Expression And Functional Characterization Of PKA Of Taenia Solium

Taenia solium is an important zoonotic parasite that causes a great threat to public health and economic loss in developing countries.Pig and humans both can be infected with metacestode by ingesting eggs of the adult T.solium,causing porcine or human cysticercosis.cAMP-dependent protein kinase?PKA?plays vital role in biochemical process in eukaryotic cells.PKA has been represented a potentially new class of therapeutic targets for the treatment of cancer,inflammation,parasitic diseases and other immune regulation disorder complicated diseases.In addition,it is worth noting that PKA regulatory subunit possesses highly specific regions that make it suitable candidates for cysticercosis diagnosis.This study not only provides valuable insight into T.solium PKA regulatory subunit?TsPKA-r?as diagnostic antigen potentiality,but also clarify characteristics of enzymology about catalytic subunit?TsPKA-c?.The results are as follows:1.Though database searches of T.solium genome and transcriptome data,the full-length sequences encoding TsPKA-r and TsPKA-c were amplified from T.solium metacestodes cDNA by PCR and identified by bioinformatics.Detailed sequence and structural analyses showed that TsPKA-r and TsPKA-c had characteristic features of PKA.The cloned full-length cDNA?1 104 nucleotides?of Tspka-r encoded a polypeptide of 367 amino acids.The cloned full-length cDNA?1 032 nucleotides?of Tspka-c encoded a polypeptide of 343 amino acids.2.TsPKA-r fusion-protein with His-tags was expressed in E.coli,purified by Ni Sepharose^TM 6 Fast Flow and used to test reactionogenicity by immunoblotting.TsPKA-r based indirect enzyme-linked immunosorbent assays?iELISA?recognized sera of pigs experimentally infected with Cysticercus cellulosae.The iELISA detected specific antibodies from pigs experimentally infected with T.solium with93.88%sensitivity and 96.40%specificity.It showed no cross-reactions against the sera samples from pigs experimentally infected with Cysticercus tenuicollis,Toxoplasma gondii or Trichinella spiralis.These results indicate that the TsPKA-r is a promising immunodiagnostic antigen for developing an iELISA to detect porcine cysticercosis.3.The optimized TsPKA-c gene was inserted into the expression vector pPIC9 K and was successfully expressed in P.pastoris.The target protein was detected by Western-blotting and mass spectrometry.The purified TsPKA-c was used to immunize New Zealand white rabbits,and the high titer specific IgG antibodies were obtained after 3 immunization.Immunohistochemical results showed that TsPKA-c was predominantly expressed in larval suckers and adult ovaries,eggs and tegument,suggesting that TsPKA-c may participate in the growth and development and nutrient absorption.4.The enzymatic activity of TsPKA-c was detected utilizing the crebtide as substrate.Phosphorylated crebtide was monitored following the increase of absorbance value at 450 nm.The results showed that the purified TsPKA-c was active with a Vmax value of 0.765±0.013 nMol/min/?g and Km value of 36.37±2.64?M.Biochemical analysis of inhibitory kinetics indicated that H89 and PKI?14-22?can inhibit TsPKA-c effectively with IC₅₀ of 14.55±1.5?M and 23.09±0.5?M,respectively.This study mainly suggested that TsPKA-c may be explored as a novel drug target for porcine cysticercosis.5.Taenia pisiformis metacestode were cultured in vitro were random divided into four groups,two groups of which were treated with H89 and PKI?14-22?,respectively.The levels of parasites glucose uptake was detected after the activity of TsPKA-c was inhibited.The results showed that the inhibitors H89 and PKI?14-22?can significantly inhibit the activity of cysticercus in a dose-dependent manner in vitro.Cysticercus treated with H89 at concentration 5?M resulted in 100%morality within 36 h,while PKI?14-22?at concentration 25?M.Parasites glucose uptake of the treatment groups were significantly decreased than the control group?P<0.05?,suggesting that TsPKA-c plays an important role in cysticercus glucose metabolism.The inhibitory experiments in vitro suggest PKA activity is required for cysticercus viability.