【作者】 包黎娟；

【导师】 郑惠玲；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2009， 硕士

【摘要】 钙是家畜体内最重要的常量矿物元素之一,在体内发挥着重要功能。动物体内流通的钙浓度始终保持相对恒定,这种恒定是由家畜体内钙代谢平衡机制精细调控实现的。泌乳期母畜为了保证乳汁中具有充足的Ca2+并保护自己不至于患低血钙症,机体钙代谢机制发生变化,母畜的骨钙流向血液,并通过乳腺上皮细胞分泌到乳汁,以此补充乳汁中的钙离子。这个过程的调控机制国外已有许多研究报道。近年来研究表明Δ2ΔFosB基因(FosB双截短亚型)与动物骨钙的吸收、转运与沉积密切相关,但是Δ2ΔFosB基因是否与乳腺上皮细胞向乳汁中转运钙离子有关,尚未见报道,GenBank中已登录了人、牛、犬、鼠等物种的FosB基因序列,但至今山羊Δ2ΔFosB基因仍未被克隆。因此,本试验旨在克隆得到山羊的Δ2ΔFosB基因cDNA全长,并构建其重组腺病毒,从而为研究Δ2ΔFosB基因在乳腺上皮细胞中的功能提供理论基础。主要研究方法:1、本研究以山羊Δ2ΔFosB基因为研究对象,通过提取山羊乳腺组织总RNA,应用逆转录PCR(RT-PCR)的方法,合成cDNA第一条链。根据GenBank上已发表的牛FosB基因序列,设计特异性引物,采用Long and Acute PCR扩增的方法从山羊cDNA中扩增目的产物,1.0 %的琼脂糖凝胶电泳检测,回收目的产物。将目的基因经过T-A克隆,构建中间载体pMD-19T-FosB,将质粒送往TaKaRa公司测序。采用DNASTAR、Lasergene-Protean、ncbi-ORF Finder等软件对测序结果进行生物信息学分析。2、根据序列分析结果,设计带有EcoRV/HindIII酶切位点及HA标签序列的引物,以质粒DNA pMD-19T-FosB为模板, PCR扩增Δ2ΔFosB ,构建克隆载体pMD-19T-Δ2ΔFosB,用EcoRV/HindIII双酶切已构建好的pMD-19T-Δ2ΔFosB载体。回收小片段Δ2ΔFosB。同时用EcoRV/HindIII双酶切商品化的穿梭质粒pAdTrack-CMV,并回收片断AdTrack-CMV。然后将小片段Δ2ΔFosB与AdTrack-CMV用T4DNA连接酶连接,从而构建穿梭质粒pAdTrack-CMV-Δ2ΔFosB。以EcoRV/HindIII双酶切穿梭载体,并进行鉴定、测序。穿梭质粒经PmeI酶切线性化后在BJ5183菌内与pAdeasy-2进行同源重组,筛选阳性克隆,酶切鉴定。3、将重组腺病毒质粒pAd-Δ2ΔFosB用PacI酶切线性化后,回收病毒骨架。将纯化的病毒骨架采用脂质体LipofectamineTM2000介导法转染HEK293细胞,进行病毒的包装、扩增与鉴定,并根据报告基因GFP来测定含有Δ2ΔFosB基因重组腺病毒的病毒滴度。主要研究结果:1、成功克隆山羊Δ2ΔFosB基因,获得长度为734 bp的目的片段,提交GenBank(登录号为FJ 493226),生物信息学分析表明其中包含由489个碱基组成的完整开放读码框,编码163个氨基酸,其为FosB的第二个转录本即Δ2ΔFosB。山羊Δ2ΔFosB基因与牛、人、犬及小鼠的Δ2ΔFosB基因的同源性分别为98 %、92 %、91 %和86 %。这说明Δ2ΔFosB基因在不同物种间具有高度同源性。2、获得携带有HA标签的重组穿梭载体pAdTrack-CMV-Δ2ΔFosB、重组腺病毒载体pAd-Δ2ΔFosB,分别经EcoRV/HindIII双酶切鉴定以及PacI线性化检测后,证明山羊Δ2ΔFosB腺病毒载体构建成功。3、pAd-Δ2ΔFosB经脂质体介导法转染HEK293细胞后,倒置荧光显微镜下观察到大量绿色荧光蛋白表达。经病毒DNA的PCR鉴定,可以扩增出Δ2ΔFosB基因目的条带。这说明Δ2ΔFosB基因已经重组进入腺病毒,从而为研究Δ2ΔFosB基因在乳腺上皮细胞中的功能奠定了基础。更多还原

【Abstract】 Calcium is the most important constant mineral elements in animal , playing an important function. The circulating calcium concentration in terrestrial organisms remains relatively constant, Such stability relies on the mechanism of calcium metabolism. In order to ensure adequate Ca2+ and protect lactating mother from hypocalcemia, large quantities of calcium transfers from the skeleton and released into the milk through mammary epithelial cells as a supplement to breast milk calcium. Many studies have been reported the regulatory mechanism of this process abroad. In recent yearsΔ2ΔFosB Gene studies show thatΔ2ΔFosB (FosB double truncated subtype) is closely related to calcium absorption, transport and deposition, but it is unknown in the field of breast biology whetherΔ2ΔFosB gene regulates calcium level in the milk. GenBank have logged in the FosB gene sequences of human, cattle, dogs, rats and other species, butΔ2ΔFosB gene of goat has not yet been cloned. Therefore, this test was aimed at cloning full-length of goatΔ2ΔFosB cDNA and constructing its recombinant adenovirus, so as to provide test and theoretical basis for the regulation ofΔ2ΔFosB gene in milk calcium level.The main methods:1. In this study, through the extraction of goat mammary tissue total RNA and reverse transcript PCR (RT-PCR) method, we get the first chain of goat cDNA. Using self-designed primers, Long and Acute PCR amplification method, we amplifiedΔ2ΔFosB gene from goat cDNA, confirmed the purpose product by 1.0% agarosegel electrophoresis and recycled the product,Δ2ΔFosB gene. Then we insertedΔ2ΔFosB gene into pMD19-T Simple cloning vector construction of intermediate vector pMD19-T-Δ2ΔFosB. After sequencing, the sequencing results carried out bioinformatics analysis.2. According to the results of sequence analysis, we designed EcoRV/HindIII restriction site and HA tag sequence primer, and digested the pMD19-T-Δ2ΔFosB vector by EcoRV and HindIII, recycled small fragmentsΔ2ΔFosB. At the same time, the commercialization of the shuttle plasmid pAdTrack-CMV was digested by EcoRV and HindIII, and recover fragments AdTrack-CMV.Δ2ΔFosB small fragments connected with AdTrack-CMV by T4DNA ligase, thereby building a shuttle plasmid pAdTrack-CMV-Δ2ΔFosB. Shuttle vector double-digested with EcoRV/HindIII , and identificated, sequencing. Shuttle plasmid linearized by PmeI digestion, then homologous recombinated with pAdeasy-2 in BJ5183 bacteria, screened of positive clones, confirmed by restriction enzyme digestion.3. The recombinant plasmid pAd-Δ2ΔFosB linearized with PacI enzyme digestion and recovered of virus skeleton. Using liposome-mediated method, the purified virus skeleton transfected of HEK293 cells for virus packaging, amplification and identification, and we measured recombinantΔ2ΔFosB adenovirus titer in accordance with GFP reporter gene.The main results:1. GoatΔ2ΔFosB gene was successfully cloned a length of 734 bp fragment submitted GenBank (accession number for the FJ 493226), which contains 489 base pairs from the composition of the complete open reading frame encoding 163 amino acids, It is justΔ2ΔFosB the second transcript of FosB. Bioinformatics analysis showed that the homologous genes of goat between cattle, people, dog and mice inΔ2ΔFosB gene were 98%, 92%, 91% and 86%, respectively. This shows thatΔ2ΔFosB genes in different species are highly homologous.2. After subcloningΔ2ΔFosB gene, we obtained recombinant shuttle vector pAdTrack-CMV-Δ2ΔFosB and recombinant adenovirus vector pAd-Δ2ΔFosB carrying the HA tag. The EcoRV/HindIII double digestion and linearization PacI post-test proved that adenovirus vector was constructed successfully.3. After pAd-Δ2ΔFosB transfection of HEK293 cells mediated by liposome, the observed green fluorescent cells were infected with high titer of recombinant adenovirus. Identified by PCR, It amplifiedΔ2ΔFosB gene. This suggests thatΔ2ΔFosB gene has been recombined into the adenovirus vector, which lay the foundation for studying the mechanism in the regulation and expression ofΔ2ΔFosB gene expression in mammary epithelial cells .更多还原