【作者】 刘杰；

【导师】 李景鹏；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2007， 博士

【摘要】 枯草芽孢杆菌224(Bacillus subtilis 224，BS224)是从人体中分离出来的一种非致病菌。它不但是用途广泛的工程菌株，而且具有抗菌消炎的作用，能够用于烧、烫伤的治疗。根据微生态学原理，利用微生物间的拮抗作用，由枯草芽孢杆菌BS224活菌体制成的生物制品抑菌生和白天鹅气雾剂都具有改善创面微生态环境、抗菌消炎和促进创面愈合的作用。然而，由于存在溶血性，使这两种药物在用于烧、烫伤的治疗时，对创面造成了一定程度的损害。因此，大大限制了这两种药物的使用。自从枯草芽孢杆菌168基因组全碱基测序完成以后，有8个可能与溶血有关的基因被发现，分别为yqhB(1329bp)，yrkA(1305bp)，yugS(1305bp)，yhdT(1386bp)，yhdP(1335bp)，yqxC(810bp)，yplQ(642bp)，ytjAα(228bp)，其中前5个基因同源性很高，为69.83％，而)，ytjAα为α溶血素样基因。溶血样基因的发现使得对枯草杆菌溶血性的研究(包括确定溶血基因和溶血发生的分子机理)成为可能。本实验是根据枯草杆菌168基因组的序列设计引物，以枯草杆菌224的染色体DNA为模板，利用PCR技术扩增了yhdP、yugS、yhdT和yqhB 4个基因，并将它们分别插入克隆载体pMD18-T中，构建了重组质粒pMD18-T-yhdP、pMD18-T-yugS、pMD18-T-yhdT和pMD18-T-yqhB。连接产物转化入大肠杆菌JM109感受态中，并从含IPTG和X-gal的青霉素抗性平板上筛选出阳性克隆。应用酶切的方法对阳性重组子进行了鉴定，最后对阳性重组子进行测序分析。结果表明克隆的这4个基因yhdP、yugS、yhdT和yqhB的核苷酸序列与Genbank上已发表的序列的同源性分别为99.9％、99.6％、99.9％和99.9％，因此可以确定枯草杆菌224的基因组含有这4个基因。为了构建基因打靶载体的骨架质粒，根据载体pEGFP-N1基因全序列设计引物，并以pEGFP-N1质粒DNA为模板，通过高保真的Ex Taq酶扩增出含有自身启动子的新霉素抗性基因的DNA片段，并将其也连入克隆载体pMD18-T中，阳性重组质粒经酶切鉴定后进行测序分析。结果表明克隆的新霉素抗性基因的核苷酸与已发表序列完全一致，证明重组质粒pMD18-T-neo构建成功。然后克隆打靶载体的长、短臂，在yhdP、yugS、yhdT、yqhB四个基因的同源短臂引物的5′端分别引入SalⅠ和PstⅠ酶切位点，在yhdP、yugS和yqhB三个基因的同源长臂引物的5′端上引入KpnⅠ和BamHⅠ酶切位点，而yhdT基因的同源长臂引物的5′端引入KpnⅠ和XbaⅠ酶切位点，这样扩增出的目的片段连入pMD18-T simple载体，从获得的阳性重组质粒上切下目的片段将携带引入的酶切位点，以便于连入打靶载体。选择两种不同的方法将长、短臂连入打靶载体。其一：首先将骨架质粒pMD18-T-neo用SalⅠ、PstⅠ分别单酶切，接着把用碱性磷酸酶CIAP处理过的骨架质粒与短臂连接，连接产物转化入大肠杆菌JM109扩培，然后按照类似的方法再连入前臂，这样就构建好了打靶载体。yhdP基因的打靶载体就是通过这种方式构建的；其二：将骨架质粒pMD18-T-neo用KpnⅠ和PstⅠ双酶切，回收大片段pMD18-T，然后再用BamHⅠ(XbaⅠ)和SalⅠ双酶切小片段neo~r基因，最后将这两个片段与长、短臂用T4DNA连接酶连接，这样就构建好了打靶载体。yugS、yhdT和yqhB的打靶载体就是通过第二种连接方法构建的。将构建好的基因打靶载体用KpnⅠ和PstⅠ双酶切线性化后，电转化入枯草杆菌224中，新霉素抗性平板筛选。抗性转化子利用PCR鉴定方法筛选yhdP、yugS、yhdT和yqhB基因缺失株。鉴定yhdP基因缺失株时，利用在前臂的上游序列和后臂的下游序列设计引物进行PCR检测：而鉴定yugS、yhdT和yqhB基因缺失株时，是利用在新霉素抗性基因的下游和同源后臂的外侧设计引物进行PCR检测。对yhdP、yugS、yhdT和yqhB分别检测96、163、105和87个抗性转化子检测出1株基因缺失株。将yqhB，yugS，yhdT，yhdP四个基因缺失株接种到5％的绵羊血琼脂平板上进行溶血性检测，结果这四个基因缺失株在血平板上仍能够引起溶血，这说明单独敲除枯草芽孢杆菌224上染色体的4个基因yhdP、yugS、yhdT和yqhB的任何一个基因并不影响菌株的溶血。本实验在枯草杆菌168完成全序列分析之后，首次对枯草杆菌224的溶血样进行克隆，而且利用同源重组的方法构建yhdP、yugS、yhdT和yqhB基因缺失株并进行溶血性检测以确定溶血基因或引起溶血的主效基因，探讨溶血作用发生的分子机理，最后改造BS224使其在医学方面得到更广泛的应用。同时也为枯草杆菌基因组中未知功能基因的的研究提供了一套行之有效的方法，为今后开展枯草杆菌蛋白组研究迈出重要一步。更多还原

【Abstract】 Bacillus subtilis 224 was separated from human body and it was non-pathogenic. BS224 wasnot only widely used as engineer strain, but also could diminish inflammation, resist germs and cureburn and scald wounds. On the grounds of the Microecology principle, Yi jun sheng and WhiteSwan aerosol made of live BS224 could improve Microecology environment of wounds, resistgerms, diminish inflammation and improve wound healing by antagonistic action among microbe.But they impaired wounds in a way because of hemolysis, when they were used for curing burn andscald wounds. Therefore their uses was much limited.Since Bacillus subtilis 168 genome sequencing was finished, eight genes possibly related tohemolysis have been found. They were yqhB (1329bp) ,yrkA (1305bp) ,yugS (1305bp) ,yhdT(1386bp) ,yhdP (1335bp), yqxC (810bp), yplQ (642bp) and ytjAα(228bp), the homologyof the fore five genes was 69.83 %, and expression product of ytjAαwas similar to alpha-hemolysin.The finding of unconfirmed hemolytic genes made it possible that hemolytic genes and molecularmechanism of hemolysis were ascertained.Here, yhdP, yugS, yhdT and yqhB DNA fragments were amplified by PCR with chromosomalDNA of BS224 as the template and inserted into cloning vector pMD18-T to construct therecombinant plasmid pMD 18-T-yhdP, pMD 18-T- yugS, pMD 18-T-yhdT and pMD 18-T-yqhB. Theprimers of PCR amplification reaction were designed according to the genome sequence of Bacillussubtilis 168. The recombinant plasmids were transformed into competent cells of E.coli JM109.Positive clones were screened from the LB solid medium plate including Ampicillin, IPTG andX-gal. The positive recombinants were identified by restriction enzyme digest and finallysequenced .The results suggested that the homology between the nucleotide sequence of the clonedyhdP, yugS, yhdT and yqhB and the reported one were separately 99.9%, 99.6%, 99.9% and 99.9%.So it could be made sure that yhdP, yugS, yhdT and yqhB existed in the genome of BS224.To construct skeleton plasmid of targeting vector, the neomycin resistance gene includingself-promoter were amplified by PCR with pEGFP-N1 plasmid DNA as the template . DNApolymerase needed in the reaction was high fidelity Ex Taq and primers were designed according tothe sequence of pEGFP-N1. The amplified DNA fragments were inserted into cloning vectorpMD18-T. The positive recombinants were identified by restriction enzyme digest and finallysequenced. The results suggested that the nucleotide sequence of the cloned neomycin resistancegene was identical with the reported one in Genbank. Thus recombinant plasmid pMD18-T-neo was successfully constructed.Then long and short arms of targeting vector were cloned. Restriction enzyme cutting siteSalⅠand PstⅠwere introduced in the 5’flank of the primers of homologous short arms of yhdP,yugS, yhdT and yqhB genes. KpnⅠand BamHⅠwere separately introduced in the 5’flank of theprimers of homologous long arms of yhdP, yugS and yqhB genes, and KpnⅠand XbaⅠwereintroduced in that of yhdT gene. These homologous arms were amplified by PCR and inserted intovector pMD18-T simple, and objective fragments carried restriction enzyme cutting site wereobtained from the above constructed recombinant plasmids. Then these DNA fragments wereligated into targeting vector in certain order.Two ligation methods were chosen. The first method: at first,skeleton plasmid pMD18-T-neowith single enzyme digestion of SalⅠand PstⅠseparately, were treated with Alkaline Phosphatase(CLAP) ,then it was ligated with homologous short arm digested from the above constructedrecombinant plasmid with SalⅠand PstⅠ. Ligation products were transformed into competentcells of E.coli JM109 to increase, In a similar way , homologous long arms were ligated ,thustargeting vector was successfully constructed. The targeting vector of yhdP gene was constructed inthis method; The second method: big linear pMD18-T fragment were recovered after skeletonplasmid pMD18-T-neo was digested with KpnⅠand PstⅠ, then small linear neomycin resistancegene fragment was recovered after skeleton plasmid pMD18-T-neo was digested with SalⅠandBamHⅠ(XbaⅠ). The tow recovered fragments were ligated with homologous long and short armsdigested from the formerly constructed recombinant plasmid. thus targeting vector wassuccessfully constructed. The targeting vectors of yugS, yhdT and yqhB gene were constructed inthe second method.The constructed gene targeting vector was linearized with KpnⅠand PstⅠ, and electroporatedinto competent cells of BS224. Positive transformers were screened from the neomycin resistanceplate, yhdP, yugS, yhdT and yqhB gene deletion were identified by PCR. Primers of identificationreaction of yhdP gene deletion were designed according to the upstream sequence of the fore armand the downstream sequence of the back arm. Whereas primers of identification reaction of yugS,yhdT and yqhB gene deletion were designed according to the downstream sequence of neomycinresistance gene and the downstream sequence of back arm.96,163,105 and 87 transformers wereanalyzed by PCR to identify yhdP, yugS, yhdT and yqhB gene deletion, and finally single yhdP,yugS, yhdT and yqhB gene deleted mutant were separately found.Hemolysis of yqhB, yugS,yhdT and yhdP gene deleted mutants was separately identified on5% sheep blood agar plate .The results suggested that the four gene deleted mutants could allinduce hemolysis, It indicated that knockout of single gene had no influence on hemolysis ofBS224.Possible hemolytic genes of BS224 were first cloned in this experiment, since Bacillus subtilis168 genome sequencing was finished. Moreover yhdP, yugS, yhdT and yqhB gene deleted mutantswere constructed by homologous recombination and their hemolysis were identified to make sure hemolytic gene or main gene inducing hemolysis and to discuss molecular mechanism of hemolysisof Bacillus subtilis, finally modified BS224 was widely used in medicine. At the same time, aneffective method in which unknown functional genes in Bacillus subtilis genome were researchedwere provided, and one important step was forward taken for studies on proteomics of BacillusSubtilis in the future.更多还原