The changes in cytokines levels are typically associated with the changes in the levels of specific antibodies. the present study suggest that immunization with rEgGST in mice is able to successfully reduce the PSC-induced formation of cysts and to stimulate an immune response, suggesting that rEgGST possesses potential value as a candidate vaccine for PSC infection. Brevianamide F glutathione S-transferase Introduction Human HERPUD1 cystic echinococcosis, also known as cystic hydatid disease (CHD), affects humans and livestock and is caused by infection with the larval stage of (4C9). In our previous study, a Chinese strain of glutathione S-transferase (EgGST) was cloned and sequenced (10), and the capacity of EgGST to induce an immune response and immunoprotection was tested in an experimental model of hydatidosis in mice. In the present study, recombinant EgGST (rEgGST) was expressed in and purified for antigen preparation (11). Following the vaccination of mice with rEgGST, the resulting immunoprotection was analyzed and the protective mechanisms were investigated to assess the potential of rEgGST as a novel molecular vaccine. Materials and methods Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from freshly isolated protoscoleces (PSCs) using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The protoscoleces were extracted aseptically from fertile cysts from the livers and lungs of infected sheep. The EgGST gene was amplified by RT-PCR (Promega Corporation, Madison, Brevianamide F WI, USA) Brevianamide F using two primers according to the sequence of DNA polymerase (Promega Corporation), 5 l of each primer, 5 l RNA and 20 l diethylpyrocarbonate-treated water. The reaction protocol for RT-PCR was as follows: 48C for 45 min, 94C for 2 min, followed by 40 cycles of 30 sec at 94C, 60 sec at 60C and 2 min at 68C, with a final extension for 7 min at 68C. RT-PCR products were identified using 1% agarose gel electrophoresis (Liuyi Instrument Factory, Beijing, China). Subcloning of EgGST gene into expression plasmid vector The target fragment was purified using a gel cleanup kit (SBS Genetech, Co., Ltd.) and inserted between the BL21 (DE3) pLysS, provided by Dr Xiao Wei (University of Saskatchewan, Saskatoon, Canada) was transformed for induced expression of His6-tagged EgGST protein. Expression and purification of rEgGST Protein expression was induced at 25C by cultivation of the transformed BL21 overnight in the presence of isopropyl–D-thiogalactoside (IPTG; Promega Corporation) at a final concentration of 0.6 mmol/l. The recombinant His6-tagged rEgGST was purified from the extract of transformed BL21 (DE3) by Ni2+ chelate affinity chromatography (Novagen) according to the manufacturer’s instructions. Purified His6-tagged protein was analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Protein concentrations were determined using the Bradford method (12). Immunization A total of 84 male 6-week old ICR mice were obtained from the Experimental Animal Centre of Ningxia Medical University (Yinchuan, China). Mice were allocated at random into two groups containing 42 mice each. Mice in group A received three subcutaneous immunizations with 10 g rEgGST in 100 l phosphate-buffered saline (PBS) emulsified in Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA). The three immunizations were delivered at 2-week intervals, starting at week 0 in Freunds complete adjuvant and followed by two booster immunizations in Freund’s incomplete adjuvant at weeks 2 and 4. Mice in the control group B were injected with the corresponding adjuvant and PBS. This study was approved by the Ningxia Medical Brevianamide F University Ethical Committee. Challenge infection and protective immunity Six weeks after the final vaccination, on week 10, a challenge infection was induced in the mice via the intraperitoneal injection of 1 1,500 PSCs. Six mice in each group were sacrificed at different 0, 2, 4, 6, 10, 18 and 30 weeks following the initial vaccination, in order to obtain sera and spleen cells. Mice were sacrificed by cervical vertebra dislocation. Subsequently, the carcasses were dissected and examined superficially for visible hydatid cysts. The percentage of protection was determined according to the method and formula described by Dempster (13): Protective immunity in vaccinated mice (%) = (average number of cysts in the test group/average number of cysts in the control group) 100. Serum collection Six mice in each group were sacrificed at 0, 2, 4, 6, 10, 18 and 30 weeks following the initial vaccination in order to observe the macroscopic and microscopic effects of parasite development. Serum samples were collected and stored individually at ?84C. Cytokine measurements using ELISA The optical density values of a number of cytokines were determined using ELISA. Spleens were isolated from mice and splenocytes were harvested. A suspension of single splenocytes was prepared after removing erythrocytes via hypotonic lysis and resuspending the samples in RPMI 1640 (Gibco Life Technologies, Carlsbad, CA, USA) by vigorous pipetting..