本文關鍵詞： SRY基因 核酸疫苗 性別控制 性別決定　出處：《塔里木大學》2017年碩士論文　論文類型：學位論文

【摘要】：在哺乳動物的Y染色體上,特有的SRY基因是雄性特征體現(xiàn)的睪丸決定基因,它的表達與否決定著性別分化的方向。SRY基因決定雄性發(fā)育的生物學功能是靠核心HMG盒與D NA結合發(fā)揮作用,通過對Y染色體上的SRY基因設計疫苗,進而產生抗Y精子的SRY基因抗體,使SRY基因的功能不再起作用以達到性別控制。本研究利用西門塔爾奶牛的凍精提取DNA擴增出SRY基因,構建成真核表達載體pcDNA3.1-His-C-SRY制備性別控制核酸疫苗,并進行小鼠的免疫實驗,為后期制備奶牛的性控疫苗對研究奶牛的性別控制提供理論基礎。采用高鹽法和改進試劑盒法提取西門塔爾牛凍精DNA,兩者提取的DNA經檢測濃度分別為151.16±25.09 ng/μL和74.57±31.53 ng/μL,相比差異顯著(P0.05),高鹽法獲得的DNA擴增后得到的SRY基因條帶較亮,效果較好。將純化回收的目的片段,克隆到p M D-19T載體上,經菌液PCR和XhoⅠ、BamhⅠ雙酶切及測序正確的重組質粒pMD-19T-SR Y為模板,以帶酶切位點的引物擴增后回收SRY目的基因與線性載體pET28a構建原核重組質粒,誘導后檢測其表達。結果顯示:克隆測序所得的SRY基因序列與Gene Bank上公布的SRY基因序列同源性為100%;構建的pET28a-SRY表達載體經SDS-PAGE電泳檢測出在30-33 KD之間有特異性蛋白,而陰性對照pET28a未出現(xiàn)該蛋白,確定為SRY蛋白。根據(jù)真核表達質粒pcDNA3.1-His-C設計帶有酶切位點的PCR引物。經菌液PCR、雙酶切鑒定正確的重組質粒pc DNA3.1-His-C-SRY進行測序。提取pc DNA3.1-His-C-SRY重組質粒,將其轉染至HeLa細胞中,48 h后裂解蛋白并提取RNA,Western blotting檢測SRY蛋白表達情況,RNA反轉錄后擴增SRY基因。結果顯示:在蛋白水平檢測到一特異性條帶,大小約27 KD,與SRY蛋白的預期蛋白大小一致,證明重組質粒能在哺乳動物He La細胞中表達;在RNA水平,能特異性的擴增出SRY基因,為后續(xù)實驗的順利進行奠定了基礎。將測序正確的真核重組質粒pc DNA3.1-His-C-SRY大量提取,制備成性別控制核酸疫苗,測量其濃度為1μg/μL。肌肉注射100μg重組質粒免疫性成熟的雌鼠40只,1周加強免疫1次,共免疫3次。對照組性成熟雌鼠用空質粒和PBS以相同的免疫方式和劑量免疫雌鼠各15只。免疫后每周摘眼球采血1次,分離血清-20℃保存待測。通過間接ELISA檢測血液中的特異性抗體,之后將免疫小鼠與未免疫雄鼠合籠,計算后代仔鼠雌雄性別比例。結果可知:重組質粒pcDNA3.1-His-C-SRY肌肉注射小鼠后,產生了抗SRY抗體;與其他組相比,重組質粒免疫組后代仔鼠的雌雄比例64:58(1.11:1)可能發(fā)生了向雌性的偏移。
[Abstract]:On the mammal Y chromosome, the unique SRY gene is the testicular determinant of male characteristics. Its expression or not determines the orientation of sex differentiation. SRY gene determines the biological function of male development by combining core HMG cassette with DNA, and designing vaccine against SRY gene on Y chromosome. Then the SRY gene antibody against Y spermatozoa was produced, and the function of SRY gene was no longer effective to achieve sex control. In this study, SRY gene was amplified by extracting DNA from Simmental dairy cow. The sex-controlled nucleic acid vaccine was prepared by constructing eukaryotic expression vector pcDNA3.1-His-C-SRY and the mice were immunized. The DNA extracted from Simmental cattle by high salt method and improved kit were 151.16 鹵25.09 ng / 渭 L and 74.57 鹵31.53 ng / 渭 L, respectively. Compared with P0.05, the SRY gene bands obtained by DNA amplified by high salt method were brighter. The purified and recovered fragment was cloned into pMD-19T vector. The recombinant plasmid pMD-19T-SR Y was digested by PCR and Xho 鈪，

本文編號：1509703