猪源嗜性衣原体HB1043株主要外膜蛋白（MOMP）基因的分析及重组表达.docVIP

猪源嗜性衣原体HB1043株主要外膜蛋白（MOMP）基因的分析及重组表达 摘要：根据GenBank中衣原体主要外膜蛋白（MOMP）全基因序列设计特异性引物，以临床分离的衣原体病原中提取的衣原体全基因组为模板，利用特异性引物PCR扩增得到MOMP全基因序列。通过BLAST比对，结果显示其与已提交的猪流产衣原体CG1株的MOMP序列（EU531729）有1个碱基不同。根据对测序结果进行信号肽序列预测及内切酶位点分析，选用BamHⅠ和SalⅠ双酶切将目的蛋白序列克隆到表达载体pET-28a上，得到重组质粒pET-28a-MOMP。将重组质粒转化入E. coli BL21进行表达，表达产物使用镍离子亲和层析柱进行纯化得到目的蛋白rMOMP。目的蛋白经Western-blot检测，证明其具有免疫学活性。 关键词：猪流产嗜性衣原体；主要外膜蛋白（MOMP）；重组表达 中图分类号：S852.67 文献标识码：A 文章编号：0439-8114（2013）04-0949-04 Cloning and Expression of Major Outer Membrane Protein of Chlamydophila Clinical Strain HB1043 from Swine YAN Shao-xia1，2，TIAN Yong-xiang1，LIU Ze-wen1，YUAN Fang-yan1，GUO Rui1，DUAN Zheng-ying1， YANG Ke-li1，MENG li1，ZHOU Dan-na1，LI Shao-wen2 （1. Institute of Animal Husbandry and Veterinary， Hubei Academy of Agricultural Sciences， Wuhan 430064，China； 2. College of Animal Science and Technology， Huazhong Agricultural University， Wuhan 430070，China） Abstract： Chlamydophila was an obligate intracecullar parasites and could infect human and multiple animals. According to the published complete nucleotide sequence of Chlamydophila major outer membrane protein （MOMP） in the GenBank and the whole genome of Chlamydophila isolated from clinical samples， the whole genome sequence of MOMP gene were obtained by PCR. The PCR product was sequenced and blasted with the committed MOMP sequence （EU531729） from strain CG1 of swine Chiamydophila abortus. The results showed that it had one base different from the CG1 strain. The extinction enzymes sites BamHⅠand SalⅠwere selected for cloning the MOMP sequence to the pET-28a. The restructuring plasmid pET-28α-MOMP were then transfered into E. coli BL21 and expressed. The target protein rMOMP was purified by the Ni Sepharose 6 Fast Flow and proved with immunocompetence by Western-blot. Key words： Chlamydophila； major outer membrane protein； recombined expression 衣原体是一类细胞内专性寄生的、大小介于细菌和病毒之间的革兰氏阴性微生物。衣原体感染在世界范围内均有流行，是一种能够引起人和动物患病的人畜共患病原体。依据衣原体核糖体rRNA的序列比对以及多态性限制性酶切位点的分析，将衣原体科分为衣原体属和嗜性衣原体属。 嗜性衣原体属包含鹦鹉热嗜性衣原体、流产嗜性衣原体、肺