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

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。

Construction and Immunogenicity of Recombinant Adenovirus Tandem Expressing M and GP5 of Porcine Reproductive and Respiratory Syndrome Virus

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.