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

【摘要】：在哺乳動(dòng)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(dá)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(xué)功能是靠核心HMG盒與D NA結(jié)合發(fā)揮作用,通過(guò)對(duì)Y染色體上的SRY基因設(shè)計(jì)疫苗,進(jìn)而產(chǎn)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(dá)到性別控制。本研究利用西門塔爾奶牛的凍精提取DNA擴(kuò)增出SRY基因,構(gòu)建成真核表達(dá)載體pcDNA3.1-His-C-SRY制備性別控制核酸疫苗,并進(jìn)行小鼠的免疫實(shí)驗(yàn),為后期制備奶牛的性控疫苗對(duì)研究奶牛的性別控制提供理論基礎(chǔ)。采用高鹽法和改進(jìn)試劑盒法提取西門塔爾牛凍精DNA,兩者提取的DNA經(jīng)檢測(cè)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(kuò)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(jīng)菌液PCR和XhoⅠ、BamhⅠ雙酶切及測(cè)序正確的重組質(zhì)粒pMD-19T-SR Y為模板,以帶酶切位點(diǎn)的引物擴(kuò)增后回收SRY目的基因與線性載體pET28a構(gòu)建原核重組質(zhì)粒,誘導(dǎo)后檢測(cè)其表達(dá)。結(jié)果顯示:克隆測(cè)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(gòu)建的pET28a-SRY表達(dá)載體經(jīng)SDS-PAGE電泳檢測(cè)出在30-33 KD之間有特異性蛋白,而陰性對(duì)照pET28a未出現(xiàn)該蛋白,確定為SRY蛋白。根據(jù)真核表達(dá)質(zhì)粒pcDNA3.1-His-C設(shè)計(jì)帶有酶切位點(diǎn)的PCR引物。經(jīng)菌液PCR、雙酶切鑒定正確的重組質(zhì)粒pc DNA3.1-His-C-SRY進(jìn)行測(cè)序。提取pc DNA3.1-His-C-SRY重組質(zhì)粒,將其轉(zhuǎn)染至HeLa細(xì)胞中,48 h后裂解蛋白并提取RNA,Western blotting檢測(cè)SRY蛋白表達(dá)情況,RNA反轉(zhuǎn)錄后擴(kuò)增SRY基因。結(jié)果顯示:在蛋白水平檢測(cè)到一特異性條帶,大小約27 KD,與SRY蛋白的預(yù)期蛋白大小一致,證明重組質(zhì)粒能在哺乳動(dòng)物He La細(xì)胞中表達(dá);在RNA水平,能特異性的擴(kuò)增出SRY基因,為后續(xù)實(shí)驗(yàn)的順利進(jìn)行奠定了基礎(chǔ)。將測(cè)序正確的真核重組質(zhì)粒pc DNA3.1-His-C-SRY大量提取,制備成性別控制核酸疫苗,測(cè)量其濃度為1μg/μL。肌肉注射100μg重組質(zhì)粒免疫性成熟的雌鼠40只,1周加強(qiáng)免疫1次,共免疫3次。對(duì)照組性成熟雌鼠用空質(zhì)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(cè)。通過(guò)間接ELISA檢測(cè)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(jì)算后代仔鼠雌雄性別比例。結(jié)果可知:重組質(zhì)粒pcDNA3.1-His-C-SRY肌肉注射小鼠后,產(chǎn)生了抗SRY抗體;與其他組相比,重組質(zhì)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(hào)：1509703