【作者】 江龙海；

【导师】 祁克宗；

【作者基本信息】 安徽农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 β-防御素属抗菌肽,因具有广谱抗微生物和增强免疫作用以及对病原微生物不易产生抗性等优点而成为当前生物医学的研究热点之一。为探讨β-防御素在家禽体内的表达分布情况和抗菌活性,本研究以家禽β-防御素Gal-6为研究对象,运用分子生物学技术,从安徽三黄鸡、皖西白鹅、巢湖麻鸭不同组织中克隆出Gal-6基因片段,并构建pGEM-TEasy-Gal-6克隆载体和pGEX-4T-1-Gal-6表达载体,诱导表达后纯化得融合蛋GST-Gal-6,对纯化产物的生物活性进行了初步研究。具体内容和结果如下:1.应用RNA Trizol Reagent试剂盒抽提法,从安徽三黄鸡、皖西白鹅的骨髓和巢湖麻鸭不同组织中提取总RNA。总RNA经1%琼脂糖凝胶电泳后得到两条清晰的条带（28s和18s）,经分光光度计检测OD₂₆₀/OD₂₈₀值在1.8-2.0之间,RNA纯度符合实验要求。2.参考GenBank注册的鸡β-防御素cDNA序列（NM₀01001193）,设计两对引物P₁/P₂、P₃/P₄,P₁/P₂扩增Gal-6基因片段,P₃/P₄扩增编码成熟肽基因。抽提总RNA用随机引物逆转录合成cDNA第一链,运用反转录聚合酶链式反应（RT-PCR）,从安徽三黄鸡、皖西白鹅的骨髓和巢湖麻鸭不同组织中克隆出目的基因,对PCR产物进行纯化回收,构建pGEM-T Easy-Gal-6克隆载体。提取纯化重组的克隆质粒并测序,经GenBank的BLAST比对:克隆安徽三黄鸡Gal-6获得的249bp碱基在第246位有一碱基变异,同源性达99%;克隆皖西白鹅Gal-6所获得的249个碱基完全相同,同源性达100%;克隆巢湖麻鸭Gal-6获得247个碱基在第12bp处缺少一个T碱基,在242bp处缺少一个T碱基,同源性达99%。分析Gal-6编码框分别由20个氨基酸残基的信号肽、5个氨基酸的原片段、42个氨基酸的成熟肽组成。以pGEM-T Easy-Gal-6质粒为模板,P₃/P₄为引物PCR扩增出126bp碱基的成熟肽基因片段。3.检测Gal-6在巢湖麻鸭小肠、肾脏、肝脏等16个不同组织的表达分布情况。结果表明,除皮肤、肾脏、法氏囊外,而其他组织均检测到Gal-6的表达。4.首次将所克隆的巢湖麻鸭、皖西白鹅Gal-6基因序列提交GenBank,获得巢湖麻鸭Gal-6注册序列号（EU366148）、皖西白鹅Gal-6注册序列号（EU606039）。5.巢湖麻鸭Gal-6成熟肽基因和pGEX-4T-1载体双酶切后连接,构建pGEX-4T-1-Gal-6表达载体。PCR鉴定的阳性转化子增菌后经IPTG诱导表达,融合蛋白经SDS-PAGE鉴定。结果表明,GST-Gal-6融合蛋白分子量约为33kD,可存在于上清和沉淀中,最佳诱导时间为4h,30℃培养时可溶性融合蛋白在上清液中的浓度高于37℃培养。融合蛋白上清液经谷胱甘肽-琼脂糖树脂柱亲和层析纯化得GST-Gal-6融合蛋白。分光光度计检测GST-Gal-6融合蛋白含量为0.2613mg/mL。单层琼脂平板扩散法检测表达产物体外生物活性,对大肠杆菌具有一定的抑菌性。以上研究表明:本研究成功的运用分子生物学技术从家禽组织中克隆Gal-6基因片段,构建Gal-6克隆载体和成熟肽基因表达载体,IPTG诱导并纯化出目的融合蛋白,并初步探讨了纯化蛋白的活性。本论文的研究为防御素抗菌肽在畜牧业上的应用与开发提供了一定的研究基础。更多还原

【Abstract】 Beta-defensin,an antimicrobial peptide in poultry,showes activity of antimicrobial and function of enhances immunity and little is known about resistivity for pathogenic microrganism,it is one of the focus for many scholar.To investigate the distribution expression information and antimicrobial activity in poultry,the study mainly applied molecular biology and molecular cloning technology.The fragment of Gal-6 was cloned from the tissues of poultry（Three Yellow broile,Wan Xi White goose and Chao Hu duck）.The cloning vector of pGEM-T Easy-Gal-6 and expression vector of pGEX-4T-1-Gal-6 were constructed,the primary biologic activity of purified product was investigated.The results were showed as followed:1.Trizol Reagent was adopted to extract the RNA in the marrow of Three Yellow Broiler, Wan Xi White goose and sixteen different tissues of Chao Hu duck.28s and 18s RNA bands were found by 1.0%agarose gel electrophoresis,The OD values of RNA at wavelenghs of 260nm and 280nm were determinated by spectrophotometer,The ratios of OD₂₆₀/OD₂₈₀ were between 1.8 and 2.0.The results suggested that the purity of obtained RNA was high enough to satisfy the requirement of experiment.2.According to the reported cDNA gene sequence（NM₀01001193） of Gallinacin beta-defensin,two pairs of primers were designed,P₁/P₂ amplified Gal-6 gene and P₃/P₄ amplified the gene of mature peptide.RNA from the tissues was reverse transcribed into a first-strand cDNA with random primers.The objective gene fragment were cloned successfully from the tissues of Three Yellow broiler,Wan Xi White goose and Chao Hu duck,by reverse transcription-polymerase chain reaction（RT-PCR）,then the produces purified and recovered, then the cloning vector of pGEM-T Easy-Gal-6 was constructed.The recombinant plasmid that was extracted and sequenced was compared by BLAST of www.ncbi.com.Compared with gene fragments registered in GenBank,there was a difference in the 246^th bp of the sequenced 249 bases of Three Yellow Broiler,T mutated C,the highest degree of identity in nucleotide was 99%;the gene sequence of Wan Xi White goose was completely homology,the highest degree of identity in nucleotide was 100%;two bases were lack in the sequenced fragment of ChaoHu duck,only 247bp,there were lack of T in the 12^th bp and 242^th respectively,the highest degree of identity in nucleotide was 99%;The cDNA fragment coded 67 amino acid residues,comprised of signal peptide with amino acid residues,propriece peptide with 5 amino acid residues and mature peptide with 42 amino acid residues.The mature peptide sequences,126bp,were amplified by nested PCR from a pair of primer of P₃/P₄ and the mould of recombinant plasmid pGEM-T Easy-Gal-6.3.To detect Gal-6 distribution information of Chao Hu duck in the sixteen tissues,including intestine,kidney and liver.The results showed that object gene fragments amplified by PCR, were not in kidney,skin and bursa of fabricius,other 13 samples was positive.4.The gene sequences Gal-6 of duck and goose were submitted to GenBank,the GenBank accession number were EU366148 and EU606039,respectively.It is the first reported in GenBank.5.The expression vector was constructed with mature peptide sequence of Gal-6 gene in the tissues of Chao Hu duck.The mature peptide sequence,which was purified and double enzyme cured,was inserted into the gene point of plasmid pGEX-4T-1- Gal-6 vector which was also double enzyme cured,then positive recombinant plasmid was identified by PCR.The amplified positive recombinant plasmid was optimized for induce with IPTG expression of time and temperature,following SDS-PAGE detected the expression information.The result were showed:the fusion protein of GST-Gal-6 was found in the point of 33kD,which meant the expression vector was constructed successfully,it was the best to induce expression with IPTG for four hours,there were fusion protein in the supematant and sediment at 30℃and 37℃,but there were more soluble fusion protein in the supernatant at 30℃.The supernatant product was purified by Glutathione Sepharose chelation affinity chromatography and upper purified GST-Gal-6 fusion protein was received.The analysis of spectrophotometer revealed that expression level of GST-Gal6 was 0.2613mg/mL.The biological activity of expression production in vitro was detected by monlayer agar plate diffusion method it appeared activity of antimicrobial.The above results showed:the objective gene fragments were cloned from the tissues of laboratory animals and constructed the cloning vector and expression vector of mature peptide successfully.The objective fusion protein was induced with IPTG,the bacteriostasis study revealed that purified fusion protein had antibacterial,hese results provides scientific guidance for development and application of antibiotic of defensin in animal husbandry.更多还原