Cloning And Expression Of Three Low-molecularweight Antigen Genes Of Cysticercus Cellilosae And Establishment Of Elisa Using Recombinant Protain

Cysticercosis is one of the important parasite zoonosis. It not only leads to the descent of pork quality, but also severely threatens the health of human worldwide. The role of low-molecular-weight antigen genes in Cysticercosis immunodiagnosis and immunoprevention are hot topics in research. Genetic engineering techology was used in this study to clone three low-molecular-weight antigen genes which are members of the Cysticercus cellilosae 8kDa antigen family and construct expression vector, respectively. After efficient expression, recombinant protein were purified by GST affinity chromat-ography technology. Then the sensitivity and specificity were examined with serum samples. According to the result of immunogenicity test, we developed the method of antibody detection and provided an important reference for detection Swine cysticercosis test paper and the kit.First, three low-molecular-weight antigen genes of Cysticercus cellilosae, Excret-ory/Secretory antigens(E/S Ag) genes M13h(258bp) and Ts8B2(270bp), lentil lectin purified glyc-oprotein (LLGP) antigens gene TsRSl(321bp),were obtained using total RNA extractive technique, reverse transcription technique and PCR technique. Then the three genes were cloned into pMD19-T and sequenced, respectively.Secondly, three target genes sequenced were inserted into prokaryotic expression vector pGEX-6p-1. Recombinant plasmid pGEX-6p-1/TsRS1,pGEX-6p-1/M13h,pGEX-6p-1/Ts8B2 were transformed into bacteria of BL21 (DE3) host after being detected by PCR amplification, restriction enzyme digestion and sequencing, which have the correct open reading frame(ORF). Then the expression products were detected by SDS-PAGE and Western blot. Results demonstrated these genes were correctly inserted into prokaryotic expression vector pGEX-6p-1, and three recombinant expression vectors pGEX-6p-1/TsRS1,pGEX-6p-1/M13h,pGEX-6p-1/Ts8B2 were developed successfully. Proteins of interest were efficiently expressed in E.coli. Specificities of three recombinant proteins were identified with rabbit anti-cysticercus cystic fluid polyantibody in Western blot, and this findings suggested that these recombinant proteins could be used as useful diagnostic antigens.Third, three recombinant proteins were soluble that confirmed by SDS-PAGE after ultrasonic lysis. Three recombinant proteins were purified through GST chromatography columns, and GST proteins, as a tag in the pGEX-6p-1, was puified through GST chromatography columns as control protein. Then three recombinant proteins and GST tags were used as the coating antigen on ELISA plate. The immunoreactivity of three expression proteins and GST tags were compared. Based on the results, the optimal antigen in immunoreactivity was selected for developing indirect ELISA method of the rapid diagnosis of cysticercosis.Last but more importantly, according to screening results, the M13h recombinant protein was used as a coating antigen, and indirect ELISA method was developed for rapid detection of swine cysticercosis antibody. Concentration of M13h recombinant antigen coated on the ELISA plate was 1μg·mL-1,1:1 000 diluted goat anti-pig HRP, incubated 37℃of 45 min, substrate solution colored 5 min at room temperature. The validity of ELISA plate after being coated in 4℃remained above six months. Data showed that this method has a high sensitivity and specificity. Then the diagnostic test strip was developed with the M13h expression protein. These provides a useful method that is simple, rapid and low price for the cysticercosis detection.