Construction And Immunifaction Of Inhibin-C3d DNA Vaccine

Inhibin, a glycoprotein hormone from gonad, acts as a negative feedback regulator of FSH.Inhibin DNA Vaccine increased FSH, ovulation rate and litter size rate. The inhibin DNA Vaccine as a fecundity vaccine was becoming more and more attentive. To improve the humoral immunity of inhibin gene vaccine and neutralize endogenic inhibin, the B cell epitope of the inhibin alpha subunit, construction, immunoreactivity, the safety of DNA vaccine and its effects on animal follicular development and reproductive hormones reproduction were studied in this paper from these aspects:(1)The 3-dimensional structure of pig inhibinαprotein was predicted by the methods of I-TASSER.The B cell epitopes of pig inhibinαprotein was predicted by different parameters. The secondary structure was predicted by the methods of Psipred and PredictProtein. The hydrophilicity, surface probability, flexibility and antigenic index were predicted by the methods of Kyte-Doolittle, Emini and Jameson-Wolf, respectively. The results of the B cell epitopes of pig inhibinαprotein by different methods were compared. According to the above methods, the B cell epitopes for inhibinαprotein were predicted. The B cell epitopes were probably located at or adjacent to the N-terminal No. 1～30,43～51,61～75,87～100 and105～125 regions.(2)The fragment of INHα(1～32) was amplified by PCR and inserted into pMDT-18 plasmid and then sub-cloned into eukaryotic expression vector pcDNA-DPPISS. As a result, a eukaryotic expressing plasmid of pcDNA-DPPISS-DINH was formed.C3d3 cDNA was obtained by cutting the presented plasmid pSG5.C3d3.YL with proper restriction endonucleases. Three copies of C3d were troduced nto pcDNA-DPPISS-DINH to construct pcDNA-DPPISS-DINH-C3d3 nd restriction endonuleases digestion was sed to identify the fused insertion.Conclusion:The constructed DNA vaccine can be expressed in vitro, which may pave a way for further studies in animals.(3)A total of 48 rats at the age of 60 days were divided into 6 groups and immunized (intramuscularly injection)with pcDNA3.1 50ug(control 1), recombinant expression plasmid pcDNA-DPPISS-DINH 50ug(trial 1), pcDNA-DPPISS-DINH-C3d3 50ug(trial2),pcDNA-DPPISS-INH(trial 3) ,pcDNA-DPPISS-DINH-C3d3(trial 4)and 0.9%saline(control 2).Booster was given w on day 20, 40 after primary immunization. The results showed that the weight of ovaries in trail group was much weightier than that in the control. But the weight of ovaries in the trial group between 1 and 2 had no remarkable differences The number of matured follicular (MF) in trial 2 group was more than that in the trial 2 group ,control 1 and 2 group (p<0.05) after three immunizations. On 30 days after booster of pcDNA-DPPISS-DINH,pcDNA-DPPISS-DINH-C3d3,pcDNA-DPPISS-INH, pcDNA-DPPISS-INH-C3d3, The concentration of blood plasma FSH,E2 respectively were remarkably higher level of than that on the days of the primary immunization ,but the concentration of blood plasma LH had no remarkable differences at the same immunizing time. In conclusions, inhibin gene immunization can stimulate follicular development of rats; raised the level of plasma FSH, E2, which set up the basis of inhibin gene immunizations on large animals;(4)The complement C3d gene was cloned from the lever tissue in sheep (Ovis aries), its nucleotide sequence, deduced sequence of amino acid and protein structure were analyzed. There were 909 nucleotides in coding region of the complement C3d gene in sheep, which coded 303 amino acid residues. Entering number in Genbank was EF681138. As for deduced sequences of amino acid in Ovis aries, Homo sapiens, Bos Taurus, Capra hircus, Sus scrofa, Mesocricetus auratus, Mus musculus, Rattus norvegicu, Macropus eugenii, Oryctolagus cuniculus and Gallus gallus, their consistency was 80.4%, 94.7%, 96.9%, 82.8%, 81.1%, 80.6%, 80.2%, 71.0%, 75.2% and 60.3%, respectively. In higher animals, the consistency of sheep and Gallus gallus was 60.3%, comparing with other animals, the consistency was from 71.0% to 96.9%. By biological software analyzing, there were 2 phosphorylation loci of protein kinase C, 1 phosphorylation loci of casein kinaseⅡ, 1 phosphorylation loci of tyrosine kinase, 1 myristoylation loci. In the secondary structure of C3d in sheep, the helix and turn appeared alternately, in them,α-helix accounted for 55.12%,β-folding 2.31%, turn region 42.57%, this structure is beneficial to form barrel-shaped structure. By homogenous model of ESyPred3D anticipating, the tertiary structure of C3d in sheep resembled to one of human, it can form the barrel-shaped structure composed of core and cortex.(5)The fragment of C3dwas amplified by RT-PCR and inserted into pMDT-18 plasmid and then sub cloned into eukaryotic expression vector pcDNA-DPPISS-DINH-mC3d3.The recombinant plasmid pcDNA-DPPISS-DINH-sC3d3 was transfected into BHK-21 cells and the expressed product was detected by Western blot. Results: Western blot demonstrated that DINH and sC3d3 existed in the cultural supernatant.Conclusion: The constructed DNA vaccine can be expressed in vitro, which may pave a way for further studies in animals.