according to the results of screening with purified protein expression M13h as coating antigen coated microtiter plates, initially established indirect ELISA method for the rapid detection of porcine cysticercosis. Antigen concentration 1μg · mL-1; the antibody dilution 1:100,37 ℃ role in 45min; goat anti-pig / HRP anti-dilution 1:1000,37 ℃ role 45min; substrate solution at room temperature was color 5min; coated ELISA plate 4 ° C can be stored for 6 months. After specificity, sensitivity test further establish indirect ELISA method specificity, high sensitivity. Diagnostic test strip marked. Provides a simple, fast, inexpensive technological means for the diagnosis and detection of cysticercosis.

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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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