奶牛γ精子特异表达SRY基因制备性控DNA疫苗的初步研究

发布时间：2018-02-14 02:48

  本文关键词： SRY基因 核酸疫苗 性别控制 性别决定　出处：《塔里木大学》2017年硕士论文　论文类型：学位论文

【摘要】：在哺乳动物的Y染色体上,特有的SRY基因是雄性特征体现的睾丸决定基因,它的表达与否决定着性别分化的方向。SRY基因决定雄性发育的生物学功能是靠核心HMG盒与D NA结合发挥作用,通过对Y染色体上的SRY基因设计疫苗,进而产生抗Y精子的SRY基因抗体,使SRY基因的功能不再起作用以达到性别控制。本研究利用西门塔尔奶牛的冻精提取DNA扩增出SRY基因,构建成真核表达载体pcDNA3.1-His-C-SRY制备性别控制核酸疫苗,并进行小鼠的免疫实验,为后期制备奶牛的性控疫苗对研究奶牛的性别控制提供理论基础。采用高盐法和改进试剂盒法提取西门塔尔牛冻精DNA,两者提取的DNA经检测浓度分别为151.16±25.09 ng/μL和74.57±31.53 ng/μL,相比差异显著(P0.05),高盐法获得的DNA扩增后得到的SRY基因条带较亮,效果较好。将纯化回收的目的片段,克隆到p M D-19T载体上,经菌液PCR和XhoⅠ、BamhⅠ双酶切及测序正确的重组质粒pMD-19T-SR Y为模板,以带酶切位点的引物扩增后回收SRY目的基因与线性载体pET28a构建原核重组质粒,诱导后检测其表达。结果显示:克隆测序所得的SRY基因序列与Gene Bank上公布的SRY基因序列同源性为100%;构建的pET28a-SRY表达载体经SDS-PAGE电泳检测出在30-33 KD之间有特异性蛋白,而阴性对照pET28a未出现该蛋白,确定为SRY蛋白。根据真核表达质粒pcDNA3.1-His-C设计带有酶切位点的PCR引物。经菌液PCR、双酶切鉴定正确的重组质粒pc DNA3.1-His-C-SRY进行测序。提取pc DNA3.1-His-C-SRY重组质粒,将其转染至HeLa细胞中,48 h后裂解蛋白并提取RNA,Western blotting检测SRY蛋白表达情况,RNA反转录后扩增SRY基因。结果显示:在蛋白水平检测到一特异性条带,大小约27 KD,与SRY蛋白的预期蛋白大小一致,证明重组质粒能在哺乳动物He La细胞中表达;在RNA水平,能特异性的扩增出SRY基因,为后续实验的顺利进行奠定了基础。将测序正确的真核重组质粒pc DNA3.1-His-C-SRY大量提取,制备成性别控制核酸疫苗,测量其浓度为1μg/μL。肌肉注射100μg重组质粒免疫性成熟的雌鼠40只,1周加强免疫1次,共免疫3次。对照组性成熟雌鼠用空质粒和PBS以相同的免疫方式和剂量免疫雌鼠各15只。免疫后每周摘眼球采血1次,分离血清-20℃保存待测。通过间接ELISA检测血液中的特异性抗体,之后将免疫小鼠与未免疫雄鼠合笼,计算后代仔鼠雌雄性别比例。结果可知:重组质粒pcDNA3.1-His-C-SRY肌肉注射小鼠后,产生了抗SRY抗体;与其他组相比,重组质粒免疫组后代仔鼠的雌雄比例64:58(1.11:1)可能发生了向雌性的偏移。
[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 卤25.09 ng / 渭 L and 74.57 卤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，

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