KISS1基因免疫去势效果及机制的研究

发布时间：2018-04-25 05:38

本文选题：KISS1 + 免疫去势　；参考：《华中农业大学》2015年博士论文

【摘要】：沿袭千年的手术去势正受到规模化标准化畜牧生产需求的挑战,基因免疫去势新技术最有希望成为替代者。下丘脑分泌的kisspeptin蛋白由KISS1基因编码,处于生殖轴控制最上游,通过调控Gn RH的分泌影响繁殖,对动物青春期起始和繁殖的控制起着至关重要的作用。本研究应用基因克隆、RT-PCR、Western blot、酶联免疫测定、免疫组化、实时荧光定量PCR、组织HE染色等技术,选择生殖轴最上游的KISS1基因作为靶基因构建非抗性筛选真核表达质粒p KS-asd,在细胞水平测试融合抗原KS的表达,将其免疫公羊,旨在探讨KISS1基因免疫去势的效果、机制、对生长的影响、可逆性和否安全。本研究为开发高效和低成本的免疫去势新技术和新方法提供了理论基础和技术支撑。1.非抗性筛选KISS1真核表达质粒p KS-asd的构建与鉴定将合成的人KISS1基因克隆到p VAX1载体上,构建p VAX1-KISS1质粒,然后再将HBs Ag-S基因克隆到p VAX1-KISS1质粒上,构建p KS质粒,最后利用天冬氨酸β-半乳糖脱氢酶(asd基因)营养筛选标记取代p KS质粒中的卡那霉素(kan)抗性基因筛选标记,构建非抗性筛选KISS1真核表达质粒p KS-asd。融合基因KS的序列、插入位点和方向经测序和酶切鉴定表明是完全正确的;p KS-asd质粒在转染He La细胞24 h后经RT-PCR法检测到了融合基因KS的正确转录;p KS-asd质粒转染He La细胞48 h后,通过Western blot方法检测到了融合蛋白KS的正确表达。2.KISS1基因免疫去势的效果将构建的非抗性筛选KISS1基因疫苗p KS-asd和空质粒p VAX-asd分三次,间隔三周免疫6头8周龄公羊,到初免后的第14周进行屠宰。初免后的第4-14周,疫苗免疫组公羊可检测到特异性的抗kisspeptin抗体;疫苗免疫组公羊睾酮浓度显著低于对照组公羊(P0.05);初免后第6-14周,疫苗免疫组公羊阴囊周长均显著低于对照组公羊(P0.05),疫苗免疫组公羊睾丸重和睾丸长均显著低于对照组公羊(P0.05),疫苗免疫组公羊睾丸内精原细胞、精母细胞和精细胞的数量均明显低于对照组公羊;疫苗免疫组公羊的性行为显著低于对照组公羊(P0.05)。结论,KISS1基因免疫公羊后,诱导了特异性的抗kisspeptin抗体应答,抑制了公羊的性腺功能和性行为,达到了去势的目的,同时,证明KISS1基因是开发免疫去势基因疫苗的一个新靶标。3.KISS1基因免疫去势的机制①抗kisspeptin抗体特异性中和外周血中的kisspeptin在初免后的第4-14周,抗kisspeptin抗体在疫苗免疫组公羊中均能检测到,同时,在初免疫后的第6-14周,疫苗免疫组公羊血清中kisspeptin的浓度显著低于空质粒对照组公羊(P0.05),表明KISS1基因免疫产生的抗kisspeptin抗体能够特异性中和外周血中内源性的kisspeptin,从而导致血清中kisspeptin浓度的急剧下降,达到去势的目的。②抗kisspeptin抗体穿过血脑、血睾屏障直接发挥免疫中和作用在睾丸和脑组织中未能检测到抗kisspeptin抗体的存在,表明KISS1基因免疫产生的抗kisspeptin抗体可能不能穿过血睾或血脑屏障而在睾丸或脑内直接发挥免疫中和作用。③KISS1基因免疫对KISS1基因在下丘脑-垂体-性腺轴(HPG轴)中表达的影响疫苗免疫组公羊下丘脑、垂体和睾丸组织中KISS1基因的表达量与对照组没有显著差异,表明KISS1基因免疫可能不是通过影响HPG轴中KISS1的合成而达到去势的目的。④KISS1基因免疫对HPG轴中GPR54基因表达的影响疫苗免疫组公羊下丘脑、垂体和睾丸组织中GPR54基因的表达量与对照组没有显著差异,表明KISS1基因免疫可能不通过影响HPG轴中GPR54的合成而达到去势的目的。结论,KISS1基因免疫去势的机制可能是通过抗kisspeptin抗体特异性中和外周血中的kisspeptin,从而达到去势的目的。4.KISS1基因免疫去势对生长的影响在初次免疫后的第4-14周,疫苗免疫组公羊血清中的睾酮浓度显著低于空质粒对照组公羊(P0.05),而疫苗免疫组公羊血清中的雌激素、IGF-1和GH浓度与对照组差异不显著。疫苗免疫组公羊胰脏和舌组织中KISS1基因的表达量显著小于对照组,疫苗免疫组公羊胰脏组织中GPR54基因的表达量显著小于对照组,说明KISS1基因免疫公羊后会在一定程度上影响KISS1及受体GPR54基因在消化系统中的表达。疫苗免疫组公羊的体重与对照组公羊无论在免疫前还是免疫后差异均不显著。综上所述,KISS1基因免疫去势不会影响动物的生长。5.KISS1基因免疫去势的可逆性将构建的非抗性筛选KISS1基因疫苗p KS-asd和空质粒p VAX-asd分三次,间隔三周免疫免疫6头8周龄公羊,到初免后第30周进行屠宰。疫苗免疫组公羊均能发现抗kisspeptin的抗体,此外,疫苗免疫组公羊血清中的睾酮水平显著低于对照组公羊(P0.05),随着时间的推移,疫苗免疫组抗体水平逐步下降,血清中睾酮的浓度也逐步提高。在初免后第6-22周,疫苗免疫组公羊阴囊周长均显著低于对照组公羊(P0.05),然而,在初免后第30周,疫苗免疫组公羊阴囊周与对照组公羊差异不显著。在初免后第30周,疫苗免疫组公羊睾丸重、睾丸长和睾丸宽与对照组差异不显著,疫苗免疫组公羊睾丸内精子、精细胞、精母细胞和精原细胞的数量与对照组公羊差异不明显。结论,KISS1基因免疫对动物睾丸生长发育的抑制具有可逆性。6.KISS1基因免疫去势的安全性将可逆性试验末期的公羊处死后,采集下丘脑、睾丸、心、肝、肺、脾、肾、肌肉和小肠组织,HE染色结果表明各个组织器官均正常,没有明显的病理变化,表明该疫苗没有引起公羊毒性反应。用PCR方法检测质粒p KS-asd与提取的基因组DNA发生整合的情况,检测结果没有发现质粒p KS-asd与基因组DNA的整合。这些结果证明KISS1基因疫苗是安全的。
[Abstract]:The millennial surgical castration is being challenged by large-scale standardized animal production demand, and the new technology for genetic immune castration is the most promising alternative. The kisspeptin protein secreted by the hypothalamus is encoded by the KISS1 gene and is in the upper reaches of the reproductive axis, which affects the initiation and reproduction of animal puberty through the regulation of the secretion of Gn RH. Control plays a vital role. This study uses gene cloning, RT-PCR, Western blot, ELISA, immunohistochemistry, real-time fluorescence quantitative PCR, tissue HE staining and so on. Select the most upstream KISS1 gene of the reproductive axis as the target gene to construct a non resistant eukaryotic expression plasmid P KS-asd, and test the fusion antigen KS at the cell level. This study provides a theoretical basis and technical support for the development of high efficiency and low cost immune castration technology and new methods for the development of the.1. non resistant screening KISS1 eukaryotic expression plasmid P KS-asd, which will be constructed and identified in this study. The synthesized human KISS1 gene was cloned to the P VAX1 vector, and the P VAX1-KISS1 plasmid was constructed. Then the HBs Ag-S gene was cloned to the P VAX1-KISS1 plasmid, and the P KS plasmid was constructed. Finally, the gene screening marker of the kanamycin resistant gene was replaced by the aspartate beta galactose dehydrogenase (ASD gene) nutrition screening marker, and the non resistant gene was constructed. Screening the sequence of KISS1 eukaryotic expression plasmid P KS-asd. fusion gene KS, the insertion site and direction were sequenced and identified by enzyme digestion. The P KS-asd plasmid detected the correct transcription of the fusion gene KS after the transfection of He La cells 24 h. The effect of the fusion protein KS on the correct expression of the.2.KISS1 gene immune castration was to divide the constructed non resistant screening KISS1 gene vaccine P KS-asd and empty plasmid P VAX-asd three times, and immunized 6 8 week old male rams at intervals of three weeks, and slaughtered for fourteenth weeks after the first exemption. The vaccine immune group rams could detect specific anti kis. Speptin antibody, the concentration of testosterone in the immunization group was significantly lower than that of the control group (P0.05). After the first 6-14 weeks, the male sheep scrotum Zhou Changjun was significantly lower than the control group (P0.05). The testis weight and testicular length of the vaccine immunization group were significantly lower than that of the control group (P0.05), and the spermatogonial cells in the testis of the vaccine immunization group, spermatogonial cells and spermatogonial spermatogonial cells, and spermatogonial spermatogonial cells in the vaccine immunization group The number of mother and sperm cells was significantly lower than that of the control group rams; the sex behavior of the goats in the vaccine immunization group was significantly lower than that of the control group (P0.05). Conclusion, after the KISS1 gene immunized rams, the specific anti kisspeptin antibody response was induced, the gonadal function and sexual behavior of the rams were inhibited, the purpose of the castration was reached, and the KISS1 base was proved. A new target.3.KISS1 gene immune castration mechanism for developing the immune castrated gene vaccine (1) the anti kisspeptin antibody specific neutralization and the kisspeptin in peripheral blood after the first 4-14 weeks, the anti kisspeptin antibody can be detected in the immunization group ram. At the same time, in the first 6-14 weeks after the immunization, the vaccine immunization group rams blood The concentration of kisspeptin in the Qing Dynasty was significantly lower than that of the empty plasmid control group (P0.05), indicating that the anti kisspeptin antibody produced by the KISS1 gene immunization could neutralize the endogenous kisspeptin in the peripheral blood, and thus resulted in a sharp decrease in the concentration of kisspeptin in the serum and the goal of the castration. (2) the anti kisspeptin antibody passed through the blood brain and the blood testis barrier. Immune neutralization can not detect the presence of anti kisspeptin antibodies in the testis and brain tissue. It shows that the anti kisspeptin antibody produced by KISS1 gene immunization may not be able to play a direct immune neutralization in the testis or brain through the blood testis or blood brain barrier. (3) the KISS1 based immune response to the KISS1 gene in the hypothalamus pituitary - Sex The expression of the gland axis (HPG axis) affects the hypothalamus of the immunization group of the vaccine group. The expression of KISS1 gene in the pituitary and testicular tissues is not significantly different from that in the control group, indicating that the KISS1 gene immunization may not achieve the castration by affecting the synthesis of KISS1 in the HPG axis. (4) KISS1 based immune effect on the gene expression of GPR54 in the HPG axis The expression of GPR54 gene in the hypothalamus, pituitary and testicular tissues of the vaccine group was not significantly different from that in the control group, indicating that the KISS1 gene immunization may not achieve the aim of the castration by affecting the synthesis of GPR54 in the HPG axis. The mechanism of the KISS1 gene immune castration may be through the specific neutralization of the anti kisspeptin antibody and in the peripheral blood. The effect of.4.KISS1 gene immune castration on growth was achieved in the 4-14 week after primary immunization. The concentration of testosterone in the serum of the immunization group was significantly lower than that of the empty plasmid control group (P0.05), while the estradiol, IGF-1 and GH concentrations in the serum of the vaccine immunization group were not significantly different from those in the control group. The expression of KISS1 gene in the pancreas and tongue of the immunization group was significantly smaller than that in the control group. The expression of GPR54 gene in the pancreas of the vaccine group was significantly smaller than that of the control group. It indicated that the KISS1 gene immune goats would affect the expression of KISS1 and the receptor GPR54 gene in the digestive system to a certain extent. The difference between the body weight and the control group was not significant before and after the immunization. To sum up, the KISS1 gene immune castration did not affect the invertibility of the.5.KISS1 gene immunization of the animals. The non resistant screening KISS1 gene vaccine P KS-asd and the empty plasmid P VAX-asd were divided into three times, and the immune immune system was immune to 6 8 weeks of age at the interval of immunization. The sheep was slaughtered for thirtieth weeks after the first exempting. The anti kisspeptin antibody was found in the immunization group. In addition, the testosterone level in the serum of the immunization group was significantly lower than that of the control group (P0.05). The level of antibody in the immunization group gradually decreased and the serum testosterone concentration increased gradually. After 6-22 weeks, the Zhou Changjun of the male sheep scrotum of the immunization group was significantly lower than the control group (P0.05). However, in the first thirtieth weeks after the first immunization, the difference was not significant between the male sheep of the vaccine group and the control group. In the thirtieth weeks after the first immunization, the weight of the testis, the length of the testis and the width of the testis were not significantly different from the control group, and the immunization group was not significantly different from the control group. The number of sperm, sperm cells, spermatocytes, spermatogonial cells and spermatogonial cells in the pellet was not significantly different from that of the control group. Conclusion: the inhibition of KISS1 gene immunization on the growth and development of animal testis has the safety of reversible.6.KISS1 gene immune castration. After the death of the rams at the end of the reversible test, the hypothalamus, the testis, the heart, the liver, the lungs, the spleen, the kidney, and the muscles are collected. The results of HE staining showed that all the tissues and organs were normal and there was no obvious pathological changes, which showed that the vaccine did not cause the toxic reaction of the rams. The integration of plasmid P KS-asd with the extracted genomic DNA was detected by PCR method, and the results did not find the integration of plasmid P KS-asd with genomic DNA. These results proved KI The SS1 gene vaccine is safe.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S857.129

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