本文選題：GnIH + RFRP-3　； 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：促性腺激素抑制激素(GnIH)是動物下丘腦-垂體-性腺軸(Hypothalamic pituitary gonadal,HPG)中的下丘腦抑制激素,通過抑制Gn RH調(diào)控動物繁殖。GnIH是由十二個氨基酸組成的短肽,它通過直接或間接作用于Gn RH神經(jīng)元,可降低動物機體內(nèi)促性腺激素等生殖激素的分泌;同時,GnIH在性腺中也可通過自分泌/旁分泌方式調(diào)控動物的性腺機能。本研究以GnIH為靶基因構(gòu)建pt S2GnIH-asd雙拷貝重組質(zhì)粒,在細胞水平檢測重組質(zhì)粒pt S2GnIH-asd的轉(zhuǎn)錄及表達,用構(gòu)建的質(zhì)粒免疫湖羊母羔,檢測血清中抗體滴度水平及相關(guān)激素水平,觀察母羔卵巢組織微觀結(jié)構(gòu)及公羊?qū)δ父岬男孕袨?評估GnIH基因疫苗在湖羊母羔上的免疫效果。(1)重組表達質(zhì)粒pt S2GnIH-asd的構(gòu)建與鑒定化學合成2對互補的GnIH基因單鏈核苷酸片段,通過PCR聚合獲得尾端和中端GnIH目的片段,尾端GnIH片段替換p VAX-S-Gn RH-asd載體中的Gn RH基因,構(gòu)建p SGnIH-asd單拷貝重組表達質(zhì)粒;將中端GnIH片段插入p SGnIH-asd重組質(zhì)粒中HBs Ag-S基因編碼的第112至113個氨基酸密碼子之間,構(gòu)建p S2GnIH-asd雙拷貝質(zhì)粒;將含有酶切位點的t PA基因連接至p S2GnIH-asd質(zhì)粒中HBs Ag-S基因的5'端,構(gòu)建pt S2GnIH-asd重組表達質(zhì)粒,融合目的基因記為t S2GnIH,各重組質(zhì)粒經(jīng)酶切、測序,其目的基因序列、插入位點及方向均正確。無內(nèi)毒素提取重組表達質(zhì)粒pt S2GnIH-asd及空質(zhì)粒p VAX-asd,以脂質(zhì)體包裹的方法轉(zhuǎn)染Hela細胞,24 h后通過RT-PCR成功檢測到融合基因t S2GnIH的轉(zhuǎn)錄;48 h后利用Western blot方法檢測到融合蛋白t S2GnIH的正常表達,大小約30.91 k Da,表明在真核細胞中pt S2GnIH-asd質(zhì)�？烧＿M行轉(zhuǎn)錄和表達。(2)GnIH基因疫苗免疫湖羊母羔及免疫效果評估選取56日齡健康湖羊母羔10頭,隨機分為2組,疫苗組注射pt S2GnIH-asd質(zhì)粒,對照組注射p VAX-asd空質(zhì)粒。每組分別免疫注射3次,每次間隔21 d。初次免疫(第0 d)至第98 d期間,每14 d采血和稱重,第98 d屠宰并采集組織。實驗過程中母羔生長狀況良好,兩組母羔體重之間沒有顯著差異(P0.05);用Elisa檢測血清抗體滴度,發(fā)現(xiàn)在第14 d至98 d時疫苗組母羔血清中均檢測到抗GnIH抗體(P0.05);對照組LH和E2水平在整個實驗期穩(wěn)定,而疫苗組LH和E2水平逐步上升,在第70 d至98 d時顯著高于對照組(P0.05);疫苗組血清中FSH和孕酮(P)與對照組相比沒有顯著差異(P0.05);疫苗組卵巢中大卵泡數(shù)顯著高于對照組(P0.05);試情公羊?qū)σ呙缃M持續(xù)嗅聞和追逐次數(shù)顯著高于對照組(P0.05)。
[Abstract]:Gonadotropin inhibitory hormone (GnIH) is a hypothalamic inhibitory hormone in animal hypothalamic-pituitary-gonadal pituitary. GnIH is a short peptide composed of 12 amino acids that regulate animal reproduction by inhibiting GnRH. It can reduce the secretion of gonadotropin and gonadotropin by directly or indirectly acting on the GnRH neurons, and GnIH can also regulate the gonadal function by autocrine / paracrine in the gonad. In this study, the ptS2GnIH-asd double copy recombinant plasmid was constructed with GnIH as the target gene. The transcription and expression of the recombinant plasmid pt S2GnIH-asd were detected at the cell level. To observe the microstructure of ovarian tissue of female lambs and the sexual behavior of male sheep against female lambs, to evaluate the immunological effect of GnIH gene vaccine on female lambs of Hu sheep. The recombinant expression plasmid pt S2GnIH-asd was constructed and identified. Two pairs of complementary single strand nucleotides of GnIH gene were synthesized chemically. The GnIH target fragment was obtained by PCR polymerization. The GnIH fragment at the tail end replaced the GnRH gene in the pVAX-S-Gn RH-asd vector, and the single copy recombinant plasmid of pSGnIH-asd was constructed. The intermediate GnIH fragment was inserted into the 112th to 113rd amino acid codon of HBs Ag-S gene in pSGnIH-asd recombinant plasmid, and the pS2GnIH-asd double copy plasmid was constructed, and the t PA gene containing restriction site was ligated to the 5 'end of HBs Ag-S gene in pS2GnIH-asd plasmid. The recombinant expression plasmid of pt S2GnIH-asd was constructed, and the fusion gene was recorded as tS2GnIH.The recombinant plasmids were digested by enzyme and sequenced. The sequence, insertion site and direction of the target gene were correct. Recombinant expression plasmid pt S2GnIH-asd and empty plasmid pVAX-asd were extracted without endotoxin and transfected into Hela cells by liposome for 24 h. The fusion protein was successfully detected by RT-PCR for 48 h and the fusion protein was detected by Western blot. Normal expression of t S2GnIH, The size of ptS2GnIH-asd plasmid in eukaryotic cells was about 30.91 kDa, which indicated that PT S2GnIH-asd plasmid could be transcribed and expressed normally in eukaryotic cells. Ten female lambs of Hu sheep were immunized with GnIH gene vaccine and 10 female lambs were randomly divided into two groups. The vaccine group was injected with pt S2GnIH-asd plasmid. The control group was injected with pVAX-asd empty plasmid. Each group was immunized three times every 21 days. From day 0 to day 98, the blood was collected and weighed every 14 days, and the tissues were slaughtered and collected on the 98 th day. In the course of the experiment, the female lambs grew well and there was no significant difference in body weight between the two groups (P 0.05). Elisa was used to detect the titer of serum antibodies. It was found that anti-GnIH antibody P0.05 was detected in serum of female lambs from day 14 to day 98, the levels of LH and E2 in control group were stable during the whole experimental period, but the levels of LH and E2 in vaccine group increased gradually. From day 70 to day 98, it was significantly higher than that of control group (P 0.05); the serum FSH and progesterone P in vaccine group were not significantly different from those in control group; the number of large follicles in ovary of vaccine group was significantly higher than that of control group (P 0.05); And chase times were significantly higher than that of control group (P 0.05).
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.4

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