摘要
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)又称为猪的蓝耳病,是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种高度接触性传染病,该病传播迅速,临床症状表现为母猪晚期早产、流产、死胎、木乃伊胎,仔猪存活率下降和育成猪的呼吸道疾病等。PRRSV基因组全长约为15kb,有8个读码框。ORF2、ORF3和ORF4分别编码病毒的次要结构蛋白GP2、GP3和GP4,ORF5、ORF6和ORF7分别编码病毒的主要结构蛋白E蛋白、M蛋白和N蛋白。E、M和N蛋白具有较好的免疫原性,是构成PRRSV的主要抗原。PRRSV在全世界范围内广泛流行,造成了重大的经济损失,严重危害了养猪业的发展。各国学者纷纷研制各种疫苗,来控制PRRSV的蔓延,腺病毒载体因其自身的优越性,由此受到广泛的关注和应用。
本试验利用复制缺陷型腺病毒载体首次成功构建了共表达PRRSV囊膜蛋白(EP)、基质蛋白(MP)和核蛋白(NP)的重组腺病毒。在构建重组腺病毒的试验中,首先用反转录聚合酶链反应(RT-PCR)的方法分别扩增出PRRSV的E基因、M基因和N基因,经测序正确后将三者用具有自裂解功能的口蹄疫病毒2A多肽的核苷酸序列依次串联起来,然后将连接好的E-2A-M-2A-N核苷酸序列克隆入腺病毒穿梭载体pAdTrack-CMV,经PCR鉴定和酶切鉴定后筛选出了重组质粒pAdTrack-CMV/E-2A-M-2A-N,将此重组质粒和腺病毒骨架载体pAdEasy-1在大肠杆菌BJ5183内进行同源重组,获得了含有外源基因的重组腺病毒质粒pAd/E-2A-M-2A-N;最后将重组腺病毒质粒经PacⅠ酶切线性化后转染HEK-293细胞,得到含有E-2A-M-2A-N基因的重组腺病毒。对重组腺病毒进行传代培养,之后提取4、6、8、10代病毒的基因组进行PCR扩增,证实外源基因在重组腺病毒中能够稳定遗传。最后,用蚀斑测定和TCID50的方法来检测病毒的毒价。本试验为研制共表达猪繁殖与呼吸综合征病毒E基因、M基因和N基因的复制缺陷型腺病毒活载体疫苗奠定了基础。
Porcine reproductive and respiratory syndrome(PRRS), commonly known as blue ear disease is caused by a porcine reproductive and respiratory syndrome virus(PRRSV), a highly contagious disease that can lead to sow miscarriage, premature birth, stillbirth, mummies and the decrease of viability and piglets severely respiratory symptoms.The genome of PRRSV is about 15kb and have eight open reading frames.GP2 protein, GP3 protein and GP4 protein are coded by ORF2,ORF3 and ORF4,that E protein,M protein and N protein are coded by ORF5,ORF6 and ORF7.E、M and N protein have a good immunogenicity,which make up major antigen of PRRSV. PRRSV is widely prevalent in the worldwide, causes economic losses and seriously harms the development of swine industry.The scholars have developed various vaccines to control the spread of PRRSV,adenovirus vector is received widely attention and applied for its own superiority.
In this study, the recombinant adenovirus containing the E, M and N genes of PRRSV simultaneously was obtained for the first time. To construct the recombinant adenovirus, the E,M and N genes were amplified by RT-PCR and sequenced, linked them by FMDV 2A sequence which has self-cleavage function, obtained the sequence E-2A-M-2A-N, and then this sequence was cloned into shuttle vector pAdTrack-CMV,by PCR identification and restriction endonuclease map analysis,the pAdTrack-CMV/E-2A-M-2A-N was obtained;Secondly,the pAdTrack-CMV/E-2A-M-2A-N was transformed into the E.coli strain BJ5183 contained the pAdEasy-1, the homologous recombination was occured between them and gained recombinant adenovirus plasmid pAd/E-2A-M-2A-N containing exogenous genes of E-2A-M-2A-N.Thirdly, the recombinant vector was cleaved by PacI and transfected into HEK-293 packaging cell line, recombinant adenovirus containing E, M and N genes of PRRSV was successfully obtained. After subculture of the recombinant adenovirus, 4, 6, 8, 10 generations genomes of viruses were extracted and amplified by PCR, which confirmed that foreign genes have hereditary stability in recombinant adenovirus.Finally, plaque purification and TCID50 were to detect the toxic of virus.This provides a basis for the research of replication-defective adenovirus vaccine co-expressing the EP, MP and NP of PRRSV.
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