本文選題：家蠅 + 差異基因��； 參考：《吉林農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：隨著抗生素廣泛及不合理的使用,導(dǎo)致臨床上耐藥性菌株種類不斷增多,耐藥性菌株的出現(xiàn)不僅給畜牧業(yè)帶來了巨大的經(jīng)濟(jì)損失,也給人和動(dòng)物的身體健康造成了嚴(yán)重的危害,研制和篩選出新型抗菌藥物,一定程度上替代傳統(tǒng)抗生素,是目前亟待解決的問題。家蠅長(zhǎng)期生活在雜菌橫生的環(huán)境中,能夠傳播多種疾病而自身卻鮮少發(fā)病,其所具有的這種獨(dú)特抗病能力可能源于家蠅體內(nèi)的多種抗菌活性物質(zhì),因此,越來越多的研究者將開發(fā)新型抗菌藥物的焦點(diǎn)轉(zhuǎn)向了家蠅抗菌物質(zhì)的研制。家蠅在經(jīng)病原菌誘導(dǎo)后,體內(nèi)會(huì)產(chǎn)生多種可表達(dá)出抗菌活性蛋白的差異基因,本研究利用基因工程技術(shù)對(duì)這些差異基因進(jìn)行篩選、克隆、表達(dá)及生物學(xué)活性研究以期從家蠅體內(nèi)篩選得到抗菌活性物質(zhì),為解決細(xì)菌耐藥性問題奠定基礎(chǔ)。本研究以課題組從豬肺炎支原體和致病性雞源大腸桿菌誘導(dǎo)家蠅幼蟲抑制性消減文庫(SSH)中篩選并克隆得到的三個(gè)全長(zhǎng)差異基因(家蠅差異基因1(Md-D1)、家蠅差異基因2(Md-D2)和家蠅溶菌酶1基因(MdL1))為基礎(chǔ),PCR擴(kuò)增獲得全長(zhǎng)差異基因,通過T-A克隆技術(shù)分別將三個(gè)基因克隆至pMD18-T載體中構(gòu)建陽性克隆質(zhì)粒,隨后,將Md-D1基因亞克隆至真核表達(dá)載體pGAPZαA中,構(gòu)建真核重組表達(dá)質(zhì)粒Md-D1-pGAPZαA;將Md-D2與MdL1基因亞克隆至真核表達(dá)載體pPIC9K中,構(gòu)建真核重組表達(dá)質(zhì)粒Md-D2-pPIC9K和MdL1-pPIC9K;將重組表達(dá)質(zhì)粒電轉(zhuǎn)化入畢赤酵母(P.pastoris)GS115菌株中進(jìn)行表達(dá);采用管碟法以及微量稀釋法藥敏試驗(yàn)檢測(cè)Md-D1,Md-D2與MdL1基因真核表達(dá)產(chǎn)物的抑菌活性,主要實(shí)驗(yàn)結(jié)果如下:1、對(duì)Md-D1、Md-D2以及MdL1三個(gè)差異基因進(jìn)行了PCR擴(kuò)增;成功構(gòu)建Md-D1、Md-D2以及MdL1與pMD-18T連接的克隆質(zhì)粒。2、成功構(gòu)建了真核重組表達(dá)質(zhì)粒Md-D1-pGAPZαA、Md-D2-pPIC9K和MdL1-pPIC9K,其中,Md-D2和MdL1在P.pastoris GS115中獲得表達(dá);GS115/Md-D2-pPIC9K最佳誘導(dǎo)條件:誘導(dǎo)時(shí)間為72h,培養(yǎng)基pH為6;GS115/MdL1-pPIC9K最佳誘導(dǎo)條件:誘導(dǎo)時(shí)間為72h,培養(yǎng)基pH為5。3、純化后的Md-D2和MdL1的表達(dá)產(chǎn)物經(jīng)過抑菌試驗(yàn)檢測(cè)結(jié)果顯示,Md L1基因的表達(dá)產(chǎn)物對(duì)臨床分離的雞源大腸桿菌耐藥株具有抑制作用,對(duì)雞源沙門氏菌耐藥株具有微弱的抑制作用;Md-D2基因的表達(dá)產(chǎn)物不具有抑菌活性,目前為家蠅未知功能差異基因。
[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 偽 A. The eukaryotic recombinant expression plasmid Md-D1-pGAPZ 偽 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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S859.79;Q78

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