猪肺炎霉形体p52基因重组腺病毒的构建及其免疫特性  

Construction of recombinant adenovirus expressing Mycoplasma hyopneumoniae p52 gene and its immune response

作　　者：曹培丽[1] 李媛[1] 陈超[1] 郭丹[1] 王亮[1] 乔祖建[1] 辛九庆[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室动物细菌病研究室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2009年第8期712-717,共6页Chinese Veterinary Science

基　　金：农业部院所长基金项目(2007)

摘　　要：为构建携带猪肺炎霉形体(Mycoplasma hyopneumoniae,Mhp)p52基因的重组腺病毒,从Mhp J株扩增p52基因,克隆到穿梭载体pShuttle-CMV中,经PmeⅠ线性化后,电转化入BJ5183菌内,与其含有的骨架载体pAdEasy-1同源重组,再与脂质体混合转染AD-293细胞,包装成完整的腺病毒,经RT-PCR、间接免疫荧光鉴定后扩增,收集病毒液,免疫BALB/c小鼠,并分析了体液免疫、黏膜免疫和细胞免疫效果。结果表明,重组质粒pShuttle-CMV-P52插入片段为1 202 bp;同源重组成功;重组腺病毒可以正确表达目的蛋白,效价达1×106TCID50/mL。重组病毒经肌肉注射和滴鼻均可诱导小鼠产生血清和肺匀浆液特异性IgG、SIgA,但不能诱导特异的淋巴细胞增殖。证实,成功构建了表达p52基因的重组腺病毒,该病毒可诱发小鼠产生特异的体液免疫和黏膜免疫,但不产生细胞免疫应答。To construct recombinant adenovirus carrying p52 gene of Mycoplasma hyopneumoniae,a p52 gene was amplified from the strain J and cloned into pShuttle-CMV plasmid.The recombinant plasmid pShuttle-CMV-P52 was linearized with PmeⅠ and then transformed into Escherichia coli BJ5183-AD-1 competent cells containing backbone vector pAdEasy-1 to homologous recombination.Purified recombinant adenovirus DNA was transfected into AD-293 cells to obtain complete recombinant adenovirus.The recombinant adenovirus was identified by RT-PCR and indirect immunofluorescence assay,then its titer was determined.BALB/c mice were immunized and the immunity was analyzed through humoral,mucosal and cell-mediated immunity aspects.The results showed that the inserted gene was 1 202 bp in length.Digestion with PacⅠ showed that homologous recombination was successful.The recombinant adenovirus was successful and P52 protein was expressed from it,and its titer was 1×106TCID 50/mL.Inoculation with the recombinant adenovirus via intramuscular and intranasal routes induced IgG and SIgA specific to P52 protein in blood and lung,but did not induce specific lymphocyte proliferation.It was confirmed that the recombinant adenovirus expressing the p52 gene was successfully constructed and it induced specific humoral and mucosal immunities but not cell-mediated immunity.

关 键 词：猪肺炎霉形体 p52基因 重组腺病毒 免疫应答 

分 类 号：S852.62[农业科学—基础兽医学]