Construction And Immunocontraception Experiment Of DNA Vaccine Of F_β.ECD-C3d3 In Mouse

Backgrounds and PurposeAlthough many methods have been used in birth control, there have no ideal birth control measures accepted by both man and woman. With the development of recombination of gene technique and people's deeper and deeper recognition to molecular basis and detailed mechanisms of the course of fertilization process, immunological anti-fertility vaccine will probably become the ideal measure to control population growth in the future.Blocking the binding of sperm and egg is very important in the immunological contraception study. Sperm antigens, such as fertilin, are important in the course of binding of sperm and ovum zona pellucida or membrane and in the course of fusion. As didymus tissue specific antigen, they have important latent contraceptional value. PH-30, Fertilin monoclonal antibody, can inhibit sperm-oocyte membrane fusion during the course of fertilization in vitro and binding of un-ZP ovum and sperm in guinea pig. Fertilin is a kind of alloplasm dimeride made up ofαandβsubunits.βsubunit ( F_β) is the functional unit of fertilin. Complement protein 3 (C3) is the center molecule in host innate immunity defence complement system. It is the key role in the complement-activated classical pathway, alternative pathway. C3d, a molecule adjuvant, is one of complement 3 split products. It can transduce antigen signal from specific complement receptor 2(CR2/CD21)to immunocell,enhance specific immune response,decrease immune activation threshold,make antigen without immunogenicity or lepto-immunogenicity produce enhance their immunoreaction. C3d molecule is the complement split fragment of organism themselves. So C3d has no toxic action as complete Freund's adjuvant(CFA).The aim of this study is to construct DNA vaccine of F_β.ECD-C3d3 and study the immunocontraceptional effect of this vaccine. The target antigen is F_βextracellular domain(F_β.ECD). 1168bp-2058bp in F_βECD were inserted into eukaryotic expression plasmid pSG.SS.C3d3.YL and pSG.SS.YL to construct pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD. The expression of F_βin eukaryotic cell (HEK 293 cells) was detected after HEK 293 cells were transfected by pSG.SS.C3d3.YL-F_β.ECD or pSG.SS.YL-F_β.ECD. The immunological contraception of them as DNA vaccine in female and male mouse were also observed.Methods1. The cDNA of F_β.ECD was amplified from plasmid pYA3149-F_βby PCR reaction. The products was digested by restriction enzyme. The fragment of F_β.ECD was directly cloned into pSG.SS.C3d3.YL and pSG.SS.YL.2. The pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD were confirmed by enzyme tomy and sequencing test.3. The expression of F_β.ECD - C3d3 fusion protein and F_β.ECD protein in eukaryotic cell transfected by pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD were detected by western blot and immuocytochemistry technique.4. The immunological contraception effect of pSG.SS.C3d3.YL-F_β.ECD DNA vaccine on female Kunming mouse was observed. The changes of specific F_β-IgG antibody titer in serum and specific F_β-IgA antibody titer in vaginal discharge were determined. The level of IL-2,INF-γ,IL-4,IL-10 in the serum were detected respectively.5. The immunological contraception effect on male Kunming mouse of pSG.SS.C3d3.YL-F_β.ECD DNA vaccine was observed. The changes of specific F_β-IgG antibody titer and the level of IL-2,INF-γ,IL-4,IL-10 in the serum were detected.Results1. cDNA of F_βECD was amplified by PCR. Then this fragment was directedly cloned into pSG.SS.C3d3.YL and pSG.SS.YL. pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD were constructed successfully. pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD were confirmed by sequence test.2. Eukaryotic cell can express F_β.ECD-C3d3 fusion protein and F_β.ECD protein after transfected by pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD. Both F_β.ECD-C3d3 and F_β.ECD showed biological activity.3. As DNA vaccine, the immunological contraception effect of pSG.SS.C3d3.YL- F_β.ECD DNA in female Kunming mouse are more significant than pSG.SS.YL-F_β.ECD. After vaccined with pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD, the specific F_β-IgG antibody titer in the serum and vaginal discharge of female mouses were higher and the positive time was longer in the pSG.SS.C3d3.YL-F_β.ECD group than those in the pSG.SS.YL-F_β.ECD group. The level of IL-2 and INF-γshowed lower tendency and the level of IL-4 and IL-10 were on the contrary.4. The pregnant rate and average product number of mouse were lower in the pSG.SS.C3d3.YL-F_β.ECD group than those in the pSG.SS.YL-F_β.ECD group. The comparable results were observed in male Kunming mouse. In the pSG.SS.C3d3.YL-F_β.ECD treated group, the specific F_β-IgG antibody titer in the serum was higher and the positive time was longer than those in the pSG.SS.YL-F_β.ECD group. The level of IL-2 and INF-γshowed lower tendency and the level of IL-4 and IL-10 were on the contrary.Conclusions1. pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD were constructed successfully.2. Plasmid pSG.SS.C3d3.YL-F_β.ECD and pSG.SS.YL-F_β.ECD can express F_β.ECD-C3d3 fusion protein and F_β.ECD protein respectively.3. The birth control effect of pSG.SS.C3d3.YL-F_β.ECD as DNA vaccine was more significant than pSG.SS.YL-F_β.ECD both in female and male Kunming mouse. The humoral immunological reaction of pSG.SS.C3d3.YL-F_β.ECD in the mouse was stronger than pSG.SS.YL-F_β.ECD indicated by F_β.ECD antibody and IL-2, INF-γ, IL-4 and IL-10 changes.4. C3d significantly enhanced the contraception effect of F_β.ECD. It could be used in DNA vaccine probably.