【作者】 都立辉；

【导师】 霍贵成；

【作者基本信息】 东北农业大学 ， 食品科学， 2007， 硕士

【摘要】 乳酸菌是应用最为广泛的食品级益生菌。虽然我国的乳酸菌资源十分丰富，但对其研究还处于初级阶段。这也正是给予我们食品工作者的机遇和挑战，大力推进关于乳酸菌的基础研究和开发应用，利用当今先进的生物技术（如蛋白质工程、基因工程、细胞工程等）开发新的优良工程菌株，必将从根本上改变我国传统的乳酸菌利用方式。增进乳酸菌益生作用的途径之一是使其表达一些同源或异源的目的蛋白，这就需要可靠的表达系统的协助。在这方面，国际上广泛使用的是采用Nisin启动子的NICE（Nisin-Controlled gene Expression）表达系统。启动子作为控制基因转录起始的第一个必要元件，在表达系统中占有头等重要的地位。推进启动子的相关研究，不仅为以后表达系统的构建奠定基础，还将使我们更进一步地理解原核生物基因表达调控的过程。本课题首先通过基本的形态学观察，从传统乳制品中分离乳酸菌；之后采用两倍稀释法从分离得到的乳酸菌中筛选对Nisin具有一定抗性的菌株，然后利用16S rRNA基因同源性分析对部分菌株进行鉴定。以黄色微球菌为指示菌，研究抗性菌株是否产生抑菌物质。对抑菌物质的理化性质（对热、pH和酶处理的稳定性）与Nisin的理化性质进行对比研究。综合对比研究和菌株的鉴定结果，确定Nisin产生菌。应用PCR方法，从Nisin产生菌中克隆nisA启动子片段，之后将该片段插入到启动子探针型载体pNZ273中，通过组织化学的方法检测β-葡糖苷酸酶活性，对克隆的片段进行功能验证。结果如下：本课题共分离出90株形态学观察为乳酸菌的菌株，其中50株为球菌。采用MRS固体培养基，当Nisin的添加量为0.0003 g·mL^-1时，50株球菌中有15株能正常生长。用两倍稀释法对这些菌株的Nisin抗性进行的检测表明，大部分菌株对Nisin的抗性不超过0.00125 g·mL^-1。15株能在Nisin环境中正常生长的菌中有4株能产生抑菌物质，并且其中S7菌株产生的抑菌物质在高温处理（121℃，15 min）时具有良好的稳定性；酸性条件（pH=2）下，抑菌物质的稳定性要好于中性条件（pH=7）下；抑菌物质能被α-胰凝乳蛋白酶降解。这些性质与Nisin的理化性质一致。采用16S rRNA序列同源性分析将S7菌株进行了鉴定，鉴定结果为乳酸乳球菌。用PCR方法从S7菌株中克隆nisA启动子，获得了大小为214 bp的片段。测序结果表明该片段与已经公布的Nisin启动子序列具有一致性。将获得的序列连入启动子探针型载体pNZ273中，构建了重组质粒pDU273。将pDU273转化L．lactis NZ9000之后，用20 ng·mL^-1的Nisin诱导时，gusA基因获得了表达。本课题主要得出以下结论：采用两倍稀释法，通过逐步提高环境中Nisin的含量来筛选Nisin抗性菌株的方法切实可行。16S rRNA序列用于细菌鉴定已经成为一项常规的实验技术，随着GenBank中16S rRNA序列数据的爆炸式增长，这一技术对细菌的鉴定更加具有快速准确的特点。本课题中克隆获得的启动子片段能够启动启动子探针型载体pNZ273下游报告基因gusA的表达，证明克隆片段具有诱导型启动子的作用，可以作为nisA启动子使用。更多还原

【Abstract】 Lactic acid bacteria （LAB） are no dout the most widely used probiotics with food grade status.Though China abounds in the resources of LAB, their research is still at the primary stage,expecially on bacteriocins produced by them, such as Nisin. Fueled up by the basic researches andcommercially useages of LAB, the construction of new industrial strains with excellent properties,specifically employing the advanced biotechnology （such as Protein Engineering, GeneEngineering and Cell Engineering et al.）, not only confers food scientists’ good opportunity aswell as big challenge, but also will fundamentally change the traditional applying of LAB inChina.A reliable expression system, like worldwide popular NICE （Nisin-Controlled geneExpression） using the Nisin promoter, is essencial in trying to enhance the beneficial functions ofLAB when employing them to express certain homo- or heterologous proteins. Researches relatedto promoter serve for the following construction of gene expression system, and also lead usnearer to the understanding of the prokaryotic expression and regulation process, becausepromoter, as the first necessary element that controls initiation of gene transcription, plays themost important role in an expression system.LABs were first isolated from traditional dairy products through basic morphologicalobservation. Then the isolates resisted to certain amount of Nisin were selected using twofolddilution method and some of them were identified by the 16S rRNA sequencing method.These Nisin-resisted stains were screened about their inhibiting substances production usingMicrococcus flavus as an indicator. And comparative studies on the physico-chemical properties（stability when treated by heat, pH and enzymes） were conducted between the inhibitingsubstances and Nisin. The Nisin-producing strain was determined by combining the two results ofthe 16S rRNA characterization and the comparative Studies.The nisA promoter fragment was amplified from Nisin-producing strain by PCR and insertedinto promoter-probe vector pNZ273, followed by histochemical experiment to check theβ-glucuronidase activity, aiming at verifying the function of the cloned fragment.The results were as follows:First, 90 strains of LAB were morphologically selected from traditional dairy productscollected from Inner Mongolia, of which, 50 strains were coccobacteria.Second, 15 out of 50 strains grew well on MRS agar when the Nisin conceration was 0.3×10^-3g·mL^-1. Most of the Nisin-resisted strains did not grow at the Nisin conceration of 1.25×10^-3g·mL^-1, as easily seen from the twofold dilution method detection experiment. Third, 4 out of the 15 Nisin-resisted strains produced some kind of antimicrobial substancesinhibiting the growth of Micrococcus flavus. And the inhibiting substance produced by a straindesignated S7 was stable when treated by heat （121℃, 15 min）. The substance was more stablein acid environment （pH=2） than in neutralized condition （pH=7）. Andα-chymotrypsin candegrade the substance. All of these charateres were the same as Nisin.Fourth, the S7 strains mentioned above were identified to be Lactococcus.lactis by 16S rRNAsequencing.Fifth, a 214 bp fragment exactly the same to the published Nisin promoter sequence wasamplified from S7 by PCR.Sixth, a recombined plasmid pDU273 was constructed by inserting the cloned fragment intopromoter-probe vector pNZ273.Seventh, after transformed pDU273 into L. lactis NZ9000, the gusA gene was expressedwhen induced by Nisin at the conceration of 20 ng·mL^-1.From this study, it was concluded:First, the twofold dilution method was feasible in selecting Nisin-resisted strains by graduallyincreasing the Nisin conceration.Second, the 16S rRNA sequence identification has become a conventional technology inbacteria identification. And with the explosive increasing of 16S rRNA sequences submitted to theGenBank, this technology will be more and more prevalent with its fast and precise character.Third, it was verified that the cloned fragment could induce expression of the reporter gusAgene in pNZ273, indicating suitable usege as a nisA promoter.更多还原