家蝇幼虫差异基因Md-D1、Md-D2和MdL1在毕赤酵母中的表达及产物活性分析

发布时间：2018-04-23 10:34

本文选题：家蝇 + 差异基因　；参考：《吉林农业大学》2016年硕士论文

【摘要】：随着抗生素广泛及不合理的使用,导致临床上耐药性菌株种类不断增多,耐药性菌株的出现不仅给畜牧业带来了巨大的经济损失,也给人和动物的身体健康造成了严重的危害,研制和筛选出新型抗菌药物,一定程度上替代传统抗生素,是目前亟待解决的问题。家蝇长期生活在杂菌横生的环境中,能够传播多种疾病而自身却鲜少发病,其所具有的这种独特抗病能力可能源于家蝇体内的多种抗菌活性物质,因此,越来越多的研究者将开发新型抗菌药物的焦点转向了家蝇抗菌物质的研制。家蝇在经病原菌诱导后,体内会产生多种可表达出抗菌活性蛋白的差异基因,本研究利用基因工程技术对这些差异基因进行筛选、克隆、表达及生物学活性研究以期从家蝇体内筛选得到抗菌活性物质,为解决细菌耐药性问题奠定基础。本研究以课题组从猪肺炎支原体和致病性鸡源大肠杆菌诱导家蝇幼虫抑制性消减文库(SSH)中筛选并克隆得到的三个全长差异基因(家蝇差异基因1(Md-D1)、家蝇差异基因2(Md-D2)和家蝇溶菌酶1基因(MdL1))为基础,PCR扩增获得全长差异基因,通过T-A克隆技术分别将三个基因克隆至pMD18-T载体中构建阳性克隆质粒,随后,将Md-D1基因亚克隆至真核表达载体pGAPZαA中,构建真核重组表达质粒Md-D1-pGAPZαA;将Md-D2与MdL1基因亚克隆至真核表达载体pPIC9K中,构建真核重组表达质粒Md-D2-pPIC9K和MdL1-pPIC9K;将重组表达质粒电转化入毕赤酵母(P.pastoris)GS115菌株中进行表达;采用管碟法以及微量稀释法药敏试验检测Md-D1,Md-D2与MdL1基因真核表达产物的抑菌活性,主要实验结果如下:1、对Md-D1、Md-D2以及MdL1三个差异基因进行了PCR扩增;成功构建Md-D1、Md-D2以及MdL1与pMD-18T连接的克隆质粒。2、成功构建了真核重组表达质粒Md-D1-pGAPZαA、Md-D2-pPIC9K和MdL1-pPIC9K,其中,Md-D2和MdL1在P.pastoris GS115中获得表达;GS115/Md-D2-pPIC9K最佳诱导条件:诱导时间为72h,培养基pH为6;GS115/MdL1-pPIC9K最佳诱导条件:诱导时间为72h,培养基pH为5。3、纯化后的Md-D2和MdL1的表达产物经过抑菌试验检测结果显示,Md L1基因的表达产物对临床分离的鸡源大肠杆菌耐药株具有抑制作用,对鸡源沙门氏菌耐药株具有微弱的抑制作用;Md-D2基因的表达产物不具有抑菌活性,目前为家蝇未知功能差异基因。
[Abstract]:With the extensive and unreasonable use of antibiotics, the variety of drug-resistant strains is increasing in clinic. The emergence of drug-resistant strains has not only brought huge economic losses to animal husbandry, but also caused serious harm to the health of human beings and animals. It is an urgent problem to develop and screen new antimicrobial agents to replace traditional antibiotics to some extent. Musca domestica has long lived in an environment in which it can spread a variety of diseases but seldom develop them. Its unique resistance to disease may originate from a variety of antimicrobial active substances in the housefly, therefore, More and more researchers have turned their attention to the development of new antimicrobial agents in Musca domestica. A variety of differentially expressed genes of antimicrobial activity proteins can be produced in housefly induced by pathogenic bacteria. In this study, these differentially expressed genes were screened and cloned by genetic engineering. The aim of this study is to screen antimicrobial active substances from Musca domestica, and to lay a foundation for solving the problem of bacterial resistance. In this study, three full-length differentially expressed genes (Musca domestica 1 Md-D1, Musca domestica 2md-D2) were screened and cloned from Mycoplasma pneumoniae and pathogenic chicken Escherichia coli induced suppression subtractive library (SSHs) of housefly larvae. The full-length differentially expressed gene was amplified by PCR from Musca domestica lysozyme 1 gene (MdL1). Three genes were cloned into the pMD18-T vector by T-A cloning technique to construct the positive clones. Then, the Md-D1 gene was subcloned into the eukaryotic expression vector pGAPZ 伪 A. The eukaryotic recombinant expression plasmid Md-D1-pGAPZ 伪 A was constructed, the Md-D2 and MdL1 genes were subcloned into the eukaryotic expression vector pPIC9K, the eukaryotic recombinant expression plasmids Md-D2-pPIC9K and MdL1-pPIC9K were constructed, and the recombinant expression plasmid was transformed into Pichia pastoris GS115 for expression. The antimicrobial activity of the eukaryotic expression products of Md-D1Md-D2 and MdL1 gene was detected by tube-disc method and microdilution method. The main results were as follows: 1. The PCR amplification of Md-D1Md-D2 and Md-D1Md-D2 and MdL1 were carried out. The induction time was 72 h, the medium pH was 5.3. The expression products of purified Md-D2 and MdL1 were tested by bacteriostasis test. The results showed that the expression products of Md L1 gene could inhibit the drug resistant strains of chicken Escherichia coli isolated from clinic. The expression product of Md-D2 gene has no bacteriostatic activity and is unknown functional difference gene of Musca domestica.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S859.79;Q78

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