| Due to the dramatically increasing of global population and mobility in recentdecades, dengue fever (DF) has expanded geographically to the majority of regionsaround the world as the most widely-spread arbovirus-borne disease. Since effectivevaccines for DF are not available yet, the elevated disease burden of DF has been aworldwide problem of public health. Besides vector control, timely identification ofdengue infections, patient isolation and prompt treatment are important strategies inpreventing the spread of disease. In particular, early laboratory diagnosis of DF is acritical step in prior to DF prevention and control measures.The early diagnosis of dengue infection has always been a hotspot in the field of DFprevention and control, a variety of emerging diagnostic technologies, includingvirus-specific nucleic acid detection, viral antigen and serological antibodies tests, hasbeen developed and gradually used in routine diagnosis at clinical laboratories. Severalforeign biotechnological companies have successfully commercialized theirenzyme-linked immunosorbent assay (ELISA) kits and immune colloidal gold techniques(ICT) etc. These kits are predominant in the DF diagnostic reagent market because oftheir rapidness, ease of use in clinical laboratories with limited equipment. Although a lotof institutions in China have tried to develop DF antibody detection kits and have claimedsignificant progress in recent years, only a few of domestic DF diagnostic kits aresatisfactory in clinical specimens.The purpose of this study is cloning and expression of the domain Ⅲ fragments ofthe envelope gene in dengue type2virus. Eight primers were designed in the upstreamand downstream, respectively with NdeI and XhoI recognition sites. Hence， thecrossed-matched combination of64DⅢ segments were amplified, subsequently clonedand expressed in the pET30a(+) expression system with E.coli BL21(DE3) as the hostcell. The expression and immune activity of recombinant proteins were verified SDS-PAGE and Western blotting assays. Constructs with high expression and strongimmune activity were screened, the purified recombinant proteins were used as antigensin establishment of indirect ELISA tests for IgM antibodies against DF.To assess the ELISA test results in this study, a total of112clinical serum specimenspre-tested by Panbio DF IgM kits were selected. According to tests in confirmativeindirect fluorescent assays (IFA) as the gold standard, it was revealed that the sensitivityof current IgM-ELISA was94.64%, and the specificity was91.07%. Compared withPanbio IgM-ELISA kit, this newly-established ELISA assay obtained a positivecoincidence rate of92.85%and a negative coincidence rate of89.29%, in addition to atotal coincidence rate of91.07%and a Kappa value of0.8214. Based on theabovementioned higher consistency and no statistically significant difference between thetwo assays (chi-square=0.3, P>0.05), it is concluded that recombinant proteins in thepresent study is prospective in developing DF diagnostic kits. |
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