Research And Application On Inhibin As Gene Engineering Vaccine

Techniques such as bioinformatic, molecular clone, protein analysis, gene immunization, enzyme-linked immunoassay (ELISA), radio immunoassay (RIA) and so on were applied to prepare the tandem inhibin as gene engineering vaccine with inhibinα-subunit (1～32) of pig and complement 3d of sheep, and to study the immune response of tandem inhibin gene vaccine, effects on the reproduction and reproductive endocrinology in sheep, and investigate its mechanism. At the same time, to search the technique of tandem inhibin gene immunization for the best way to increase the reproduction of sheep, reduce the costs and popularize the new technolgoy. The main content presents as follows:1. The epitopes prediction of pig inhibinα-subunitThe secondly structure, epitopes, hydrophobicity, hydrophilicity, coil regions, solvent accessibility and so on were analyzed by use of on-line predictprotein, DNAStar and Antheprot softwares to predict the antigen locus and epitopes of inhibinα-subunit of pig. The results demonstrated that the epitopes of inhibin were probably at or adjacent to amino acids sequence 1～32 and 105～130 of its N-terminal, suggesting that there are some Pro in the N-terminal. The epitopes of inhibin antigen were possibly localized in amino acide sequence 1～32 of its N-terminal.2. Preparation and identification of tandem inhibin gene vaccineUsing to the technology of molecular clone, the recombinant plasmid of tandem inhibin were prepareted with inhibinα-subunit (1～32) of pig, complement 3d of sheep and dog preproinsulin signal sequence (DPPISS). The results showed that the recombinant plasmid, pcDNH-DPPISS-DINH and pcDNH-DPPISS-DINH-sC3d3 were constructed successfully. After BHK-21 cells were transfected with the recombinant plasmid pcDNH-DPPISS-DINH and pcDNH-DPPISS-DINH-sC3d3, secreted expression was obtained. All these were the basis of developing the inhibin gene as vaccine for sheep.3. Effect of tandem inhibin gene immunization on reproductive hormones of sheepAccording to the technology of gene immunization, 60 sheep were immunized with pcDNA-DPPISS-DINH and pcDNA-DPPISS-DINH-sC3d3, respectively. The P/N values of antibody against inhibin were detected by ELISA (enzyme-linked immunoassay) and the hormone levels were detected in different stage by RIA (radioimmunoassay) after gene immunization of tandem inhibin in sheep. The results showed the P/N values of antibody against inhibin in gene immunization groups of pcDNA-DPPISS-DINH and pcDNA-DPPISS-DINH-sC3d3 were higher than those in control group (P < 0.05), respectively, and the levels of antibody against inhibin were rose significantly after the third immunization. The levels of follicle stimulating hormone (FSH) in gene immunization groups were higher than those in control group after the first immunized, and the difference was significant after the second and third immunization (P<0.05), respectively. The levels of LH, E2 and P were all higher than those in control group after the second immunized, but there is no difference (P>0.05). After the third immunization, the levels of E2 and P were higher than those in control group(P < 0.05), respectively. All the results demonstrated that the recombinant plasmid of tandem inhibin gene as vaccine for sheep can stimulate FSH secretion, so as to effect follicle development and induce sheep twinning.4. Tandem inhibin gene immunization to induce sheep twinningTo study the effect of tandem inhibin gene immunization on sheep twinning, 120 sheep were immunized with pcDNA-DPPISS-DINH and pcDNA-DPPISS-DINH-sC3d3, respectively. After immunization, the twining rate of sheep was 22.2% and 30.0%, respectively, which was significantly higher (P<0.5) than that in the control group. Molecular adjuvant, sC3d (sheep complement 3d) can promote immune response for gene immunization of tandem inhibin on sheep. The study of tandem inhibin gene immunization successfully to induce sheep twinning made theoretical foundation and technical basis for developing the inhibin gene as vaccine for sheep.5. Research on safety of tandem inhibin gene immunizationIn order to make sure the safety after tandem inhibin gene immunization on sheep, 8 sheep were immunized with 1.5 mg, 3.0 mg pcDNA-DPPISS-DINH and 2.0 mg, 4.0 mg pcDNA-DPPISS-DINH-sC3d3, respectively. The results showed that the observations of mental conditions, appetite and movement were normal, and the programs of body temperature, pulse and breathing rate were all insignificant change. At the same time, toxicity and adverse reaction were not observed after gene immunization for sheep. So, the gene vaccines of tandem inhibin, pcDNA-DPPISS-DINH and pcDNA-DPPISS-DINH-sC3d3 were safe and reliable.