Immune Response Of PVAX1-AgB Recombinant Plasmid Against Taenia Solium Cysticercus Cellulosae

Cysticercosis is an important zoonotic disease caused by Taenia Solium larvae. The larvae can infect intermediate host such as human or pig. The conventional treatment using drugs against cysticercosis plays an important role, however it bears danger of food safety caused by drug residues and drug-resistance. This urges us having to change our thoughts to use economic, safe and efficient vaccine to prevent the disease. Cysticercus antigen B known as paramyosin B, is a protein in the wall cells of cysticerci, which secretes to the outside of parasite, and can inhibit the complement activation pathway of its host. It plays a protective role in cysticercosis and in avoiding the host's immune and it has been pointed out as one of most promising vaccine candidates of schistosomiasis or other worms by WHO.In this study, the primers were designed according to cDNA sequence of AgB strain in GenBank and used to amplify AgB gene from total RNA by RT-PCR. The PCR products were ligated into pGEM-T Easy vector and sequenced. Sequence analysis indicated AgB gene of 2590 bp was successfully obtained with 99.7% similarity to those registered in the GenBank. Besides, specific prokaryotic expression primers were designed to amplify the recombinant plasmid pGEM-AgB. The target fragments were digested by EcoRⅠand NotⅠand inserted into pGEX-4T-1vector to construct recombinant eukaryotic expression vector pGEX-AgB. The pGEX-AgB was expressed in E.coli and purified. The expressed product were detected by SDS-PAGE. The results appeared a size of clear protein band in the 121 ku consistent with the expected molecular weight.The product can be identified by cysticercosis positive serum by Western blot detection, indicating the expression of foreign protein has good immunoreactivity.Meanwhile, the mammalian expression vector primers were also designed to construct recombinant plasmid pVAX1-AgB by using plasmid pGEM-AgB as a PCR template, and the products were digested by BamHⅠ, HindⅢ. Balb/c mice at 5-week-old were intramuscularly inoculated using 100μg of recombinant plasmid of pVAX1-AgB, with PBS and pVAX1 plasmid group as control. The serum samples were collected at different times after immunization and specific antibodies were detected by ELISA. The results showed that the mice produced detectable specific antibody at 2 weeks after the initial immunization and reach to the peak value at 6 weeks. The persistence of the antibody was lasting longer than 8 months: antibody in the PBS and the pVAX1 control groups was negative.To further study the pVAX1-AgB recombinant plasmid immunogenicity, the health pig were intramuscularly inoculated using 1000μg of recombinant plasmid of pVAX1-AgB to conduct a comprehensive evaluation of the DNA vaccine, with AgB recombinant protein group (200μg per head), pVAX1 plasmid group (1000μg per head) and PBS group as control. Antibody levels were measured with ELISA in each group, while serum IL-4 and IFN-γexpression lever were detected. The results showed that the specific antibody of pVAX1-AgB recombinant plasmid group could be detected at 4 weeks after the immunization and reached the peak at the 8th week: of the AgB recombinant protein group was at 2 weeks after the immunization and reached the peak at 6 week. The antibody titer of pVAX1-AgB plasmid group was less than AgB recombinant protein group. The IL-4 and IFN-γexpression levels of pVAX1-AgB plasmid group and AgB recombinant protein group were increased after initial immunization, and booster immunization higher than initial immunization. Both of two immunizations were significantly higher than preimmunization (P<0.01). There were not significantly different between pVAX1-AgB and AgB recombinant plasmid group with IL-4 and IFN-γexpression levels. It demonstrates that the pigs were induced effective immune response by pVAX1-AgB plasmid and AgB recombinant protein, the times to produce antibodies of recombinant plasmid later than the recombinant protein group. The actually immune effect of the pVAX1-AgB DNA vaccine and AgB recombinant protein vaccine need to be further varificated by challenged infection test with eggs. This study lay the foundation for the development of safe and effective Cysticercus candidates vaccine.