以木糖为选择标记的乳酸菌表达载体的构建及猪细小病毒VP2基因的表达  

Construction of expression vector with selection marker of xylose in lactobacillus and expression of porcine parvovirus VP2 gene

作　　者：唐丽杰[1] 欧笛[2] 杨云清[3] 葛俊伟[2] 乔薪媛[2] 姜艳萍[2] 杨丽娟[1] 李一经[2] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030 [3]吉林省安图县畜牧兽医局,吉林安图133600

出　　处：《中国预防兽医学报》2010年第6期432-436,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：高等学校科技创新工程重大项目培育基金项目(706019);东北农业大学创新基金(CX2008)

摘　　要：为构建以木糖为选择标记的乳酸杆菌表达载体,根据GenBank登录的E.coli JM109 xylA和xylB基因序列设计引物,以E.coli JM109基因组为模板,扩增xylAB基因,与人工合成的多克隆位点序列MCS基因、去除氯霉素基因的乳酸菌载体质粒pPG2片段及以pPG2载体质粒为模板扩增的repA基因连接,电击转化于L.casei 393感受态细胞,获得以木糖为选择标记的乳杆菌表达载体并命名为pOXM2。为鉴定pOXM2载体表达蛋白的效果,以含猪细小病毒VP2基因的重组质粒pMD-VP2为模板,扩增VP2基因序列,与载体质粒pOXM2连接,产物电击转化L.casei393感受态细胞,获得阳性克隆子pOXM2-VP2。重组菌经诱导,并进行SDS-PAGE及western blot分析。结果表明,有约180ku重组蛋白得到了表达,而且表达的重组蛋白具有与天然病毒蛋白一样的免疫反应特异性。本研究结果表明已成功构建了以木糖为选择标记的乳杆菌表达载体并实现了外源基因的表达,为进一步研究以重组干酪乳杆菌作为口服疫苗或表达功能性蛋白奠定了基础。In order to construct the expressing vactor with Xylose as the selection Marker in Lactobacillus casei, the xylA and xylB genes of E.coli JM109 were amplified by PCR, ligated with pPG2 plasmid fragment and repA gene to generate the pOXM2 expression vector with Xylose as selection Marker instead of chloromycetin resistance. To verified the selective efficiency, an 1 800 bp VP2 gene porcine parvovirus （PPV） was inserted into pOXM2 vector and transformed into L.casei 393 by electroporation. Expression of recombinant protein VP2 was detected by SDS-PAGE and western blot. The recombinant protein retained the antigenicity of PPV as expected. This pOXM2 without any drug resistant gene provided a food-grade expression vector for oral vaccine development and the functional proteins expression.

关 键 词：干酪乳杆菌 食品级载体 xylAB基因 VP2表达 

分 类 号：S852.61[农业科学—基础兽医学]