Cloning And Expression M13h ES Antigen Gene Of Asian Cysticercus Cellulosae

Cysticercosis cellulosae is one of the important parasite zoonosis.It not only leads to the descent of pork quality,but also poses great harm to our health.Its immunodiagnosis and immunoprophylaxis evokes much attention of scholar in domestic and abroad.Ten 344bp coxI partial sequences of Cysticercus cellulosae from partial areas of Henan Province were obtained using total RNA extractive technique, reverse transcription technique and PCR technique.Gene was cloned into pMD19-T and sequenced,aligned using Clustal X 1.81 Program.Phylogenetic relationships were reconstructed using MP and NJ of PAUP 4.0 Program,and ML of PUZZLE 5.2 Program.Homology analysis were done using WDANSIST 2.5 Program and Megalign of DNAstar 5.0 Program.M13h ES antigen gene of Cysticercus cellulosae was cloned by RT-PCR.Its sequence was analyzed by Megalign and Protean program. Prediction of signal peptide was done by Anthprot 5.0 program and signalP server. Homology search was done using Blast program.Sequences were aligned using Clustal X and WDNASIST.Phylogenetic relationships were constructed as cox I gene. Then M13h gene was cloned into pET43a and pcDNA3.0 to construct recombinate expression vector pET43M13h and pc3M13h.Prokaryotic expression were done by transforming pET43M13h into BL21(DE3)host cell.The expression products were detected by SDS-PAGE and Western blot.Eukaryotic expression was done by trancfecting pc3M13h into COS-7 cell.The products were detected by Double Antibodies Sandwich ELISA and immunohistochemistry.Results indicates that 10 sequences of of Cysticercus cellulosae from partial areas of Henan Province were completely uniformity,belonging to Asian genetype.Cysticercus cellulosae coxI partial sequences can be used to distinguish Asian or American/African genetype and to be used for differential diagnosis of different Taeniidae tapeworm.,suggesting that it will be a differential gene for PCR differential diagnosis of taeniasis and cysticercosis.M13h ES antigen gene which is quiet in local variability was succeessful cloned,possible located in oncosphere stage and was a specific antigen. Therefore,it will be a diagnostic antigen in the future.Prokaryotic expression vector pETM13h was constructed successfully and expressed in E.coli.The expression protein presented to be cytorrhyctes and could react with RAT pAB specificity. Eukaryotic expression vector pc3M13h was constructed successfully and expressed in COS-7 cell.The products could be detected in cultural supernatant and inside cell, which could react with RAT pAb serum and IgG.These results indicate that the expressive products had immunoreactivity.