本文選題：KISS1 + 免疫去勢　； 參考：《華中農業(yè)大學》2015年博士論文

【摘要】：沿襲千年的手術去勢正受到規(guī)模化標準化畜牧生產需求的挑戰(zhàn),基因免疫去勢新技術最有希望成為替代者。下丘腦分泌的kisspeptin蛋白由KISS1基因編碼,處于生殖軸控制最上游,通過調控Gn RH的分泌影響繁殖,對動物青春期起始和繁殖的控制起著至關重要的作用。本研究應用基因克隆、RT-PCR、Western blot、酶聯(lián)免疫測定、免疫組化、實時熒光定量PCR、組織HE染色等技術,選擇生殖軸最上游的KISS1基因作為靶基因構建非抗性篩選真核表達質粒p KS-asd,在細胞水平測試融合抗原KS的表達,將其免疫公羊,旨在探討KISS1基因免疫去勢的效果、機制、對生長的影響、可逆性和否安全。本研究為開發(fā)高效和低成本的免疫去勢新技術和新方法提供了理論基礎和技術支撐。1.非抗性篩選KISS1真核表達質粒p KS-asd的構建與鑒定將合成的人KISS1基因克隆到p VAX1載體上,構建p VAX1-KISS1質粒,然后再將HBs Ag-S基因克隆到p VAX1-KISS1質粒上,構建p KS質粒,最后利用天冬氨酸β-半乳糖脫氫酶(asd基因)營養(yǎng)篩選標記取代p KS質粒中的卡那霉素(kan)抗性基因篩選標記,構建非抗性篩選KISS1真核表達質粒p KS-asd。融合基因KS的序列、插入位點和方向經測序和酶切鑒定表明是完全正確的;p KS-asd質粒在轉染He La細胞24 h后經RT-PCR法檢測到了融合基因KS的正確轉錄;p KS-asd質粒轉染He La細胞48 h后,通過Western blot方法檢測到了融合蛋白KS的正確表達。2.KISS1基因免疫去勢的效果將構建的非抗性篩選KISS1基因疫苗p KS-asd和空質粒p VAX-asd分三次,間隔三周免疫6頭8周齡公羊,到初免后的第14周進行屠宰。初免后的第4-14周,疫苗免疫組公羊可檢測到特異性的抗kisspeptin抗體;疫苗免疫組公羊睪酮濃度顯著低于對照組公羊(P0.05);初免后第6-14周,疫苗免疫組公羊陰囊周長均顯著低于對照組公羊(P0.05),疫苗免疫組公羊睪丸重和睪丸長均顯著低于對照組公羊(P0.05),疫苗免疫組公羊睪丸內精原細胞、精母細胞和精細胞的數量均明顯低于對照組公羊;疫苗免疫組公羊的性行為顯著低于對照組公羊(P0.05)。結論,KISS1基因免疫公羊后,誘導了特異性的抗kisspeptin抗體應答,抑制了公羊的性腺功能和性行為,達到了去勢的目的,同時,證明KISS1基因是開發(fā)免疫去勢基因疫苗的一個新靶標。3.KISS1基因免疫去勢的機制①抗kisspeptin抗體特異性中和外周血中的kisspeptin在初免后的第4-14周,抗kisspeptin抗體在疫苗免疫組公羊中均能檢測到,同時,在初免疫后的第6-14周,疫苗免疫組公羊血清中kisspeptin的濃度顯著低于空質粒對照組公羊(P0.05),表明KISS1基因免疫產生的抗kisspeptin抗體能夠特異性中和外周血中內源性的kisspeptin,從而導致血清中kisspeptin濃度的急劇下降,達到去勢的目的。②抗kisspeptin抗體穿過血腦、血睪屏障直接發(fā)揮免疫中和作用在睪丸和腦組織中未能檢測到抗kisspeptin抗體的存在,表明KISS1基因免疫產生的抗kisspeptin抗體可能不能穿過血睪或血腦屏障而在睪丸或腦內直接發(fā)揮免疫中和作用。③KISS1基因免疫對KISS1基因在下丘腦-垂體-性腺軸(HPG軸)中表達的影響疫苗免疫組公羊下丘腦、垂體和睪丸組織中KISS1基因的表達量與對照組沒有顯著差異,表明KISS1基因免疫可能不是通過影響HPG軸中KISS1的合成而達到去勢的目的。④KISS1基因免疫對HPG軸中GPR54基因表達的影響疫苗免疫組公羊下丘腦、垂體和睪丸組織中GPR54基因的表達量與對照組沒有顯著差異,表明KISS1基因免疫可能不通過影響HPG軸中GPR54的合成而達到去勢的目的。結論,KISS1基因免疫去勢的機制可能是通過抗kisspeptin抗體特異性中和外周血中的kisspeptin,從而達到去勢的目的。4.KISS1基因免疫去勢對生長的影響在初次免疫后的第4-14周,疫苗免疫組公羊血清中的睪酮濃度顯著低于空質粒對照組公羊(P0.05),而疫苗免疫組公羊血清中的雌激素、IGF-1和GH濃度與對照組差異不顯著。疫苗免疫組公羊胰臟和舌組織中KISS1基因的表達量顯著小于對照組,疫苗免疫組公羊胰臟組織中GPR54基因的表達量顯著小于對照組,說明KISS1基因免疫公羊后會在一定程度上影響KISS1及受體GPR54基因在消化系統(tǒng)中的表達。疫苗免疫組公羊的體重與對照組公羊無論在免疫前還是免疫后差異均不顯著。綜上所述,KISS1基因免疫去勢不會影響動物的生長。5.KISS1基因免疫去勢的可逆性將構建的非抗性篩選KISS1基因疫苗p KS-asd和空質粒p VAX-asd分三次,間隔三周免疫免疫6頭8周齡公羊,到初免后第30周進行屠宰。疫苗免疫組公羊均能發(fā)現(xiàn)抗kisspeptin的抗體,此外,疫苗免疫組公羊血清中的睪酮水平顯著低于對照組公羊(P0.05),隨著時間的推移,疫苗免疫組抗體水平逐步下降,血清中睪酮的濃度也逐步提高。在初免后第6-22周,疫苗免疫組公羊陰囊周長均顯著低于對照組公羊(P0.05),然而,在初免后第30周,疫苗免疫組公羊陰囊周與對照組公羊差異不顯著。在初免后第30周,疫苗免疫組公羊睪丸重、睪丸長和睪丸寬與對照組差異不顯著,疫苗免疫組公羊睪丸內精子、精細胞、精母細胞和精原細胞的數量與對照組公羊差異不明顯。結論,KISS1基因免疫對動物睪丸生長發(fā)育的抑制具有可逆性。6.KISS1基因免疫去勢的安全性將可逆性試驗末期的公羊處死后,采集下丘腦、睪丸、心、肝、肺、脾、腎、肌肉和小腸組織,HE染色結果表明各個組織器官均正常,沒有明顯的病理變化,表明該疫苗沒有引起公羊毒性反應。用PCR方法檢測質粒p KS-asd與提取的基因組DNA發(fā)生整合的情況,檢測結果沒有發(fā)現(xiàn)質粒p KS-asd與基因組DNA的整合。這些結果證明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.

【學位授予單位】：華中農業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S857.129

【參考文獻】

相關期刊論文 前10條

1 王軍,王云芳;促黃體素釋放激素免疫哺乳動物對其繁殖生理的影響[J];國外畜牧學(草食家畜);1990年06期

2 李維紅,吳建平,王欣榮;靖遠灘羊體脂脂肪酸與肉品質關系的研究[J];中國草食動物;2005年01期

3 張利平,吳建平;肉羊體脂脂肪酸與肉品質關系的研究[J];甘肅農業(yè)大學學報;2000年04期

4 岳海寧，，吳潤;GnRH主動免疫對綿羊血清睪酮含量的影響[J];甘肅農業(yè)大學學報;1995年02期

5 王雪松;湯軍;崔毓桂;;新的生殖激素調控系統(tǒng)——KiSS-1/GPR54[J];國外醫(yī)學(計劃生育/生殖健康分冊);2006年04期

6 劉倩;;去勢在養(yǎng)豬生產中的應用及對豬生長性能的影響——物理去勢與疫苗去勢的比較[J];國外畜牧學(豬與禽);2010年05期

7 張興隆;汪先友;何陽;薛志杰;楊瑩;曹兵海;;去勢對12~18月齡皖南牛血液睪酮含量、生長性能和外貌特征的影響[J];動物營養(yǎng)學報;2014年06期

8 唐璐;江明鋒;王永;;羊肉膻味物質的研究進展[J];動物營養(yǎng)學報;2014年08期

9 王云芳，張永和，王軍，王新軍;激素免疫去勢公羊的研究[J];黑龍江動物繁殖;1994年02期

10 楊錦波,劉天佳,李繼遙;抗齲基因疫苗pcDNA3-gtfB整合宿主細胞基因組DNA的可能性研究[J];華西口腔醫(yī)學雜志;2003年03期

相關博士學位論文 前3條

1 張洪英;口蹄疫多表位DNA疫苗的免疫原性和安全性研究[D];第二軍醫(yī)大學;2003年

2 韓麗;抑制素新型基因工程疫苗的構建及其免疫效果研究[D];華中農業(yè)大學;2009年

3 曹曉涵;GnRH主動免疫動物去勢機制及其可逆性研究[D];四川農業(yè)大學;2013年

相關碩士學位論文 前3條

1 朱杰青;促黃體素釋放激素在大腸桿菌中的融合表達及抗原性鑒定[D];南京農業(yè)大學;2000年

2 楊仕群;GnRH一次性免疫對豬生殖、生產性能和胴體品質的影響[D];四川農業(yè)大學;2004年

3 王慶玲;卵泡抑制素基因疫苗pXAIS在小鼠體內的分布及安全性研究[D];華中農業(yè)大學;2009年

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