枯草杆菌224 yhdP、yugS、yhdT和yqhB基因敲除及对其溶血性的影响
|
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。
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.
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。
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.
引文
白光兴，孙志伟，黄莺，俞炜源．2005．利用Red重组系统对大肠杆菌ClpP基因的敲除．中国生物化学与分子生物学报．21(1)35—38
白雪源，陈香美．2000．基因打靶技术及其在生物医学中的应用．中华肾病杂志．16(6)403—405
宾文，刘墨青，彭俊平，孙立连，徐星哗，张景海，金奇．2006．痢疾志贺菌A1型IroN，ShuA单、双突变体的构建及功能分析．中国科学C辑生命科学．36(3)255—260
陈兵，戴文佳，王正敏，李忠明．2006．肺炎球菌溶血素基因的克隆．中国耳鼻咽喉头颈外科．13(4)237—239
陈芳，陈策实，李越，尹光琳．2000．欧文氏菌2—酮基醛糖还原酶基因敲除的研究．微生物学报．40(5)475—481
褚晓红，杨公社，郑新民．2001．基因打靶技术及其影响因素．黄牛杂志．27(5)1—4
窦薇．2005．基因打靶技术及其发展和应用．国外医学：遗传学分册．28(4)193—195
杜秉海，姜巨全，李小红，王磊，杨苏声．2005．苜蓿中华根瘤菌042BM noeA基因的克隆、敲除与功能的初步分析．微生物学报．45(2)195—200
高玉强，牟玉清，刘忠科．1996．枯草芽孢杆菌气雾剂治疗深浅烧伤的临床观察．中国急救医学．16(6)33—34
高正琴，邢华，李厚达．2002．基因打靶技术的应用研究．动物科学与动物医学．19(2)9—13
高正琴．2002．基因打靶技术在实验动物中的研究进展．实验动物科学与管理，19(2)37—39
顾永清．Hitoshi Shimoi．2003．通过基因敲除研究HSP1 2的酒精耐受功能．农垦医学．25(3)157—161
郭志儒，金宁一．2001．2000年动物克隆及相关领域研究进展．中国兽医学报．21(2)208-212
韩聪，张惟材，游松等．2004．大肠杆菌ptsG基因敲除及其缺陷株生长特性研究．生物工程学报．20(1)16—20
杭晓明，蔺兆星，王关林，陈国强．2003．大肠杆菌fadB缺失突变株构建及对聚羟基脂肪酸酯积累影响．大连理工大学学报．43(5)595—598
何亦祥，齐涵．1991．抑菌生在防治创、烧伤创面感染中的应用．中国微生态杂志．3(4)12-13
胡玲美，李忠义，刘江秋，沈宝军，何亦祥，齐涵．1991．抑菌生的急性毒性试验．中国微生态杂志．3(4)21
黄维，钟辉，李平，曹诚，马清钧．2004．大肠杆菌胸腺嘧啶合成酶thyA缺失突变菌株的构建．微生物学报．44(1)62—67
金涛，吴补领，苏凌云，高杰．2005．远缘链球菌基因敲除菌株的构建．牙体牙髓牙周病学 杂志．15(5)251—254
李瑞国，安晓荣，苟克勉，陈永福．2002．基因打靶技术及其应用前景．生物技术通报．5：6—9
刘刚，翟朝阳．2004．一种通用的从少量培养液中快速提取细菌染色体DNA的方法．西部医学．16(2)111—113
刘红全，戴继勋，于文功，杨堃峰．2002．基因打靶技术的研究进展．遗传．24(6)707—711
刘建忠，敖敬，田为宇，马季骅．2006．基因敲除技术研究进展．化学与生物工程．23(2)4—6
鲁润铭，李万军，董占双，张铜，郭羡华．1991．枯草杆菌BS_(224)菌培养物体外抑菌效果观察．中国微生态杂志．3(4)20
毛晓华，汪道涌，刘芳．2002．一种粘细菌发育相关基因的分离及敲除分析．微生物学报．42(3)316—320
毛晓华．2002．粘细菌转录因子FruA结合蛋白敲除分析．东南大学学报．21(1)20—23
倪艳秀，何孔旺，何家惠，王继春，吕立新．2002．猪链球菌2型溶血素基因的检测及其序列分析．中国兽医科技．32(11)11—14
彭清忠，张惟材，朱厚础．2001．枯草杆菌表达系统的研究进展．生物技术通讯．12(3)220—225
彭文要，谢举临．2002．白天鹅气雾剂对Ⅱ°烧伤创面愈合的作用．青海医药杂志．32(2)5—6
齐涵，胡玲美，李忠义，刘江秋，沈宝军，何亦祥．1991．抑菌生的安全性试验．中国微生态杂志．3(4)22—23
齐涵，胡玲美，李忠义，刘江秋，沈宝军，何亦祥．1991．抑菌生防治大鼠烫伤创面大肠杆菌感染的实验研究．中国微生态杂志．3(4)30—31
齐涵，刘江秋，侯晓娜，王淑芬，何亦祥．1991．枯草杆菌BS_(224)菌体外拮抗试验．中国微生态杂志．3(4)18—19
齐涵，马忠良，王淑芬，王占明，邸文学，于跃林，何亦祥，胡玲美，李忠义，刘江秋，沈宝军．1991．抑菌生的研究总结报告．中国微生态杂志．3(4)1—11
齐涵，王淑芬，任秋生，明月，牟玉清，张美东．1998．枯草芽孢杆菌BS_(224)菌活菌制剂(白天鹅气雾剂)治疗烧伤创面的环境细菌学监测．中国微生态学杂志．10(3)174—176
齐涵，王占明，王淑芬，胡玲美，李忠义，何亦祥．1991．抑菌生抗大鼠烫伤创面金葡菌感染的实验研究．中国微生态杂志．3(4)27—29
齐涵，于跃林，王淑芬，王占明，沈维林．1991．抑菌生活菌制剂的研制．中国微生态杂志．3(4)14—15
钱锋，潘卫庆，杜景伶．2001．Cre／loxP介导的伤寒杆菌染色体上插入基因的切除．生物化学与生物物理进展．28(5)732—735
邱云青，鲍朗，赵计林，赵明才，张会东，吴悦涵．2004．采用基因打靶技术构建结核分枝杆菌新基因Rv0901缺失株的研究．中国病理生理杂志．20(8)1507—1510
邱云青，鲍朗，赵计林，赵明才，张会东．2003．结核分支杆菌Rv0901基因置换型打靶 载体的构建．四川生理科学杂志．25(1)21—24
饶志明，张君胜，沈微，方慧英，诸葛健．2006．谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除．中国生物工程杂志．27(1)59—63
萨姆布鲁克等著．2002．分子克隆实验指南．黄培堂等译．第三版．科学出版社，96—99．
生秀杰．基因打靶的策略及其发展．2001．国外医学·遗传学分册．24(1)8—10
宋浩雷，郭晓贤，杨月梅，江贤章，黄建忠．2006．酿酒酵母ADH3基因的敲除．工业微生物．36(4)28—32
宋秀凯，王蔚，汝少国．2006．藻类溶血素研究进展．海洋科学．30(6)82—86
苏桂栋，苏建新，聂军．2003．副溶血性弧菌直接溶血毒素和溶血相关毒素的研究进展．广东医学．24(12)1378—1380
苏建新，聂军，吴振龙．2002．副溶血性弧菌耐热直接相关溶血素基因的克隆与序列分析．第一军医大学学报．22(6)515—519
孙国凤．1998．枯草杆菌基因组全碱基序列的确定．生物技术通报．5：47—48
孙晶，李景鹏，千敖全，唐国敏 王华明．2004．黑曲霉pepB基因缺失菌株的构建及其功能分析．微生物学报．44(6)766—770
孙新艳，宁玉英，金一和，王国柱，康一强，王胜军．1991．抑菌生的致突变性试验．中国微生态杂志．3(4)24—26
唐雪明，邵蔚蓝，王正祥，方慧英，诸葛健．2003．整合型碱性蛋白酶基因工程菌中抗性基因的敲除．微生物学通报．30(3)1—5
田三德，孙鹏．2003．基因打靶技术及其应用．陕西科技大学学报．21(4)94—98
汪道涌，谢维，毛晓华．2003．棕色固氮菌nifS敲除菌株的构建．微生物学通报．30(6)34—38
汪亚平，朱作言．1999．基因靶位操作的原理与策略．遗传．21(3)46—50
王宏，沈子龙，奚涛，成国祥．2001．条件性基因打靶技术研究进展．药物生物技术．8(1)54—57
王磊，谭谦，宁官森，周栩．1999．枯草杆菌微生态制剂在度烧伤创面的应用．铁道医学．27(6)417
王连秀，赵维勇，牛桓彩，张才，王彦彬．2001．食品中单核细胞增生李斯特氏菌调查及毒力研究．中国食品卫生杂志．13(2)16—18
王勤，周颂，陈江野．1999．白色念珠菌CRK1基因的敲除及其功能研究．生物化学与生物物理学报．31(5)545-552
王淑芬，齐涵，吴秋梅．1991．抑菌生的药物敏感试验．中国微生态杂志．3(4)16—17
王万东，高庆祥，何士平．2002．白天鹅气雾剂治疗面、颈深Ⅱ度烧伤疗效观察．宁夏医学院学报．24(5)363—364
王又红，余新炳．1999．基因敲除技术的应用现状与发展前景．国外医学寄生虫病分册．26(5)196—199
王志强，崔玉东，余丽芸，朴范泽．2006．金黄色葡萄球菌α溶血素基因的克隆与序列分 析．黑龙江畜牧兽医．16—17
吴丹莉，王艳飞，谷庆阳，吴祖泽．2002．碱性成纤维细胞因子在枯草芽孢杆菌BS224中的表达．生物技术通讯．13(6)439—442
吴振龙，聂军，鲍慧宁．2001．副溶血性弧菌直接溶血毒素基因的原核表达及产物的性质．细胞与分子免疫学杂志．17(5)426—430
吴振龙，聂军，鲍慧宁．2001．副溶血弧菌直接溶血毒素65位色氨酸的突变．中华微生物学和免疫学杂志．21(1)13—17
薛猛．2002．基因打靶技术的研究进展．生物学杂志．18(1)7—8
杨晓，邓初夏，叶鑫生．2001．基因打靶和功能基因组学．北京：军事医学科学出版社，60—69；82—83
杨晓，黄培堂，黄翠芬．2003．基因打靶技术．北京：科学出版社，18
叶苓．2001．基因打靶技术及其在寄生虫学研究中的应用．国外医学·寄生虫病学分册．28(2)56—58
殷月兰，焦新安，潘志明，张晓明，顾志强．2004．李斯特菌溶血素基因的原核表达及其生物学特性．微生物学报．44(6)752—755
余志强，杨明明，杨朝霞，林峰，张西锋，张炜炜，樊俊华，姜发堂．2004．同源重组法构建枯草杆菌sp00A基因缺失突变株．武汉大学学报．50(2)229—233
张帆，宋辉，班容．2006．hprK基因敲除及对其枯草芽孢杆菌核黄素发酵的影响．生物工程学报．22(4)534—538
张延平，刘铭，曹竹安．2005．醛脱氢酶基因敲除的K．pneumoniae重组菌的构建．中国生物工程杂志．25(12)34—38
张怡轩，赵国屏，吴春福．2002．致病性钩端螺旋体溶血素基因功能的研究进展．国外医学·流行病学传染病学分册．26(9)359—362
钟穗生，胡全胜，张林香．1997．枯草杆菌α-淀粉酶的活性研究．太原工业大学学报．28(4)22—27
周立雄，张兆山．2004．大肠杆菌α溶血素分泌系统基因表达调控研究进展．生物技术通讯．15(4)374—378
周倩，刘文忠．2003．基因打靶技术及其应用．动物科学与动物医学．20(10)25—27
朱大玲，李爱华，钱冬，汪建国．2004．嗜水气单胞菌毒力基因的研究进展．水生生物学报．28(1)80—84
朱焕章，史景泉．2000．Cre／LoxP系统在转基因小鼠上的应用策略．生物化学与生物物理进展．27(3)235—238
朱武洋，贾青．2002．基因打靶用于哺乳动物体细胞核移植技术研究进展．畜牧兽医杂志．21(6)10—12
朱越雄，曹广力．2000．嗜水气单胞菌溶血毒素对草鱼血细胞的溶血作用．水产养殖．6：27—30
邹运明，邹丽红，任艳红，李一经．2006．单核增生性李斯特杆菌感染和李氏溶血素．东北农业大学学报．37(1)120—124
Agaisse H, Lereclus D. 1994. Expession in Bacillus Subtilis of the Bacillus Thuringiensis CryIIIA Toxin Gene is Not Dependent on a Sporulation-Specific Sigma Factor and is Increased in a SpoOA MutantJ Bacterial. 176(15)4734-4741
Andreeva ZI, Nesterenko VF, Fomkina MG, Ternovsky VI, Suzina NE, Bakulina AY, Solonin AS, Sineva EV. 2007. The properties of Bacillus cereus hemolysin II pores depend on environme -ntal conditions. Biochim Biophys Acta. 1768(2)253-263
Aoki T, Hirono I .1991. Cloning and characterization of the hemolysin determinants from Aeromonas hydrophila. J Fish Dis .14:303-312
Bakas L, Veiga MP, Soloaga A, Ostolaza H, Goni FM. 1998.Calcium-dependent conformation of E. coli a-hemolysin. Implications for the mechanism of membrane insertion and lysis. Biochim. Biophys. Acta Biomembr. 1368:225-234
Bernd R. 2003.Homologous recombination and gene targeting in plant cells. Int. Rev. Cytol. 228: 85-139
Bhakdi S, Bayley H, Valeva A, Walev I, Walker B, Kehoe M, Palmer M.1996.StaphyIococcal alpha-toxin, streptolysin-0,and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins. Arch Microbiol. 165 : 73-79
Bin Wang, Jiangfeng Zhou. 2003. Specific genetic modifications of domestic animals by gene targeting and animal cloning. Reprod Biol Endocrinol. 1:103
Braun I, Ghebrehiwet B, Cossart P. 2000.gClq-R/p32, a C1-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes .EMBO J. 19: 1458—1466
Bu Z, Ye L, Skeen M J. 2003. Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listerioIysinO. AIDS Res Hum Retroviruses. 19(5)409-420
Cheng HR, Jiang N. 2006. Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol lett. 28:55-59
Cluff CW, Ziegler HK. 1987. Inhibition of macrophage-mediated antigen presentation by hemolysin-producing Listeria monocytogenes. J Immunol. 139(11)3808-3812
Cossart P, Bierne H. 2001.The use of host cell machinery in the pathogenesis of Listens monocytogenes. Curr Opin Immun. 13: 96—103
Craig A. Pritzlaff, Jennifer C. W. Chang, Shrin P. Kuo. 2001.Genetic basis for the β-hemolytic / cytolytic activity of group B Streptococcus. Molecular Microbiology .39(2) 236—247
De Vuyst L, Foulquie Moreno MR, Revets H. 2003. Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int J Food Microbiol.84 (3) 299—318
Decatur A L, Portnoy D A. 2000. A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity. Science. 290(5493)992-995
Doganay M. 2003. Listefiosisxlinical presentation. FEMS Immunol Med Microbiol. 35: 173 —175.
Ecker A, Moon R, Sinden RE, Billker O. 2005. Generation of gene targeting constructs for Plasmodium berghei by a PCR-based method amenable to high throughput applications. 10 (6) 1604—1610
Evans M J, Carlton M B L, Russ A P. 1997. Gene trapping and functional genomics. Trends Genet. 13:370-374
Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P. 1996. Ligand-activated site-specific recombination in mice . Proc Natl Acad Sci USA. 93(20)10887-10890
Felmlee T, Pellett S, Welch R. 1985. Nucleotide sequence of .Escherichia coli chromosomal hemolysin. J Bacteriol. 163(1)94
Feucht A, Lewis PJ. 2002.Improved Plasmid Vectors for the Production of Multiple Fluorescent Protein Fusion in Bacillus Subtilis.Gene. 264:289-297
Gedde MM, Higgins DE, Tilney LG, Portnoy DA. 2000. Role of listeriolysin in cell-to-cell spread of Listeria monocytogenes. Infect Immun. 68(2)999-1003
Giammarini C, Andreoni F, Amagliani G, Casiere A, Barocci S, Magnani M. 2003. High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein. Protein Expr Purif. 28(1)78-85
Gldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH.1996.A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acid Res. 24(13)2519-2524
Gldener U, Heinisch J, Koehler G.J, Voss D, Hegemann JH. 2002.A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.NucleicAcidsRes. 30(6):e23
Gormley E, Mengaud J, Cossart P. 1989. Sequences homologous to the listeriolysin O gene region of of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria. Res Microbiol. 140(9)631-643
Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. 1994. Deletion of a DNA polymerase β gene segment in T cells using cell type-specific targeting.Science. 265:103-106
Hasty P, Reuer-perez J, Bradley A. 1991.The length required for gene targeting in embryonic stem cells. Mol.Cell Bio. 11:5586-5591.
Herlax V, Bakas L. 2003. Acyl chains are responsible for the irreversibility in the Escherichia coli alpha-hemolysin binding to membranes. Chem Phys Lipids. 122 (2) 185—190
Hess J, Grode L, Gentschev I, Fensterle J, Dietrich G, Goebel W, Kaufmann SH.2000.Secretion of different literiolysin cognates by recombinant attenuated Salmonella typhimurium:superior efficacy of haemolytic over non-hemolytic constructs after or vaccination. Microbes and Infection. 2(15)1799-1806
Hirono I, Aoki T . Nucleotide sequence and expression of an extracellular hemolysin gene of Aeromonas hydrophila. Microb Pathog .1991, 11:189-197
Hsu T, Hutto DL, Minion FC, Zuerner RL, Wannemuehler MJ. 2001. Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodysenteriae and its expression in Escherichia coli. Infect Immun. 69(2)706—711
Huang IH, Waters M, Grau RR, Sarker MR. 2004 Disruption of the gene (spoOA) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A. FEMS Microbiol Lett.233(2)233—240
Huang ZW, Liu Z. 2006 .Construction of Streptococcus mutans luxS gene allelic exchange plasmid. Hua Xi Kou Qiang Yi Xue Za Zhi. 24(1)70-72
Janniere L, Bruand C, Ehrlich SD . 1996. Structurally Stable Bacillus Subtilis Clonnig Vector. Gene. 87:53—61
Jau-Der , Chen · Shau-Yan Lai , Shiang-Long Huang. 1996. Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda. Arch Microbiol. 165 :9-17
Juranka P, Zhang F, Kulpa J, Endicott J, Blight M, Holland IB, Ling V. 1992.Characterization of the hemolysin transporter, HlyB, using an epitope insertion. J Biol Chem . 267:3764-3770
Jurick WM, Rollins JA. 2006.Deletion of the adenylate cyclase (sac1) gene affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum. Fungal Genet Biol. [Epub ahead of print]
Justice M, Zheng B, Woychik RP, Bradley A. 1997.Using targeted large deletions and high-efficiency N-en-thyl-N-nitrosourea mutagenesis for functional analyses of the mammalian genome. Methods. 13(4)423-436
Kalt wasser M, Wiegert T, Schumann W. 2002.Construction and Application of Epitope and Green Fluorescent Prtein-Tagging Integration Vectors for Bacillus Subtilis.Appl Environ Microbiol. 68:2624—2632
Kaltwasser M, Wiegert T, Schumann W. 2002. Constructio and Application of Epitope and Green Fluorescent Protein Tagging Integration Vectors for Bacillus Subtilis. Appl Environ Microbiol. 68:2624-2622
Kelly R, Miller S M, Kurtz M B, Kurtz MB, Kirsch DR. 1987. Directed mutagenesis in Candida albicans : One-step gene disruption to isolate URA3 mutant. Mol Cell Biol. 7:199-207.
Kenane N, Brachet M, Galoisy-Guibal L, Boutin JP. 2002. Leptospirosis microepidemic in Martinique. Med Imp(Mays).62(2)205—206
Kenny B, Chervaux C, Holland IB. 1994. Evidence that residues-15 to -46 of the hemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator. Mol Microbiol. 11:99-109
Kishishita M, Matsuoka N, Kumagai K, Yamasaki S, Takeda Y, Nishibuchi M. 1992.Sequence variation in thethermostable direct hemolysin-related hemolysin(trh) gene of vibrio parahaemoly-ticus. Appli& Environm Microbiol. 58(8)2449—2456
Kuhn R, Schwenk F, Aguet M. 1995. Inducible gene targeting in mice.Science. 269:1427-1429
Lee S H, Kim K A, Park Y G, Seong I W, Kim M J, Lee Y J. 2000. Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai. Gene. 254: 19—28
Lee SH, Kim S, Park SC, Kim M J. 2002. Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells. Infect Immun. 70(1):315—322
Lety M A, Frehel C, Dubail I, Beretti JL, Kayal S, Berche P,Charbit A. 2001. Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes. Mol Microbiol. 39(5)1124-1139
Li SH, Wang J, Li JZ, Huang CF, Zhou JG. 2005 . Construction of recombinant plasmid using Neo/E Technology. Sheng Wu Gong Cheng Xue Bao. 21(4)520-523
Li XX, Colombini M. 2002.Catalyzed insertion of proteins into phospholipid membranes: specificity of the process. Biophys J. 83 (5) 2550—2559
Liu Rongrong, Wang Lixia,Lin Zhongping.2005.Effect of deletion mutation on the recombination activity of Cre recombinase. Acta Biochimica Polonica. 52(2)541-544
Lyytikainen O. Autio T, Maljala R. 2000. An outbreak of Listeria monocytogenes serotype 3a infections from butter in Finland. J Infect Dis. 181:1838—1841
Menestrina G, Serra MD, Prevost G. 2001 .Mode of action of beta-barrel pore-forming toxins of the staphylococcal alpha-hemolysin family. Toxicon. 39(11)1661—1672
Mengaud J, Vicente MF, Cossart P. 1989.Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation. Infect Immun. 57(10)3695-3701
Mengaud J, Vicente M F, Chenevert J, Pereira JM, Geoffroy C, Gicquel-Sanzey B,Baquero F, Perez-Diaz JC, Cossart P.1988.Expression in Escherichia coli and sequence analysis of the listeriolysin O deter-minant of Listeria monocytogenes.Infect Immun. (56)766—772
Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, Ram AF. 2007.Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol. 128(4)770-775
MITSUAKI N1SHIBUCHI, JAMES B. KAPER. 1995. Thermostable Direct Hemolysin Gene of Vibrio parahaemolyticus: a Virulence Gene Acquired by a Marine Bacterium. INFECTION AND IMMUNITY. 63(6)2093-2099
Moralejo F J, Cardoza R E. Gutierrez S, Lombrana M, Fierro F, Martin JF.2002.Silencing of the aspergillopepsin B (pepB) gene of Aspergillus awamorl by antisense RNA expression or protease removal by gene disruption results in a large increase in thaumatin production. Appl Environ Microbiol. 68(7)3550—3559
Morona R, Yeadon J, Considine A, Morona JK, Manning PA. 1991 .Construction of plasmid vectors with a non-antibiotic selection system based on the E.coli thyA+ gene: application to cholear accine development. Gene. 107:139-144
Nguyen HD, Nguyen QA, 2005.Ferreira RC, et al. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid .54(3)241-248
Ostolaza H, Soloaga A, Goni FM. 1995. The binding of divalent cations to Escherichia coli a-hemolysin. Eur. J. Biochem.,228:39-44
Palmer M. 2001. The family of thiol-activated. cholesterol-binding cytolysins. Toxieon. 39: 1681-1689
Peter Igarashi. 2004.Kidney-Specific Gene Targeting. J Am Soc Nephrol. 15:2237-2239
Peter Stanley, Vassilis Koronakis, Colin Hughes. 1998.Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function. Microbiol Mol Biol Rev. 62(2)309-333
Raimondi F, Kao JP, Fiorentini C, Fabbri A, Donelli G, Gasparini N, Rubino A, Fasano A. 2000. Enterotoxicity and cytotoxicity of vibrio parahaemolyticus thermostable direct hemolysin in in - vitro systems. Infect Immun. 68(6) 3180—3186
Ramirez-Solis R, Liu P, Bradley A. 1995.Chromosome engineering in mice. Nature. 378: 720 -724
Robert B Norgren. 2004. Creation of non-human primate neurogenetic disease models by gene targeting and nuclear transfer. Reprod Biol Endocrinol. 2: 40
Rohlmann A, Gotthardt M, Willnow TE, Hammer RE, Herz J. 1996.Sustained gene inactivation by viral transfer of Cre recombinase. Nature Biotechnology. 14:1562-1565
Rossolillo P, Albertini AM. 2001. Functional analysis of the Bacillus subtilis y shD gene, a mutS paralogue. Mol Gene Genet. 264 (4) 809—818
Saito G, Amidon G L, LeeK-D. 2003.Enhanced cytosolic delivery of plasmid DNA by a Sulfhydryl-activatable listeriolysinO? Protamin conjugate utilizing cellular reducing potential. Gene Therapy. 10:72-83
Sambrook J, Russell DW. 2002.Molecular Cloning A Laboratory Mannual (3rded). NewYork: Cold Spring Harbor Laboratory Press, 96—99
ShenY, NaujokasM, Park M, Ireton K. 2000.InIb-dependent internalization of Listeria is mediated by the met receptor tyrosine kinase. Cell. 103: 501—510
Stanley P, Koronakis V, Hughes C . 1991. Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli hemolysin. Mol Microbiol. 5:2391-2403
Stefureac R, Long YT, Kraatz HB, Howard P, Lee JS. 2006 Transport of alpha-helical peptides through alpha-hemolysin and aerolysin pores. Biochemistry. 45(30)9172—9179
Sun J, Li JP, Wang AQ, Tang GM, Wang HM. 2004.Construction and functional detection of pepB gene disruptant mutant in Aspergillus niger. Acta Microbiologica Sinica. 44 (6) 765-770
Suthienkul O, Iida T, Park KS, Ishibashi M, Supavej S, Yamamoto K, Honda T. 1996. Restricti -on fragment length polymorphism of the tdh and trh genes in clinical vibrio parahaemolyticus strains. Clin Microbiol. 34(5)1293—1295
Suzuki N, Ueda Y. 1997.Incidence of kanagawa-positive and-negative vibrio parahaemolyticus strains isolated from traveler' s diarrhea and their relation to tdh and trh genes. Kansenshogaku Zasshi. 71(5)417—423
Takahashi A, Sato Y, Shiomi Y, Cantarelli VV, Iida T, Lee M, Honda T.2000. Mechanism of chloride secreation induced by thermostable direct hemolysin of vibrio parahaemolyticus in human colonic tissue and a human intestinal epithelial cell line. Med Microbiol. 49(9)801—806
Tang G, Iida T, Yamamoto K, Honda T. 1997.Analysis of functional domains of vibrio parahaemolyticus thermostable direct hemolysin using monoclonal antibodies.FEMS Microbioi Lett. 150(2)289—296
Tatum FM, Briggs RE. 2005 .Construction of in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus by using a new temperature -sensitive plasmid. Appl Environ Microbioi. 71(11)7196-7202
Terada R. Urawa H. Inagaki Y. Tsugane K. Iida S. 2002. Efficient gene targeting by homologous recombination in rice. Nat. Biotechnol. 20: 1030-1034
Tham W, Ericasson H, Loncarevie D. 2000.Lessons from an outbreak of listeriosis related to vacuum-packed gravid and cold-smoked fish. Int J Fiid Mierobiol, 62: 173-175.
Tucker AD, Parker MW, Tsernoglou D . 1990. Crystallization of a proform of aerolysin, a hole-forming toxin from Aeromonas hydrophila. J Mol Biol. 212:561-562
Van Deursen JV, Fornerod M, van Rees B. 1995.Cre-midiated site-specific translocation between nonhomologous mouse chromosomes. Proc Natl Acad Sci USA, 92:7376-7380.
Vazquez-Boland JA, Dominguez-Bernal G, Gonzalez-Zom B, Kreft J, Goebel W.. 2001 .Pathogenicity islands and virulence evolution in Listeria. Microbes lnfect.3: 571—584
Volokhov DV, Duperrier S, Neverov AA, George J, Buchrieser C, Hitchins AD. 2007 .The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes. 73(6)1928-1939
Welch R, Hull R, Falkow S. 1983.Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli. Infact Immun .42(1)178
Wong HC, Lu KT, PanTM, Lee CL, Shih DY. 1996. Subspecies typing of Vibrio parahaemolyticus by pulsed -field gel electrophoresis. Clin Microbioi. 34(6)1535—1540
Ye L, Bu Z, Skeen M J, Ziegler HK, Compans RW, Yang C. 2003. Enhanced immunogenicity of SIV Gag DNA vaccines encoding chimeric proteins containing a C-termnal segment of Listeriolysin O. Virus Res. 97(1)7-16
Ying M (应明), Ban R (班睿) .2006 . Knockout of the ccpA gene in B . subtilis and its influence on riboflavin fermentation. Acta Microbiologica Sinica (微生物学报) .46 (1) 23 - 27
Zeigler DR.2003.Bacillus Genetic Stock Center Catalog of Strains, Seven Edition. Vol.4: Integration Vectors for Gram-Positive organisms, http:// www.bgsc.org/ Catalogs/ Catpart4. pdf.
Zhang D, Honda T. 1999.Disappearance of glyceroldehyde3-phosphate dehydrogenase from erythrocyte membrane by hemolysis with thermostable direct hemolysin of vibrio parahaemolyticus or vibrio cholerae El TOR hemolysin. Microbioi Immunol. 43(3)303—309
Zhu H, Qu F, Zhu LH. 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Research. 21 (22)52799-52801
白雪源，陈香美．2000．基因打靶技术及其在生物医学中的应用．中华肾病杂志．16(6)403—405
宾文，刘墨青，彭俊平，孙立连，徐星哗，张景海，金奇．2006．痢疾志贺菌A1型IroN，ShuA单、双突变体的构建及功能分析．中国科学C辑生命科学．36(3)255—260
陈兵，戴文佳，王正敏，李忠明．2006．肺炎球菌溶血素基因的克隆．中国耳鼻咽喉头颈外科．13(4)237—239
陈芳，陈策实，李越，尹光琳．2000．欧文氏菌2—酮基醛糖还原酶基因敲除的研究．微生物学报．40(5)475—481
褚晓红，杨公社，郑新民．2001．基因打靶技术及其影响因素．黄牛杂志．27(5)1—4
窦薇．2005．基因打靶技术及其发展和应用．国外医学：遗传学分册．28(4)193—195
杜秉海，姜巨全，李小红，王磊，杨苏声．2005．苜蓿中华根瘤菌042BM noeA基因的克隆、敲除与功能的初步分析．微生物学报．45(2)195—200
高玉强，牟玉清，刘忠科．1996．枯草芽孢杆菌气雾剂治疗深浅烧伤的临床观察．中国急救医学．16(6)33—34
高正琴，邢华，李厚达．2002．基因打靶技术的应用研究．动物科学与动物医学．19(2)9—13
高正琴．2002．基因打靶技术在实验动物中的研究进展．实验动物科学与管理，19(2)37—39
顾永清．Hitoshi Shimoi．2003．通过基因敲除研究HSP1 2的酒精耐受功能．农垦医学．25(3)157—161
郭志儒，金宁一．2001．2000年动物克隆及相关领域研究进展．中国兽医学报．21(2)208-212
韩聪，张惟材，游松等．2004．大肠杆菌ptsG基因敲除及其缺陷株生长特性研究．生物工程学报．20(1)16—20
杭晓明，蔺兆星，王关林，陈国强．2003．大肠杆菌fadB缺失突变株构建及对聚羟基脂肪酸酯积累影响．大连理工大学学报．43(5)595—598
何亦祥，齐涵．1991．抑菌生在防治创、烧伤创面感染中的应用．中国微生态杂志．3(4)12-13
胡玲美，李忠义，刘江秋，沈宝军，何亦祥，齐涵．1991．抑菌生的急性毒性试验．中国微生态杂志．3(4)21
黄维，钟辉，李平，曹诚，马清钧．2004．大肠杆菌胸腺嘧啶合成酶thyA缺失突变菌株的构建．微生物学报．44(1)62—67
金涛，吴补领，苏凌云，高杰．2005．远缘链球菌基因敲除菌株的构建．牙体牙髓牙周病学 杂志．15(5)251—254
李瑞国，安晓荣，苟克勉，陈永福．2002．基因打靶技术及其应用前景．生物技术通报．5：6—9
刘刚，翟朝阳．2004．一种通用的从少量培养液中快速提取细菌染色体DNA的方法．西部医学．16(2)111—113
刘红全，戴继勋，于文功，杨堃峰．2002．基因打靶技术的研究进展．遗传．24(6)707—711
刘建忠，敖敬，田为宇，马季骅．2006．基因敲除技术研究进展．化学与生物工程．23(2)4—6
鲁润铭，李万军，董占双，张铜，郭羡华．1991．枯草杆菌BS_(224)菌培养物体外抑菌效果观察．中国微生态杂志．3(4)20
毛晓华，汪道涌，刘芳．2002．一种粘细菌发育相关基因的分离及敲除分析．微生物学报．42(3)316—320
毛晓华．2002．粘细菌转录因子FruA结合蛋白敲除分析．东南大学学报．21(1)20—23
倪艳秀，何孔旺，何家惠，王继春，吕立新．2002．猪链球菌2型溶血素基因的检测及其序列分析．中国兽医科技．32(11)11—14
彭清忠，张惟材，朱厚础．2001．枯草杆菌表达系统的研究进展．生物技术通讯．12(3)220—225
彭文要，谢举临．2002．白天鹅气雾剂对Ⅱ°烧伤创面愈合的作用．青海医药杂志．32(2)5—6
齐涵，胡玲美，李忠义，刘江秋，沈宝军，何亦祥．1991．抑菌生的安全性试验．中国微生态杂志．3(4)22—23
齐涵，胡玲美，李忠义，刘江秋，沈宝军，何亦祥．1991．抑菌生防治大鼠烫伤创面大肠杆菌感染的实验研究．中国微生态杂志．3(4)30—31
齐涵，刘江秋，侯晓娜，王淑芬，何亦祥．1991．枯草杆菌BS_(224)菌体外拮抗试验．中国微生态杂志．3(4)18—19
齐涵，马忠良，王淑芬，王占明，邸文学，于跃林，何亦祥，胡玲美，李忠义，刘江秋，沈宝军．1991．抑菌生的研究总结报告．中国微生态杂志．3(4)1—11
齐涵，王淑芬，任秋生，明月，牟玉清，张美东．1998．枯草芽孢杆菌BS_(224)菌活菌制剂(白天鹅气雾剂)治疗烧伤创面的环境细菌学监测．中国微生态学杂志．10(3)174—176
齐涵，王占明，王淑芬，胡玲美，李忠义，何亦祥．1991．抑菌生抗大鼠烫伤创面金葡菌感染的实验研究．中国微生态杂志．3(4)27—29
齐涵，于跃林，王淑芬，王占明，沈维林．1991．抑菌生活菌制剂的研制．中国微生态杂志．3(4)14—15
钱锋，潘卫庆，杜景伶．2001．Cre／loxP介导的伤寒杆菌染色体上插入基因的切除．生物化学与生物物理进展．28(5)732—735
邱云青，鲍朗，赵计林，赵明才，张会东，吴悦涵．2004．采用基因打靶技术构建结核分枝杆菌新基因Rv0901缺失株的研究．中国病理生理杂志．20(8)1507—1510
邱云青，鲍朗，赵计林，赵明才，张会东．2003．结核分支杆菌Rv0901基因置换型打靶 载体的构建．四川生理科学杂志．25(1)21—24
饶志明，张君胜，沈微，方慧英，诸葛健．2006．谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除．中国生物工程杂志．27(1)59—63
萨姆布鲁克等著．2002．分子克隆实验指南．黄培堂等译．第三版．科学出版社，96—99．
生秀杰．基因打靶的策略及其发展．2001．国外医学·遗传学分册．24(1)8—10
宋浩雷，郭晓贤，杨月梅，江贤章，黄建忠．2006．酿酒酵母ADH3基因的敲除．工业微生物．36(4)28—32
宋秀凯，王蔚，汝少国．2006．藻类溶血素研究进展．海洋科学．30(6)82—86
苏桂栋，苏建新，聂军．2003．副溶血性弧菌直接溶血毒素和溶血相关毒素的研究进展．广东医学．24(12)1378—1380
苏建新，聂军，吴振龙．2002．副溶血性弧菌耐热直接相关溶血素基因的克隆与序列分析．第一军医大学学报．22(6)515—519
孙国凤．1998．枯草杆菌基因组全碱基序列的确定．生物技术通报．5：47—48
孙晶，李景鹏，千敖全，唐国敏 王华明．2004．黑曲霉pepB基因缺失菌株的构建及其功能分析．微生物学报．44(6)766—770
孙新艳，宁玉英，金一和，王国柱，康一强，王胜军．1991．抑菌生的致突变性试验．中国微生态杂志．3(4)24—26
唐雪明，邵蔚蓝，王正祥，方慧英，诸葛健．2003．整合型碱性蛋白酶基因工程菌中抗性基因的敲除．微生物学通报．30(3)1—5
田三德，孙鹏．2003．基因打靶技术及其应用．陕西科技大学学报．21(4)94—98
汪道涌，谢维，毛晓华．2003．棕色固氮菌nifS敲除菌株的构建．微生物学通报．30(6)34—38
汪亚平，朱作言．1999．基因靶位操作的原理与策略．遗传．21(3)46—50
王宏，沈子龙，奚涛，成国祥．2001．条件性基因打靶技术研究进展．药物生物技术．8(1)54—57
王磊，谭谦，宁官森，周栩．1999．枯草杆菌微生态制剂在度烧伤创面的应用．铁道医学．27(6)417
王连秀，赵维勇，牛桓彩，张才，王彦彬．2001．食品中单核细胞增生李斯特氏菌调查及毒力研究．中国食品卫生杂志．13(2)16—18
王勤，周颂，陈江野．1999．白色念珠菌CRK1基因的敲除及其功能研究．生物化学与生物物理学报．31(5)545-552
王淑芬，齐涵，吴秋梅．1991．抑菌生的药物敏感试验．中国微生态杂志．3(4)16—17
王万东，高庆祥，何士平．2002．白天鹅气雾剂治疗面、颈深Ⅱ度烧伤疗效观察．宁夏医学院学报．24(5)363—364
王又红，余新炳．1999．基因敲除技术的应用现状与发展前景．国外医学寄生虫病分册．26(5)196—199
王志强，崔玉东，余丽芸，朴范泽．2006．金黄色葡萄球菌α溶血素基因的克隆与序列分 析．黑龙江畜牧兽医．16—17
吴丹莉，王艳飞，谷庆阳，吴祖泽．2002．碱性成纤维细胞因子在枯草芽孢杆菌BS224中的表达．生物技术通讯．13(6)439—442
吴振龙，聂军，鲍慧宁．2001．副溶血性弧菌直接溶血毒素基因的原核表达及产物的性质．细胞与分子免疫学杂志．17(5)426—430
吴振龙，聂军，鲍慧宁．2001．副溶血弧菌直接溶血毒素65位色氨酸的突变．中华微生物学和免疫学杂志．21(1)13—17
薛猛．2002．基因打靶技术的研究进展．生物学杂志．18(1)7—8
杨晓，邓初夏，叶鑫生．2001．基因打靶和功能基因组学．北京：军事医学科学出版社，60—69；82—83
杨晓，黄培堂，黄翠芬．2003．基因打靶技术．北京：科学出版社，18
叶苓．2001．基因打靶技术及其在寄生虫学研究中的应用．国外医学·寄生虫病学分册．28(2)56—58
殷月兰，焦新安，潘志明，张晓明，顾志强．2004．李斯特菌溶血素基因的原核表达及其生物学特性．微生物学报．44(6)752—755
余志强，杨明明，杨朝霞，林峰，张西锋，张炜炜，樊俊华，姜发堂．2004．同源重组法构建枯草杆菌sp00A基因缺失突变株．武汉大学学报．50(2)229—233
张帆，宋辉，班容．2006．hprK基因敲除及对其枯草芽孢杆菌核黄素发酵的影响．生物工程学报．22(4)534—538
张延平，刘铭，曹竹安．2005．醛脱氢酶基因敲除的K．pneumoniae重组菌的构建．中国生物工程杂志．25(12)34—38
张怡轩，赵国屏，吴春福．2002．致病性钩端螺旋体溶血素基因功能的研究进展．国外医学·流行病学传染病学分册．26(9)359—362
钟穗生，胡全胜，张林香．1997．枯草杆菌α-淀粉酶的活性研究．太原工业大学学报．28(4)22—27
周立雄，张兆山．2004．大肠杆菌α溶血素分泌系统基因表达调控研究进展．生物技术通讯．15(4)374—378
周倩，刘文忠．2003．基因打靶技术及其应用．动物科学与动物医学．20(10)25—27
朱大玲，李爱华，钱冬，汪建国．2004．嗜水气单胞菌毒力基因的研究进展．水生生物学报．28(1)80—84
朱焕章，史景泉．2000．Cre／LoxP系统在转基因小鼠上的应用策略．生物化学与生物物理进展．27(3)235—238
朱武洋，贾青．2002．基因打靶用于哺乳动物体细胞核移植技术研究进展．畜牧兽医杂志．21(6)10—12
朱越雄，曹广力．2000．嗜水气单胞菌溶血毒素对草鱼血细胞的溶血作用．水产养殖．6：27—30
邹运明，邹丽红，任艳红，李一经．2006．单核增生性李斯特杆菌感染和李氏溶血素．东北农业大学学报．37(1)120—124
Agaisse H, Lereclus D. 1994. Expession in Bacillus Subtilis of the Bacillus Thuringiensis CryIIIA Toxin Gene is Not Dependent on a Sporulation-Specific Sigma Factor and is Increased in a SpoOA MutantJ Bacterial. 176(15)4734-4741
Andreeva ZI, Nesterenko VF, Fomkina MG, Ternovsky VI, Suzina NE, Bakulina AY, Solonin AS, Sineva EV. 2007. The properties of Bacillus cereus hemolysin II pores depend on environme -ntal conditions. Biochim Biophys Acta. 1768(2)253-263
Aoki T, Hirono I .1991. Cloning and characterization of the hemolysin determinants from Aeromonas hydrophila. J Fish Dis .14:303-312
Bakas L, Veiga MP, Soloaga A, Ostolaza H, Goni FM. 1998.Calcium-dependent conformation of E. coli a-hemolysin. Implications for the mechanism of membrane insertion and lysis. Biochim. Biophys. Acta Biomembr. 1368:225-234
Bernd R. 2003.Homologous recombination and gene targeting in plant cells. Int. Rev. Cytol. 228: 85-139
Bhakdi S, Bayley H, Valeva A, Walev I, Walker B, Kehoe M, Palmer M.1996.StaphyIococcal alpha-toxin, streptolysin-0,and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins. Arch Microbiol. 165 : 73-79
Bin Wang, Jiangfeng Zhou. 2003. Specific genetic modifications of domestic animals by gene targeting and animal cloning. Reprod Biol Endocrinol. 1:103
Braun I, Ghebrehiwet B, Cossart P. 2000.gClq-R/p32, a C1-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes .EMBO J. 19: 1458—1466
Bu Z, Ye L, Skeen M J. 2003. Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listerioIysinO. AIDS Res Hum Retroviruses. 19(5)409-420
Cheng HR, Jiang N. 2006. Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol lett. 28:55-59
Cluff CW, Ziegler HK. 1987. Inhibition of macrophage-mediated antigen presentation by hemolysin-producing Listeria monocytogenes. J Immunol. 139(11)3808-3812
Cossart P, Bierne H. 2001.The use of host cell machinery in the pathogenesis of Listens monocytogenes. Curr Opin Immun. 13: 96—103
Craig A. Pritzlaff, Jennifer C. W. Chang, Shrin P. Kuo. 2001.Genetic basis for the β-hemolytic / cytolytic activity of group B Streptococcus. Molecular Microbiology .39(2) 236—247
De Vuyst L, Foulquie Moreno MR, Revets H. 2003. Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int J Food Microbiol.84 (3) 299—318
Decatur A L, Portnoy D A. 2000. A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity. Science. 290(5493)992-995
Doganay M. 2003. Listefiosisxlinical presentation. FEMS Immunol Med Microbiol. 35: 173 —175.
Ecker A, Moon R, Sinden RE, Billker O. 2005. Generation of gene targeting constructs for Plasmodium berghei by a PCR-based method amenable to high throughput applications. 10 (6) 1604—1610
Evans M J, Carlton M B L, Russ A P. 1997. Gene trapping and functional genomics. Trends Genet. 13:370-374
Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P. 1996. Ligand-activated site-specific recombination in mice . Proc Natl Acad Sci USA. 93(20)10887-10890
Felmlee T, Pellett S, Welch R. 1985. Nucleotide sequence of .Escherichia coli chromosomal hemolysin. J Bacteriol. 163(1)94
Feucht A, Lewis PJ. 2002.Improved Plasmid Vectors for the Production of Multiple Fluorescent Protein Fusion in Bacillus Subtilis.Gene. 264:289-297
Gedde MM, Higgins DE, Tilney LG, Portnoy DA. 2000. Role of listeriolysin in cell-to-cell spread of Listeria monocytogenes. Infect Immun. 68(2)999-1003
Giammarini C, Andreoni F, Amagliani G, Casiere A, Barocci S, Magnani M. 2003. High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein. Protein Expr Purif. 28(1)78-85
Gldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH.1996.A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acid Res. 24(13)2519-2524
Gldener U, Heinisch J, Koehler G.J, Voss D, Hegemann JH. 2002.A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.NucleicAcidsRes. 30(6):e23
Gormley E, Mengaud J, Cossart P. 1989. Sequences homologous to the listeriolysin O gene region of of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria. Res Microbiol. 140(9)631-643
Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. 1994. Deletion of a DNA polymerase β gene segment in T cells using cell type-specific targeting.Science. 265:103-106
Hasty P, Reuer-perez J, Bradley A. 1991.The length required for gene targeting in embryonic stem cells. Mol.Cell Bio. 11:5586-5591.
Herlax V, Bakas L. 2003. Acyl chains are responsible for the irreversibility in the Escherichia coli alpha-hemolysin binding to membranes. Chem Phys Lipids. 122 (2) 185—190
Hess J, Grode L, Gentschev I, Fensterle J, Dietrich G, Goebel W, Kaufmann SH.2000.Secretion of different literiolysin cognates by recombinant attenuated Salmonella typhimurium:superior efficacy of haemolytic over non-hemolytic constructs after or vaccination. Microbes and Infection. 2(15)1799-1806
Hirono I, Aoki T . Nucleotide sequence and expression of an extracellular hemolysin gene of Aeromonas hydrophila. Microb Pathog .1991, 11:189-197
Hsu T, Hutto DL, Minion FC, Zuerner RL, Wannemuehler MJ. 2001. Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodysenteriae and its expression in Escherichia coli. Infect Immun. 69(2)706—711
Huang IH, Waters M, Grau RR, Sarker MR. 2004 Disruption of the gene (spoOA) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A. FEMS Microbiol Lett.233(2)233—240
Huang ZW, Liu Z. 2006 .Construction of Streptococcus mutans luxS gene allelic exchange plasmid. Hua Xi Kou Qiang Yi Xue Za Zhi. 24(1)70-72
Janniere L, Bruand C, Ehrlich SD . 1996. Structurally Stable Bacillus Subtilis Clonnig Vector. Gene. 87:53—61
Jau-Der , Chen · Shau-Yan Lai , Shiang-Long Huang. 1996. Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda. Arch Microbiol. 165 :9-17
Juranka P, Zhang F, Kulpa J, Endicott J, Blight M, Holland IB, Ling V. 1992.Characterization of the hemolysin transporter, HlyB, using an epitope insertion. J Biol Chem . 267:3764-3770
Jurick WM, Rollins JA. 2006.Deletion of the adenylate cyclase (sac1) gene affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum. Fungal Genet Biol. [Epub ahead of print]
Justice M, Zheng B, Woychik RP, Bradley A. 1997.Using targeted large deletions and high-efficiency N-en-thyl-N-nitrosourea mutagenesis for functional analyses of the mammalian genome. Methods. 13(4)423-436
Kalt wasser M, Wiegert T, Schumann W. 2002.Construction and Application of Epitope and Green Fluorescent Prtein-Tagging Integration Vectors for Bacillus Subtilis.Appl Environ Microbiol. 68:2624—2632
Kaltwasser M, Wiegert T, Schumann W. 2002. Constructio and Application of Epitope and Green Fluorescent Protein Tagging Integration Vectors for Bacillus Subtilis. Appl Environ Microbiol. 68:2624-2622
Kelly R, Miller S M, Kurtz M B, Kurtz MB, Kirsch DR. 1987. Directed mutagenesis in Candida albicans : One-step gene disruption to isolate URA3 mutant. Mol Cell Biol. 7:199-207.
Kenane N, Brachet M, Galoisy-Guibal L, Boutin JP. 2002. Leptospirosis microepidemic in Martinique. Med Imp(Mays).62(2)205—206
Kenny B, Chervaux C, Holland IB. 1994. Evidence that residues-15 to -46 of the hemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator. Mol Microbiol. 11:99-109
Kishishita M, Matsuoka N, Kumagai K, Yamasaki S, Takeda Y, Nishibuchi M. 1992.Sequence variation in thethermostable direct hemolysin-related hemolysin(trh) gene of vibrio parahaemoly-ticus. Appli& Environm Microbiol. 58(8)2449—2456
Kuhn R, Schwenk F, Aguet M. 1995. Inducible gene targeting in mice.Science. 269:1427-1429
Lee S H, Kim K A, Park Y G, Seong I W, Kim M J, Lee Y J. 2000. Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai. Gene. 254: 19—28
Lee SH, Kim S, Park SC, Kim M J. 2002. Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells. Infect Immun. 70(1):315—322
Lety M A, Frehel C, Dubail I, Beretti JL, Kayal S, Berche P,Charbit A. 2001. Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes. Mol Microbiol. 39(5)1124-1139
Li SH, Wang J, Li JZ, Huang CF, Zhou JG. 2005 . Construction of recombinant plasmid using Neo/E Technology. Sheng Wu Gong Cheng Xue Bao. 21(4)520-523
Li XX, Colombini M. 2002.Catalyzed insertion of proteins into phospholipid membranes: specificity of the process. Biophys J. 83 (5) 2550—2559
Liu Rongrong, Wang Lixia,Lin Zhongping.2005.Effect of deletion mutation on the recombination activity of Cre recombinase. Acta Biochimica Polonica. 52(2)541-544
Lyytikainen O. Autio T, Maljala R. 2000. An outbreak of Listeria monocytogenes serotype 3a infections from butter in Finland. J Infect Dis. 181:1838—1841
Menestrina G, Serra MD, Prevost G. 2001 .Mode of action of beta-barrel pore-forming toxins of the staphylococcal alpha-hemolysin family. Toxicon. 39(11)1661—1672
Mengaud J, Vicente MF, Cossart P. 1989.Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation. Infect Immun. 57(10)3695-3701
Mengaud J, Vicente M F, Chenevert J, Pereira JM, Geoffroy C, Gicquel-Sanzey B,Baquero F, Perez-Diaz JC, Cossart P.1988.Expression in Escherichia coli and sequence analysis of the listeriolysin O deter-minant of Listeria monocytogenes.Infect Immun. (56)766—772
Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, Ram AF. 2007.Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol. 128(4)770-775
MITSUAKI N1SHIBUCHI, JAMES B. KAPER. 1995. Thermostable Direct Hemolysin Gene of Vibrio parahaemolyticus: a Virulence Gene Acquired by a Marine Bacterium. INFECTION AND IMMUNITY. 63(6)2093-2099
Moralejo F J, Cardoza R E. Gutierrez S, Lombrana M, Fierro F, Martin JF.2002.Silencing of the aspergillopepsin B (pepB) gene of Aspergillus awamorl by antisense RNA expression or protease removal by gene disruption results in a large increase in thaumatin production. Appl Environ Microbiol. 68(7)3550—3559
Morona R, Yeadon J, Considine A, Morona JK, Manning PA. 1991 .Construction of plasmid vectors with a non-antibiotic selection system based on the E.coli thyA+ gene: application to cholear accine development. Gene. 107:139-144
Nguyen HD, Nguyen QA, 2005.Ferreira RC, et al. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid .54(3)241-248
Ostolaza H, Soloaga A, Goni FM. 1995. The binding of divalent cations to Escherichia coli a-hemolysin. Eur. J. Biochem.,228:39-44
Palmer M. 2001. The family of thiol-activated. cholesterol-binding cytolysins. Toxieon. 39: 1681-1689
Peter Igarashi. 2004.Kidney-Specific Gene Targeting. J Am Soc Nephrol. 15:2237-2239
Peter Stanley, Vassilis Koronakis, Colin Hughes. 1998.Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function. Microbiol Mol Biol Rev. 62(2)309-333
Raimondi F, Kao JP, Fiorentini C, Fabbri A, Donelli G, Gasparini N, Rubino A, Fasano A. 2000. Enterotoxicity and cytotoxicity of vibrio parahaemolyticus thermostable direct hemolysin in in - vitro systems. Infect Immun. 68(6) 3180—3186
Ramirez-Solis R, Liu P, Bradley A. 1995.Chromosome engineering in mice. Nature. 378: 720 -724
Robert B Norgren. 2004. Creation of non-human primate neurogenetic disease models by gene targeting and nuclear transfer. Reprod Biol Endocrinol. 2: 40
Rohlmann A, Gotthardt M, Willnow TE, Hammer RE, Herz J. 1996.Sustained gene inactivation by viral transfer of Cre recombinase. Nature Biotechnology. 14:1562-1565
Rossolillo P, Albertini AM. 2001. Functional analysis of the Bacillus subtilis y shD gene, a mutS paralogue. Mol Gene Genet. 264 (4) 809—818
Saito G, Amidon G L, LeeK-D. 2003.Enhanced cytosolic delivery of plasmid DNA by a Sulfhydryl-activatable listeriolysinO? Protamin conjugate utilizing cellular reducing potential. Gene Therapy. 10:72-83
Sambrook J, Russell DW. 2002.Molecular Cloning A Laboratory Mannual (3rded). NewYork: Cold Spring Harbor Laboratory Press, 96—99
ShenY, NaujokasM, Park M, Ireton K. 2000.InIb-dependent internalization of Listeria is mediated by the met receptor tyrosine kinase. Cell. 103: 501—510
Stanley P, Koronakis V, Hughes C . 1991. Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli hemolysin. Mol Microbiol. 5:2391-2403
Stefureac R, Long YT, Kraatz HB, Howard P, Lee JS. 2006 Transport of alpha-helical peptides through alpha-hemolysin and aerolysin pores. Biochemistry. 45(30)9172—9179
Sun J, Li JP, Wang AQ, Tang GM, Wang HM. 2004.Construction and functional detection of pepB gene disruptant mutant in Aspergillus niger. Acta Microbiologica Sinica. 44 (6) 765-770
Suthienkul O, Iida T, Park KS, Ishibashi M, Supavej S, Yamamoto K, Honda T. 1996. Restricti -on fragment length polymorphism of the tdh and trh genes in clinical vibrio parahaemolyticus strains. Clin Microbiol. 34(5)1293—1295
Suzuki N, Ueda Y. 1997.Incidence of kanagawa-positive and-negative vibrio parahaemolyticus strains isolated from traveler' s diarrhea and their relation to tdh and trh genes. Kansenshogaku Zasshi. 71(5)417—423
Takahashi A, Sato Y, Shiomi Y, Cantarelli VV, Iida T, Lee M, Honda T.2000. Mechanism of chloride secreation induced by thermostable direct hemolysin of vibrio parahaemolyticus in human colonic tissue and a human intestinal epithelial cell line. Med Microbiol. 49(9)801—806
Tang G, Iida T, Yamamoto K, Honda T. 1997.Analysis of functional domains of vibrio parahaemolyticus thermostable direct hemolysin using monoclonal antibodies.FEMS Microbioi Lett. 150(2)289—296
Tatum FM, Briggs RE. 2005 .Construction of in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus by using a new temperature -sensitive plasmid. Appl Environ Microbioi. 71(11)7196-7202
Terada R. Urawa H. Inagaki Y. Tsugane K. Iida S. 2002. Efficient gene targeting by homologous recombination in rice. Nat. Biotechnol. 20: 1030-1034
Tham W, Ericasson H, Loncarevie D. 2000.Lessons from an outbreak of listeriosis related to vacuum-packed gravid and cold-smoked fish. Int J Fiid Mierobiol, 62: 173-175.
Tucker AD, Parker MW, Tsernoglou D . 1990. Crystallization of a proform of aerolysin, a hole-forming toxin from Aeromonas hydrophila. J Mol Biol. 212:561-562
Van Deursen JV, Fornerod M, van Rees B. 1995.Cre-midiated site-specific translocation between nonhomologous mouse chromosomes. Proc Natl Acad Sci USA, 92:7376-7380.
Vazquez-Boland JA, Dominguez-Bernal G, Gonzalez-Zom B, Kreft J, Goebel W.. 2001 .Pathogenicity islands and virulence evolution in Listeria. Microbes lnfect.3: 571—584
Volokhov DV, Duperrier S, Neverov AA, George J, Buchrieser C, Hitchins AD. 2007 .The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes. 73(6)1928-1939
Welch R, Hull R, Falkow S. 1983.Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli. Infact Immun .42(1)178
Wong HC, Lu KT, PanTM, Lee CL, Shih DY. 1996. Subspecies typing of Vibrio parahaemolyticus by pulsed -field gel electrophoresis. Clin Microbioi. 34(6)1535—1540
Ye L, Bu Z, Skeen M J, Ziegler HK, Compans RW, Yang C. 2003. Enhanced immunogenicity of SIV Gag DNA vaccines encoding chimeric proteins containing a C-termnal segment of Listeriolysin O. Virus Res. 97(1)7-16
Ying M (应明), Ban R (班睿) .2006 . Knockout of the ccpA gene in B . subtilis and its influence on riboflavin fermentation. Acta Microbiologica Sinica (微生物学报) .46 (1) 23 - 27
Zeigler DR.2003.Bacillus Genetic Stock Center Catalog of Strains, Seven Edition. Vol.4: Integration Vectors for Gram-Positive organisms, http:// www.bgsc.org/ Catalogs/ Catpart4. pdf.
Zhang D, Honda T. 1999.Disappearance of glyceroldehyde3-phosphate dehydrogenase from erythrocyte membrane by hemolysis with thermostable direct hemolysin of vibrio parahaemolyticus or vibrio cholerae El TOR hemolysin. Microbioi Immunol. 43(3)303—309
Zhu H, Qu F, Zhu LH. 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Research. 21 (22)52799-52801