嗜麦芽寡养单胞菌D2株smp基因缺失株的构建及鉴定  

Establishment & Identification of a smp Gene Deletion Mutant of Stenotrophomonas maltophilia D2

作　　者：秦江楠 毕春霞[2] 陈秀琴[1] 罗玮[2] 张卫[1] 任莹[1] 王斌[1] 闫志勇[1] 

机构地区：[1]青岛大学医学院微生物学教研室,山东青岛266071 [2]青岛市市立医院,山东青岛266071

出　　处：《微生物学杂志》2014年第3期30-35,共6页Journal of Microbiology

基　　金：山东省优秀中青年科学家科研奖励基金(BS2011SW005);山东省科技公关基金(2007GG3WZ05009)

摘　　要：为深入研究smp基因的功能,需构建嗜麦芽寡养单胞菌D2株smp基因缺失株。首先,PCR扩增D2株smp基因上游、下游片段作为上下游同源臂,同时扩增获得氯霉素抗性(cat)基因,采用SOE-PCR方法将各片段连接,然后双酶切后克隆入自杀质粒pEX18Tc,构建获得重组自杀质粒pEX18Tc-Δsmp/cat,并转化入大肠埃希菌SM10λpir。通过接合将重组自杀质粒转入嗜麦芽寡养单胞菌D2野生株,经同源重组以cat基因替换野生株的smp基因,链霉素和氯霉素双抗培养基筛选接合子,15%蔗糖选择培养基筛选smp基因缺失株。PCR、酶切和测序验证重组自杀质粒pEX18Tc-Δsmp/cat构建正确,缺失株的分泌蛋白经12%SDS-PAGE证实嗜麦芽寡养单胞菌D2株smp基因缺失株失去表达SMP蛋白的能力。结果显示成功获得smp基因缺失的嗜麦芽寡养单胞菌D2株,为进一步研究其功能和胞外分泌途径奠定基础。In order to study the function of smp gene deeply, it is need to establish a strain of smp gene deletion mutant of Stenotrophomonas maltophilia D2. First, the upstream and downstream fragments of smp gene were amplified by PCR as homologous arms of its upstream and downstream, at the same time chloromycetin resistant gene （smp）was amplified, then the fragments were linked by SOE-PCR, and cloned into the suicide plasmid pEX18Tc after double enzyme digestion and establish to gain a recombinant plasmid pEX18Tc-Δsmp/cat and introduced into SM10λpir of E. coli,then conjugated to S. maltophilia D2 wild strain, the smp gene of wild strain was replaced by homologous recombinant smp gene. The conjugant was screened by the medium with chloromycetin and streptomycin double resistances. The smp gene deletion mutant was screened with 15% sucrose selection medium. Finally, PCR, digestion and sequencing were used to prove that the recombinant plasmid pEX18Tc-Δsmp/cat was constructed successfully, and the secreted protein was verified by 12% SDS-PAGE, and proved that the mutant of S. maltophilia D2 with smp deletion gene has lost the expression capability SMP protein. The results showed that the smp gene deletion mutant of S. maltophilia D2 was successfully obtained and it had laid down the foundation to study further on the function and extracellular secretion pathway.

关 键 词：嗜麦芽寡养单胞菌 自杀质粒 同源重组 基因敲除 

分 类 号：Q78[生物学—分子生物学]