【作者】 周雷；

【导师】 赵兴绪；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 奶牛乳房炎是严重危害奶牛业的一种常见的复杂多发性疾病,目前尚无十分有效的治疗方法。近年来,随着分子生物学的发展,对乳房炎的研究逐渐深入到分子水平,对乳房炎相关基因的研究也取得了新的进展。本研究以中国荷斯坦奶牛白细胞为材料,提取总RNA,并利用半定量RT-PCR技术,检测了乳房炎奶牛和正常奶牛白细胞中β-防御素5、巨噬细胞炎症蛋白-3、L-选择素、S100A9和S100A12基因的mRNA表达水平。另外,还克隆了MIP-3基因和β-防御素5基因,并对其同源性进行了分析。然后以重组克隆载体质粒为模板,扩增β-防御素5基因成熟肽片段。扩增产物经EcoR I和Not I双酶切后,导入pET28a原核表达载体,并转化大肠杆菌BL21(DE3),然后对重组菌进行诱导表达,表达产物用Tricine-SDS-PAGE进行检测。结果表明,患乳房炎奶牛白细胞中β-防御素5、MIP-3、S100A9和S100A12四种基因mRNA表达量明显上调,而L-选择素基因mRNA表达量变化不明显。经PCR鉴定、酶切鉴定和测序说明所克隆的两种基因是正确的。克隆的MIP-3基因与GeneBank上所登录的序列完全一致。人、黑猩猩和恒河猴MIP-3基因的同源性较高,犬次之,而牛与以上四者的同源性均较低。克隆的β-防御素5基因与GeneBank上登录的BNBD 5序列同源性为82%,8个位点的氨基酸存在变异,分别为第9位(P→L)、第10位(Q→V)、第14位(W→I)、第16位(M→R)、第18位(V→F)、第28位(M→T)、第35位(F→L)和第40位(P→K)。不同品种鸡的β-防御素5基因同源性较高,而牛的β-防御素5基因与鸡的同源性最低,人和鼠次之;β-防御素1、β-防御素4、β-防御素7、β-防御素8、β-防御素10、舌抗菌肽和气管抗菌肽同源性较高,β-防御素122、β-防御素122a和β-防御素123同源性较高,β-防御素119和β-防御素120同源性也较高,β-防御素5基因与其它防御素的同源性均较低。Tricine-SDS-PAGE分析结果证明,重组菌能够正确表达出目的蛋白质,而且在IPTG浓度为0.5mM、37℃诱导时表达量最大,主要以可溶性蛋白存在,相对分子质量约为6.4kDa。更多还原

【Abstract】 Mastitis of dairy cows is a serious disease which is of great damage to dairy industry; however, there is no efficient method to deal with. Recently, it was studied at molecular level with the development of molecular biology, so did the mastitis-associated genes.Chinese Hosltein cow leukocytes were used to extract total RNA and the mRNA expression level ofβ-defensin 5, MIP-3, L-selectin, S100A9 and S100A12 in leukocytes of dairy cows with or without mastitis was detected using semi-quantitative RT-PCR technique. In addition, MIP-3 gene andβ-defensin 5 gene were cloned, and the percent identity was analyzed. Then, theβ-defensin 5 gene mature peptide fragment was amplified according the recombinant clone vector plasmid. The gene fragment amplified was inserted into prokaryotic expressive vector pET28a after digested with EcoR I and Not I to form recombinant expressive plasmid. Furthermore, the recombinant expressive plasmid was transformed into Escherichia coli BL21(DE3) and induced with IPTG. Then, the expressive product was detected by Tricine-SDS-PAGE.The current results showed that the gene mRNA expression levels ofβ-defensin 5, MIP-3, S100A9 and S100A12 increased significantly in mastitis cow; L-selectin was not but holding on a same level. Both genes were correctly cloned and identified by PCR, restriction enzyme digestion and sequencing. And the MIP-3 gene cloned was completely consistent with that on the GeneBank. The identity of MIP-3 gene in Homo sapiens, Pan troglodytes and Mcacaca mulatta was high, but low in Canis and lowest in Bos taurus. Theβ-defensin 5 gene shared a 82% identity with the BNBD5 gene reported on the GeneBank. Eight amino acid residues might be mutation, that were: the ninth amino acid residue(P→L), the tenth amino acid residue(Q→V), the fourteenth amino acid residue(W→I), the sixteenth amino acid residue(M→R), the eighteenth amino acid residue(V→F), the twenty-eighth amino acid residue(M→T), the thirty-fifth amino acid residue(F→L) and the fortieth amino acid residue(P→K). Theβ-defensin 5 gene identity of different Gaulus species was high; however, it was the lowest between Bos taurus and Gallus, but Homo sapiens and Rattus norvegicus were higher than them. The identity betweenβ-defensin 1,β-defensin 4,β-defensin 7,β-defensin 8,β-defensin 10, LAP and TAP was high, so did it betweenβ-defensin 122 andβ-defensin 122a.β-defensin 119 andβ-defensin 120 also shared the similar identity. However, the identity betweenβ-defensin 5 gene and any other defensins was lower. The recombinant bacterial could express the aimed protein which was identified by Tricine-SDS-PAGE. It was obtained to the biggest mount of soluvable protein which weighed 6.4kDa when the recombinant bacterial were induced with IPTG at the concentration of 0.5mM, and the inducing temperature was 37℃.更多还原