Cloning And Expression 10kD Gene Of Cysticercus Cellulosa And Establishment Of ELISA Using Recombinant Protein

Cysticercosis is one of the important parasite zoonosis. It not only leads to the descent of pork quality, but also poses great harm to our health. Cysticercosis immunodiagnosis and immunoprevention are hot topics in research. Genetic engineering techology was used in this research to clone 10KD gene of Cysticercosis cystic fluid antigen and construct expression vector. After highly expression, recombinant protein was purified by Nickel affinity chromatography technology. The establishment of antibody detection method provides an important reference for detection Swine cysticercosis test paper and the kit.First, 10 kD gene of Cysticercosis cystic fluid was amplified by PCR techology, and inserted into prokaryotic expression vector pET32a. Recombinant plasmid pET32a-TS10-1 was transformed into bacteria of BL21 (DE3) host after being detected by PCR amplification, restriction enzyme digestion and sequence. Interested protein was highly expressed after induced by isopropyl thio-β-D-half-galactosidase (IPTG) and then analyzed by SDS-PAGE. Data shows that the 29KD recombinant protein can be identified specificity by rabbit anti-cysticercus cystic fluid polyantib- ody in Western blot, and so can be used as dignosis antigen.Soluble expression protein was purified through nickel chelate chromatography columns by changing concentrations of midazolam and adjust velocity or temperature in reaction, and then detected by rabbit anti-cysticercosis serum in western blot. Results show that recombinant protein was purified and had a high affinity with antibody of rabbit anti-cysticercosis, proved that fusion protein have an good immunogenicity. The results provid a fundation for the rapid diagnosis of cysticercosis.pET32a-TS10-1 protein was used as a coating antigen, coated on ELISA plate, and indirect ELISA method was developed for the initial establishment of rapid detection of swine cysticercosis antibody. Concentration of antigen package was 3.145μg?mL-1, 1:2 000 dilute goat anti-pig HRP, 37℃of 45 min, at the end of 37℃solution color 10 min. Data showed that this method has a high sensitivity and specificity. To further assembly diagnostic test paper and kit laid the material foundation for the diagnosis and cysticercosis detection provides a mathod that is simple, quick and low price.