Eukaryotic Expression Of PO Gene Of Neospora Caninum And Initial Studies On DNA Vaccine

Neosporosis is a kind of worldwide epidemic and many animals troubled protozoal diseases caused by Neospora caninum, which causes neuromuscular system diseases in dogs and serious epidemic diseases of animal reproductive barrier in cattle.A series of clinical symptom happened frequently in pregnant cattle,such as abortion,fetal death and weak fetus.This disease caused severe economic losses in livestock farming.At present,there are no effective measures to prevent and control Neosporosis.Generally,vaccine immunity is a kind of feasible measure to prevent abortion in cattle and degrade the economic losses,so the development of new vaccines has become a hot field of research.The ribosomal phosphoprotein PO of Neospora caninum (NcPO) could make a cross-reactive antigen between Neospora. caninum and Toxoplasma.gondii, the anti-rNcPO antibody could inhibit the growth of both parasites. In this test full length gene sequence of PO protein was amplified by PCR, and then cloned into the eukaryotic expression vector pVAX1, to construct a pVAX1-NcPO expression, and the PCR, restriction enzyme digestion and sequencing identification had been done.we transfected the recombinant into Vero cell by lipidosome, and RT-PCR and IFA detection have been done to determine the transcription of target genes.The results thowed that the cloned full length gene sequence of PO protein was 1 379bp,constructed expression vector was identified correctly. The RT-PCR result showed that purpose gene was succeed in transcribing. The purpose gene was successfully in expressed.by IFA test. This test was successfully constructed eukaryotic expression vector of ribosomal phosphoprotein PO of Neospora caninum, transfected Vero cells, and expressed ribosomal phosphoprotein PO of Neospora caninum (NcPO). Through largely abstraction and purification, gerbils were immuned pVAX1-NcPO plasmids with adjuvant, humoral immunity and cellular immunity response were measured in order to evaluate the immuno-effect of vaccine.The valence of antibody of immunized gerbils is determined with IFAT every week. The results show that the valence of antibody of Cp-M groups and pVAX1-NcPO group are higher than pVAX1 empty vector group and control group; the valence of antibody of Cp-M group is highest especially. The valence of antibody of Cp-M groups and pVAX1-NcPO group are more striking than pVAXl empty vector group and control group after eliminating toxic material experiment. The valence of vector group, control group is remarkable extremely(P<0.01).This experiment evaluate cell immunity of immunized gerbils by determining percentage of CD4+ and CD8+ of T lymphocyte subgroup and lymphocyte conversion test. The results show that the percentage of CD4+ T lymphocyte subgroup in vaccine group including Cp-M group, pVAX1-NcPO group is higher than empty vector group, control group, Cp-M group, pVAX1-NcPO group is distinct extremely compared to pVAX1 empty vector group, control group (P<0.01). The highest value is Cp-M group.The percentage of CD8+ T lymphocyte subgroup in Cp-M group, pVAX1-NcPO group is higher than pVAX1 empty vector group, control group. Cp-M group and pVAX1-NcPO group are distinct significant compared to control group (P<0.05) Cp-M group and pVAX1-NcPO group are quiet compared to pVAXl empty vector group.The ratio of CD4+/CD8+ T lymphocyte subgroup in Cp-M group and pVAX1-NcPO group is higher than pVAX1 empty vector group and control group, the distinction is significant between Cp-M group, pVAX1-NcPO group and control group(P<0.05). Cp-M group and pVAX1-NcPO group are quiet compared to pVAX1 empty vector group. The protection ratio of vaccine after eliminating toxic material experiment is 70% in Cp-M group,66.7% in pVAX1-NcPO group.The experiment results show that the Neospora caninum DNA vaccine could activate humoral mediated immunity reaction and cell mediated immunity reaction, the Neospora caninum subunit vaccine has homologus protection, the results could provide reference for the development of Neospora caninum new generation vaccine.