ABSTRACT
Objective:To establish the prokaryotic expression system of Taenia solium (Ts) 14-3-3.2, and observe the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus. Methods:Based on the Ts14-3-3.2 gene sequence obtained by the Department of Parasitology, Zunyi Medical University in the previous study, the whole gene was synthesized by PCR-based accurate synthesis (PAS) method. After double digestion with restriction enzymes Nde Ⅰ and Xba Ⅰ, the plasmid pCzn1 was ligated to construct a recombinant plasmid pCzn1-Ts14-3-3.2. Then it was transformed into Escherichia coli ArcticExpress competent cells to induce the expression of Ts14-3-3.2 protein. The expression products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie blue staining. The purified Ts14-3-3.2 recombinant protein was obtained by Ni-affinity chromatography. New Zealand rabbits were immunized with the recombinant protein to produce Ts14-3-3.2 polyclonal antibody. Western blotting was used to detect the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus. Results:The recombinant plasmid pCzn1-Ts14-3-3.2 was successfully constructed. After induced expression, Ts14-3-3.2 target protein bands appeared in the supernatant and precipitated at the relative molecular weight of about 29.31 × 10 3. The purified Ts14-3-3.2 recombinant protein with His label could be recognized by anti-His monoclonal antibody, and the Ts14-3-3.2 polyclonal antibody with titer of 1 ∶ 512 000 was obtained. Western blotting showed that Ts14-3-3.2 protein was expressed at the stages of Ts adult and cysticercus. Conclusions:The prokaryotic expression system of Ts14-3-3.2 is successfully established, and the Ts14-3-3.2 polyclonal antibody with relatively higher purity and titer is obtained. The Ts14-3-3.2 protein is expressed at the stages of Ts adult and cysticercus.
ABSTRACT
Parasitic diseases are diseases caused by parasites invading the human body, and 14-3-3 family plays an important role in the life activities of parasites, such as infection, growth and development, reproduction, and so on. Carrying out systematic research on it will help to understand the distribution and biological functions of the 14-3-3 family in parasites, and provide help for the immunological diagnosis and prevention of parasitic diseases. This article reviews the recent progress in the study of 14-3-3 family of important human parasites.
ABSTRACT
Objective To investigate the expression difference of 14-3-3 gene in different stages of Taenia solium (Ts),and to provide basic information for exploring the regulation mechanism of Ts14-3-3 gene in growth and development of Ts.Methods Adult worms were collected from patients with taeniasis solium in the taeniasis epidemic area of Yajiang County,Ganzi Tibetan Autonomous Prefecture,Sichuan Province.After determining the species through morphological observation under the light microscope and transcribed spacer 1 (ITS1) method,it was identified as Ts,then piglets were infected to obtain the Cysticercus cellulosae of Ts (larvae).Reverse transcription PCR was used to detect the expression of Ts14-3-3 gene in the adult stage and larval stage of Ts,and the relative expression levels of each were detected by real-time fluorescent quantitative PCR.Results Under light microscope,it could be seen that the collected adult worm scolex consisted of 4 suckers,rostellum and hooks,which was consistent with the typical characteristics of the Ts scolex;the worm sample could amplify the target fragment which was consistent with the length of the ITS1 gene by reverse transcription PCR,and was determined to be Ts.The Ts14-3-3 gene family members Ts14-3-3.1,Ts14-3-3.2,Ts14-3-3.3,Ts14-3-3.4,Ts14-3-3.5 and Ts14-3-3.6 were expressed in the Ts adult stage and larval stage.Compared with the larval stage,the expression levels of Ts14-3-3.1 (0.47 ± 0.09 vs 1.01 ± 0.23),Ts14-3-3.2 (0.31 ± 0.09 vs 1.05 ± 0.14),Ts14-3-3.3 (0.64 ± 0.23 vs 1.26 ± 0.23) and Ts14-3-3.4 (0.30 ± 0.09 vs 0.79 ± 0.23) were significantly decreased in the adult stage (t =-3.816,-7.093,-3.377,-3.481,P ＜ 0.01 or ＜ 0.05);the expression levels of Ts14-3-3.5 (3.59 ± 0.09 vs 0.99 ± 0.12) and Ts14-3-3.6 (5.74 ± 2.76 vs 1.03 ± 0.60) were significantly increased (t =30.714,9.718,P ＜ 0.01).Conclusion The expression of Ts14-3-3 gene has stage specificity and the Ts14-3-3 gene may play a special regulatory role in different stages of Ts.