A Cell Penetrating Peptide Enhances The Immune Response Of DNA Vaccine Against Theileria Sergenti

Cell-penetrating peptides(CPPs)are small molecular peptides with membrane penetrating function,which can efficiently carry nucleic acids,peptides,proteins and other biomacromolecules through the cell membrane into the cytoplasm,it has many advantages,such as high efficiency and low toxicity,in mediating the entry of biological macromolecular drugs into cells.It has been widely used in various fields,such as carrier of transport imaging agents,as antibacterial and anti-inflammatory drugs,as a delivery carrier of anti-tumor drugs and as a delivery carrier of vaccines,etc.Theileria Sergenti is a blood protozoan parasitic on bovine red blood cells,mainly transmitted by Haemaphysalis Longicornis,can cause anemia in cattle,causing harm to cattle industry.Especially the imported cattle,purebred cattle and improved hybrid cattle are seriously harmful to the healthy development of cattle industry.Although DNA vaccine has the advantages of safety and easy preparation compared with traditional vaccine,its immune effect still needs to be improved,and so far,there is no research on CPPs in enhancing immune effect of parasite DNA vaccine.Therefore,following our research group has been engaged in the research of Bovine Theileria Sergenti.In this experiment,the DNA vaccine of bovine Theileria Sergenti was taken as the research object,the effect of a cell-penetrating peptide on DNA immune response was investigated in order to provide a new strategy for future DNA vaccine research.Based on the P33 gene sequence of Theileria Sergenti(DQ078264)published in Genbank,a pair of specific primers were designed,and the anticoagulant DNA of theileria Sergenti infected cattle was used as the template,the target fragment was cloned into the eukaryotic expression plasmid pVAX1 to construct the pVAX1-p33recombinant plasmid.The pVAX1-P33 recombinant plasmid was mixed with CPPs at a certain charge ratio,named CPPs-pVAX1-P33,and transfected into vero cells.Forty female Kunming mice were randomly divided into 4 groups and inoculated with CPPs-pVAX1-P33,pVAX1-P33,pVAX1 and PBS respectively.The levels of CD3~+,CD4~+and CD8~+in each group were detected by flow cytometry and the proliferation of lymphocyte was detected by CCK-8.At the same time,mice blood was taken and serum was isolated before each immunization and every 2 weeks after the third immunization,the specific antibody against Theileria Sergenti,Ig G1,Ig G2a,IFN-γ,IL-2,IL-4 and IL-6 in each group of mice were detected and recorded by Elisa,so as to evaluate the effects of cellular and immune responses.The results showed that the constructed eukaryotic expression plasmid pVAX1-P33 had 99.49%homology with Theileria Sergenti P33 gene sequence published in Genbank.The results of transfection showed that CPPs-pVAX1-P33could be successfully transfected into vero cells and expressed in vero cells without lipofectamine.The results of flow cytometry showed that the levels of CD3~+and CD4~+in CPPs-pVAX1-P33 group and pVAX1-P33 group were significantly higher than those in pVAX1 group and PBS Group(P<0.01),and there were significant differences between CPPs-pVAX1-P33 group and pVAX1-P33 Group(P<0.01),the content of CD8~+in CPPs-pVAX1-P33 group and pVAX1-P33 group was significantly higher than that in pVAX1 group and PBS Group(P<0.05),and there was significant difference between CPPs-pVAX1-P33 group and pVAX1-P33 Group(P<0.05).In addition,there was no significant difference between CD4~+/CD8~+groups(P<0.05)Elisa showed that the proliferation index of Spleen in CPPs-pVAX1-P33 group and pVAX1-P33 group was significantly higher than that in pVAX1 group and PBS Group(P<0.05),the levels of Bovine Theileria Sergenti specific antibody,Ig G1,Ig G2a,IFN-γ,IL-2,IL-4 and IL-6 in serum of mice showed an ascending trend,and gradually decreased after the 6th week,reaching the highest level at the 6th week,and the CPPs-pVAX1-P33 group and pVAX1-P33 group were significantly different from PVAX1 group and PBS Group(P<0.01),and there were significant differences between CPPs-pVAX1-P33 group and pVAX1-P33 Group(P<0.01).In conclusion,the CPPS successfully transfected Theileria Sergenti DNA plasmid into vero cells and enhanced the humoral and cellular Immune responses induced by DNA vaccine against Theileria Sergenti.These results provide a new strategy for gene transfection and DNA vaccine research in molecular biology.