【作者】 喻江；

【导师】 李景鹏；

【作者基本信息】 东北农业大学 ， 微生物学， 2008， 硕士

【摘要】 枯草芽孢杆菌224(Bacillus subtilis 224,BS224)是一种从人体中分离出来的非致病性芽孢杆菌,根据微生态学原理,利用微生物间的拮抗作用,由其活菌体制成的生物制品白天鹅气雾剂是一种有效治疗烧伤创面感染、促进创面愈合的烧伤外用药,但由于具有溶血性能,目前,已经停止了其在医疗方面的使用。目前,国内外还没有枯草芽孢杆菌224基因组序列的相关报道,但枯草芽孢杆菌168基因组全碱基序列已经确定,其中8个基因可能与溶血有关,yplQ(642bp)、ytjAα(228bp)为其中的两个,并且ytjAα为α溶血素样基因。本实验是根据枯草芽孢杆菌168基因组序列设计引物,以枯草芽孢杆菌224染色体DNA为模板,利用PCR技术扩增了yplQ、ytjAα两个基因,并将它们分别插入到克隆载体pMD18-T中,构建了重组质粒pMD18-T-yplQ、pMD18-T-ytjAα。连接产物转化入大肠杆菌JM109感受态中,并从含氨苄青霉素抗性平板上筛选出阳性克隆,对阳性重组子进行酶切鉴定和测序分析。结果表明所克隆的yplQ、ytjAα基因核苷酸序列与Genbank上已发表的序列同源性分别为99.8%、99.4%,因此可以确定枯草杆菌224的基因组中yplQ、ytjAα基因克隆正确。以枯草芽孢杆菌224染色体DNA为模板,用PCR方法分别扩增yplQ基因上游约1.0kb和基因中的下游0.5kb的两段DNA序列作为打靶载体的同源长、短臂,将此两段序列按同一方向分别插入骨架质粒pMD18-T-neo中新霉素抗性基因(neor)表达单元的5’端和3’端,构建成重组质粒pMD18-T-neo-yplQ作为基因打靶载体。将构建好的yplQ基因打靶载体用HindⅢ单酶切线性化后,电转化至枯草芽孢杆菌224感受态中,50μg·ml-1新霉素抗性平板筛选。利用PCR鉴定方法筛选yplQ基因缺失株,检测36个抗性转化子确定基因缺失株。以枯草芽孢杆菌224染色体DNA为模板,用PCR方法分别扩增ytjAα基因下游约0.5kb和上游约0.4kb的两段DNA序列作为打靶载体的同源长、短臂,将此两段序列按同一方向分别插入骨架质粒pMD18-T-neo新霉素抗性基因(neor)表达单元的3’端和5’端,构建成重组质粒pMD18-T-neo-ytjAα作为基因打靶载体。将构建好的ytjAα基因打靶载体用PstⅠ、EcoRⅠ双酶切线性化后,电转化至枯草芽孢杆菌224感受态中,50μg·ml-1新霉素抗性平板筛选。利用PCR鉴定方法筛选ytjAα基因缺失株,检测12个抗性转化子确定基因缺失株。将yplQ、ytjAα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,结果这两个基因缺失株在血平板上仍能够引起溶血,说明单独敲除枯草芽孢杆菌224染色体的yplQ、ytjAα基因对菌株的溶血性无影响或影响不大。本实验是在已经确定yqhB,yrkA,yugS,yhdT,yhdP 5个基因单独作用对其溶血性均无影响的基础上,对枯草芽孢杆菌224溶血样基因继续研究,利用同源重组法构建枯草芽孢杆菌224yplQ、ytjAα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,对枯草芽孢杆菌224溶血基因的最终确定和溶血作用的研究迈出了关键的一步。更多还原

【Abstract】 Bacillus subtilis 224 was a non-pathogenic Bacillus that separated from human body. Accord- ing to the Microecology principle and the antagonism among the microorganism, White Swan aerosol made of live BS224 was a effective medicine for burn external using, it could treat infected burn wounds and promote wound healing. But because of hemolysis, it has been stopped using in medicine now.Up to now, no investigation was performed on the Bacillus subtilis 224 genome sequencing, but Bacillus subtilis 168 genome sequencing has been sure, eight genes were possibe related to hemolysis. yplQ(642bp)、ytjAα(228bp)were two of them, and expression product of ytjAαwas similar to alpha-hemolysin.Here, yplQ and ytjAαDNA fragments were amplified by PCR with chromosomal DNA of BS224 as the template and inserted into cloning vector pMD18-T to construct the recombinant plasmids pMD18-T-yplQ、pMD18-T-ytjAα. The primers of PCR amplification reaction were designed according to the genome sequence of Bacillus subtilis 168. The recombinant plasmids were transformed into competent cells of E.coli JM109. Positive clones were screened from the LB solid medium plates including Ampicillin. The positive recombinants were identified by restriction enzyme digest and finally sequenced. The results suggested that the homology between the nucleotide sequence of the cloned yplQ、ytjAαand the reported ones were separately 99.8%、99.4%. So it could be sure that the cloning of yplQ and ytjAαgenes is right in the genome of BS224.An integration plasmid pMD18-T-neo-yplQ for the yplQ gene disruption in Bacillus subtilis 224 was constructed. The plasmid contained upstream region of the yplQ gene 1.0kb and the yplQ gene downstream 0.5kb homologous fragments with insertion of the expression unit of the neom- ycin resistance gene between them. Upstream and downstream DNA fragments were synthesized by PCR from Bacillus subtilis 224 chromosomal DNA. The constructed gene targeting vector was linearized with HindⅢ, and electroporated into competent cells of BS224. Positive transformers were screened from the 50μg·ml-1 neomycin resistance plates. 36 transformers were analyzed by PCR to identify yplQ gene deletion.An integration plasmid pMD18-T-neo-ytjAαfor the ytjAαgene disruption in Bacillus subtilis 224 was constructed. The plasmid contained the ytjAαgene downstream 0.5kb and the ytjAαgene upstream 0.4kb homologous fragments with insertion of the expression unit of the neomycin resistance gene between them. Upstream and downstream DNA fragments were synthesized by PCR from Bacillus subtilis 224 chromosomal DNA. The constructed gene targeting vector was linearized with PstⅠ、EcoRⅠ, and electroporated into competent cells of BS224. Positive transformers were screened from the 50μg·ml-1 neomycin resistance plates. 12 transformers were analyzed by PCR to identify ytjAαgene deletion.Hemolysis of yplQ and ytjAαgene deleted mutants were separately identified on 5% sheep blood agar plates. The results suggested that the two genes deleted mutants could all induce hemolysis. It indicated that knockout of single gene had no influence or no main influence on hemolysis of BS224.Based on knockouting of single gene yqhB, yrkA, yugS, yhdT, yhdP had no influence on hemolysis of BS224, the test was futher study on Bacillus subtilis 224 other hemolytic genes. yplQ and ytjAαgenes deleted mutants were constructed by homologous recombination and their hemolysis were identified to make sure hemolytic genes or main genes inducing hemolysis, and one key step was forward taken for finally determines on Bacillus subtilis 224 hemolytic genes and studies on hemolysis.更多还原