产单核细胞李斯特菌溶血素基因的克隆及原核表达  

Cloning and expression of listeriolysin O gene of Listeria monocytogenes

作　　者：张衍海[1,2] 白艳艳 张彦明[3] 郑增忍[1] 张宝[3] 童光志[1,4] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]西北农林科技大学动物医学学院,陕西杨陵712100 [4]中国农业科学院上海兽医研究所,上海200232

出　　处：《中国预防兽医学报》2008年第11期870-874,共5页Chinese Journal of Preventive Veterinary Medicine

摘　　要：利用PCR方法扩增产单核细胞李斯特菌(LM)的溶血素基因hlyA,获得一条大小约为1600bp的目的片段,将其与pMD18-T载体连接,经酶切、PCR鉴定,命名为pMD18-T-hlyA。测序结果显示所扩增的hlyA基因与GenBank中发表的hlyA的序列同源性为99%。利用含有BamHⅠ/XhoⅠ酶切位点引物B从pMD18-T-hlyA扩增不含信号肽序列的目的片段B,获得了大小约为1500bp的基因片段,该片段与pET32a表达载体经BamHⅠ/XhoⅠ处理后进行连接,转化BL21受菌体。通过酶切、PCR鉴定及序列测定后获得阳性pET32a-hlyA表达重组质粒,将重组菌用终浓度为0.1mmol/L的IPTG进行诱导表达,结果表明重组菌在诱导2h～3h表达量最高,融合蛋白约占菌体的40%,分子量大小约为80ku;利用LM的阳性血清进行Western blot分析,证实该融合蛋白可以被LM阳性血清所识别,为今后研制针对该蛋白的单克隆抗体和建立间接ELISA诊断方法提供了条件。The hlyA of Listeria monocytogenes was amplified by PCR and cloned into pMD18-T vector. Sequence analysis showed that the hlyA gene shared 99 % homology at the nucleotide level with the published hlyA gene sequences. A 1 500 bp fragment of hlyA without signal peptide was amplified from pMD18-T-hlyA, and cloned into pET32a. The recombinant expression plasmid pET32a-hlyA was transformed to E.coli and induced with 0.1 mmol/L IPTG. The expression products were analyzed by SDS-PAGE and Western blot. The results showed that the most of the expressed protein with a molecular weight 80 ku were produced after induction for 2 to 3 hours, which accounted for 40 % of the total cellular protein. The fusion protein could be recognized by the positive serum of Listeria monocytogenes, indicating it could be used for development of monoclonal antibodies and establishment of indirect ELISA.

关 键 词：产单核细胞李斯特菌 溶血素 hlyA 克隆 表达 

分 类 号：Q786[生物学—分子生物学]