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串联表达PRRSV M与GP5蛋白重组腺病毒的构建及其免疫特性研究
引用本文:蒋文明,姜平,李玉峰,汤景元,王先炜,杜以军.串联表达PRRSV M与GP5蛋白重组腺病毒的构建及其免疫特性研究[J].生物工程学报,2006,22(4):555-560.
作者姓名:蒋文明  姜平  李玉峰  汤景元  王先炜  杜以军
作者单位:南京农业大学农业部动物疫病诊断与免疫重点开放实验室,南京,210095
基金项目:国家自然科学基金;高等学校博士学科点专项科研项目;新世纪优秀人才支持计划
摘    要:利用PCR扩增出猪繁殖与呼吸综合征病毒的M基因,按正确的读码框与GP5基因串联,成功构建穿梭载体pShuttle-CMV-M-GP5,经PCR、测序鉴定正确。PmeⅠ线性化后在BJ5183大肠杆菌内与腺病毒骨架载体pAdEasy-1同源重组,产生重组腺病毒DNA。重组腺病毒DNA经PacⅠ线性化后用脂质体转染HEK-293A细胞,在细胞内包装成完整的腺病毒,通过IFA可以检测到M与GP5串联的重组腺病毒构建成功,可以正确地表达目的蛋白。将构建好的重组腺病毒免疫小鼠,结果表明可以诱导产生较强的体液免疫应答(ELISA抗体和中和抗体)和细胞免疫应答(淋巴细胞增殖和CTL反应)。证明该重组腺病毒具有较好的免疫原性,为下一步猪体免疫试验奠定了基础。

关 键 词:PRRSV  M  GP5  重组腺病毒  体液免疫  细胞免疫
文章编号:1000-3061(2006)04-0555-06
修稿时间:2006年1月9日

Construction and Immunogenicity of Recombinant Adenovirus Tandem Expressing M and GP5 of Porcine Reproductive and Respiratory Syndrome Virus
JIANG Wen-Ming,JIANG Ping,LI Yu-Feng,TANG Jing-Yuan,WANG Xian-Wei,DU Yi-Jun.Construction and Immunogenicity of Recombinant Adenovirus Tandem Expressing M and GP5 of Porcine Reproductive and Respiratory Syndrome Virus[J].Chinese Journal of Biotechnology,2006,22(4):555-560.
Authors:JIANG Wen-Ming  JIANG Ping  LI Yu-Feng  TANG Jing-Yuan  WANG Xian-Wei  DU Yi-Jun
Institution:Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. jiangp@njau.edu.cn
Abstract:The M protein gene of porcine reproductive and respiratory syndrome virus amplified by PCR was tandem linked with its GP5 gene in shuttle vector in correct frame, resulting in shuttle vector pShuttle-CMV-M-GP5. The positive clone was identified by PCR and further confirmed by sequencing. The constructed plasmid was linearized with Pme I and co-transformed BJ5183 host bacteria with pAdEasy-1 to produce recombinant adenovirus DNA by homologous recombination. Then the adenovirus DNA was linearized with Pac I and transfected into HEK-293A cells to obtain recombinant adenovirus. The specific expression of target proteins by the recombinant adenovirus was verified by indirect immuno-fluorescence assay (IFA) with monoclonal antibodies against M and GP5.The results showed that the tandem linked M with GP5 could be co-expressed by adenovirus vector. Mice immunized with the constructed recombinant adenovirus induced strong humoral immunity (ELISA antibody and virus neutralizing antibody) and cellular immunity (lymphocyte proliferation and CTL responses). The results showed that the recombinant adenovirus has strong immunogenicity and provided the basis for the further experiments in pigs.
Keywords:PRRSV  M  GP5  recombinant adenovirus  humoral immunity  cellular immunity  
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