【作者】 郭宪；

【导师】 赵兴绪；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2010， 博士

【摘要】 本文综合应用生物信息学技术、基因克隆技术、蛋白质分析技术、基因免疫技术、酶免疫测定技术、放射免疫测定技术等,以猪抑制素α亚基(1～32)基因及绵羊补体C3d基因作为选择基因,制备串联抑制素基因工程疫苗,用于基因免疫绵羊,研究串联抑制素基因免疫对母羊生殖及生殖内分泌的影响,探讨其作用机制。通过串联抑制素基因疫苗应用研究,探索串联抑制素基因疫苗免疫方案,达到提高绵羊繁殖力、降低生产成本、推广繁育新技术的目的,并为抑制素基因疫苗的成功研制奠定基础。主要内容如下:1.猪抑制素α亚基抗原表位的预测综合应用在线软件PredictProtein、DNAStar软件、Antheprot软件和现代生物信息学技术,分析猪抑制素α亚基的二级结构、抗原表位、亲水性、疏水性、Coil区域、易溶性等理化特性,旨在预测其抗原位点和抗原表位。结果表明:猪抑制素α亚基可能的抗原位点分别是N端1～32、105～130氨基酸区段。由于N端有多个Pro,易构成Coil结构,对抗原表位的形成有一定的影响,所以猪抑制素α亚基的抗原表位可能位于1～32。2.串联抑制素基因疫苗的制备与鉴定应用基因克隆技术,以猪抑制素α(1～32)基因及绵羊补体C3d基因为选择基因,并引入狗前胰岛素原信号肽基因,制备串联抑制素重组质粒pcDNA-DPPISS-DINH和pcDNA-DPPISS-DINH-sC3d3。通过酶切鉴定,串联抑制素重组质粒pcDNA-DPPISS-DINH和pcDNA-DPPISS-DINH-sC3d3构建正确,并在BHK-21细胞中获得了分泌型表达,为羊抑制素基因工程疫苗的研制奠定了基础。3.串联抑制素基因疫苗对绵羊生殖激素的影响根据基因免疫技术,以成功制备的串联抑制素重组质粒pcDNA-DPPISS-DINH和pcDNA-DPPISS-DINH-sC3d3为免疫原,对60只绵羊进行基因免疫试验,应用酶联免疫法(ELISA)检测抗抑制素抗体、放射免疫法(RIA)测定处理后不同时期血清中激素水平。结果表明:重组质粒pcDNA-DPPISS-DINH和pcDNA-DPPISS-DINH-sC3d3免疫绵羊后,各实验组P/N值均显著高于对照组(P<0.05),且在第3次加强免疫后抗体水平明显上升。首次免疫后,促卵泡素(FSH)平均含量均高于对照组,且在第2次、第3次加强免疫阶段差异显著(P<0.05)。第2次免疫后促黄体素(LH)、雌二醇(E2)、孕酮(P)含量均高于对照组(P>0.05),第3次加强免疫后E2、P含量均显著高于对照组(P<0.05)。这些结果表明,串联抑制素基因免疫绵羊可促进FSH分泌,进而影响绵羊卵泡发育和诱导绵羊产双羔。4.串联抑制素基因免疫诱导绵羊孪生的研究为了研究串联抑制素基因免疫对绵羊孪生的影响,以制备的串联抑制素基因疫苗pcDNA-DPPISS-DINH和pcDNA-DPPISS-DINH-sC3d3为免疫原,对120只绵羊进行免疫试验。结果表明:0.6 mg pcDNA-DPPISS-DINH和0.8 mg pcDNA-DPPISS-DINH-sC3d3免疫绵羊后,双羔率分别为22.2%和30.0%,均与对照组间差异显著(P<0.05),并且分子佐剂sC3d能增加绵羊对串联抑制素基因免疫的反应性。串联抑制素重组质粒成功诱导绵羊孪生的研究,为抑制素基因疫苗的研制提供了理论依据和技术支撑。5.串联抑制素基因免疫的安全性研究用高于正常实验组5倍、10倍(重组质粒pcDNA-DPPISS-DINH 1.5 mg、3.0 mg;pcDNA-DPPISS-DINH-sC3d3 2.0 mg、4.0 mg)的免疫剂量对8只绵羊进行基因免疫试验,旨在确定串联抑制素基因免疫绵羊的安全性。结果表明:串联抑制素基因疫苗pcDNA-DPPISS-DINH和pcDNA-DPPISS-DINH-sC3d3免疫绵羊后,实验羊的精神、食欲、运动等均无异常,体温、脉搏、呼吸均无明显变化,没有观察到基因免疫的任何毒副作用与不良反应。由此得出结论,串联抑制素基因疫苗pcDNA-DPPISS-DINH和pcDNA-DPPISS-DINH-sC3d3免疫绵羊诱导其孪生是安全、可靠的。更多还原

【Abstract】 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.更多还原