Study On A Rapid Diagnostic Kit For Hydatidosis

Hydatidosis, also called Cystic Echinococcosis(CE), is an important zoonose, which is caused by Echonociccinae granulosus and damages seriously the health of people living in the western region of China. The common diagnostic antigen is sac liquid of Echinociccinae granulosus. Crossing reaction with serum of other worm's diseases and non-specific reaction were happened due to its complicated composition, which result to false positive and specificity is not satisfactory. Researchers have been searching for strong specific antigen and rapid testing method for the accurate diagnosis of Cystic Echinococcosis. Dot immunogold filtration assay (DIGFA) is more sensitive and rapid method in serum test with simple protocol and stable results. Antigens B (AgB) are known as antigens specifically of sac liquid of Echinociccinae granulosus. Five 8 KDa subunits of antigen B have been found and EgAgB8/2 is regarded as the most specifically one of the current used antigens. Therefore, cloning AgB8/2 gene cDNA from Echinociccinae granulosus and expressed in E.coli followed purifying antigens are necessary. It is significant for diagnosis of CE to combine the specificity of AgB8/2 antigen and rapid character of DIGFA assay and to develop a rapid diagnostic kit.AgB8/2 gene was amplified from Echinococcus granulosus by RT-PCR, and then was cloned into pET44a (+) to construct the prokaryotic expression vector pET-AgB8/2. The cloned fragment of AgB8/2 gene cDNA is 335 bp, containing the 273bp complete ORF and 33bp before ATG and 29bp behind TAA. AgB8/2 cDNA sequence has an identity with 100% to the sequence released in GenBank (U15001) and was connected with expression vector correctly. ORF encodes a protein with 90 amino acid residues.AgB8/2 fusion protein was expressed successfully in E.coli BL21 (DE3) at an optimized expression condition and then was purified after identification by SDS-PAGE and Western Blot. The highest expression level was achieved by inducing the bacteria with 0.05mM IPTG, at 0.6 of OD600 value for 5 h at 37℃. Purified soluble fusion protein was identified to be the correct target protein by SDS-PAGE and western blot. The recombinant fusion protein is occupied 26% of bacterium (BL21) protein.The purified AgB8/2 fusion protein was detected by Western Blot with Anti-His antibody because of the two 6 x His-tag. Western Blot showed that AgB8/2 fusion protein can identify by the patients serum, which is act as an primary antibody, but not healthy person serum. Using the purified AgB8/2 fusion protein as antigen, one patient serum and twenty healthy persons serum was detected by Dot blot. The results demonstrated that the antigen can identify the patient serum while had a 10%(2/20) false positive rate for healthy persons serum. So, AgB8/2 gene from Echinococcus granulosus is expressed exactly in E.coli.According to the principle of DIGFA, a rapid detection kit was assembled with AgB8/2 fusion protein as an antigen, nitrocellulose membrane (NC), TBS, closed liqiud, display reagent diluent liquid, and colour-display reagent (SPA), the positive control and the negative control. Results can be got in a few minutes just droping serum on the nitrocellulose membrane. A assay was carried out to detect 4 patients serum and 30 healthy persons serum with the kit. The results showed that the patients serum were detected exactly while had a 3.33%(1/30) false positive rate for healthy persons serum. Control group was negative.The rapid diagnostic kit for Hydatidosis caombined the specificity characteristic of AgB8/2 antigen and rapid characteristic of DIGFA. It will has a wide application prospect in clinical diagnosis of cystic echinococcosis and epidemiological investigation in the future, but its specificity and sensitivity need further confirmed with more patient serum.