| Giardia duodenalis,an anaerobic protozoan pathogen,which causes gastrointestinal diseases in many mammals including humans.The most common symptoms of G.duodenalis include foul-smelling diarrhea,greasy stools,flatulence,and emaciation.The infection can lead to chronic disease and extralimentary complications.With the continuous innovation and development of modern biotechnology,the genetic vaccine(DNA vaccine)of G.duodenalis has attracted increasing attention.DNA vaccines are stable,convenient and safe,and can trigger cellular and humoral immune responses in the body,which can improve the antibody response of infants.pc DNA3.1 plasmid is a mammalian cell expression vector,which has been widely used in the construction of eukaryotic expression vector and is suitable for the development of DNA vaccine.The Cp G sequence induces a strong Th1-like response and can be used as an adjuvant against a variety of targets,including infectious agents,tumor antigens,and allergens.Giardins are important elements of cytoskeleton proteins of Giardia.The giardins identified so far include ?,?,? and ?-giardins.? and ?-giardins are highly immunogenic.?-giardin is a protein with a weight of 38 k Da and may be one of the components of microribbons.There are some progress in ?-giardin at the molecular level,but its exact location is unknown.It has been reported that ? and ?-giardins molecules have high immunogenicity,and ?1-giardin is an effective protein for the development of Giardia vaccine.This has paved the way for our work on DNA vaccines for ? and ?-giardin.The aim of this study is to construct DNA vaccines of ? and ?-giardin genes and evaluate their immune protection against G.duodenalis.In this study,truncated ? and ?-giardin genes were obtained by RT-PCR.The target genes were inserted into the eukaryotic expression vector pc DNA3.1,and eukaryotic expression plasmids pc DNA3.1-?-giardin,pc DNA3.1-?-giardin and pc DNA3.1-?-?-giardin were successfully constructed.In this study,three groups of eukaryotic expression recombinant plasmids were transfected into Vero cells and their in vitro expression was identified by indirect immunofluorescence and Western Blot.Then the eukaryotic expression recombinant plasmids were intramusculally injected into BALB/c mice for three times of immunization,and the mice inoculated with empty pc DNA3.1 vector were used as a control group.Serum of mice was collected after three immunizations,and the levels of Ig G and its subtypes Ig G1 and Ig G2 a in serum were detected using ELISA.After the third immunization,CCK-8 was used for the proliferation test of spleen lymphocytes.The cytokines IFN-? and IL-4 produced by stimulating spleen lymphocytes with G.duodenalis soluble antigen were detected by ELISA,andthe percentages of CD3~+CD4~+CD8~-and CD3~+CD8~+CD4~-T cells in spleen lymphocytes were detected by flow cytometry.Mice were inoculated with Giardia duodenalis cyst and the feces of 16 days were counted after inoculation.The results showed that pc DNA3.1-?-giardin ?pc DNA3.1-?-giardin and pc DNA3.1-?-?-giardin were successfully transfected into Vero cells and expressed,and mice immunized with three recombinant plasmids induced specific humoral and cellular responses,with produce of high level of Ig G and Ig G2 a antibody.The SI values of pc DNA3.1-?-giardin,pc DNA3.1-?-giardin and pc DNA3.1-?-?-giardin were 1.09±0.022,1.07±0.021,1.05±0.023,respectively,which significantly increased compared with the control group.The percentage of CD3~+CD4~+CD8~-and CD3~+CD8~+CD4~-T cells signally increased compared to the PBS and pc DNA3.1 control.The percentages of CD4~+ T cells were 23.65±0.78,23.20±0.99,21.25±0.64 for the three groups,and the percentages of CD8+ T cells were 10.90±0.99,11.80±1.27,9.90±0.57,likewise.Compared to pc DNA3.1-?-giardin,mice immunized with pc DNA3.1-?-giardin and pc DNA3.1-?-?-giardin induced higher levels of cytokine IFN-? and IL-4,and mice immunized with pc DNA3.1-?-giardin induced higher levels of cytokine IFN-? likewise.The results showed that three types of DNA vaccines derived from ?-giardin,?-giardin,and?-?-giardin elicited 39.1%,26.1% and 18.3% reduction in cyst shedding respectively.In this study,eukaryotic recombinant expression vectors pc DNA3.1-?-giardin,pc DNA3.1-?-giardin and pc DNA3.1-?-?-giardin were successfully constructed,and the immunoprotective effect was evaluated.The above results showed that pc DNA3.1-?-giardin,pc DNA3.1-?-giardin and pc DNA3.1-?-?-giardin could induce humoral and cellular immune responses in mice,and generate accordingly certain immune protection against G.duodenalis infection.The results provide a theoretical basis for the further development of DNA vaccines against G.duodenalis. |
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