新城疫C3d-P29分子佐剂F基因疫苗的构建

Construction of DNA Vaccines Containing C3d-P29 against Newcastle Disease

摘要:

C3d是补体C3的裂解产物之一, 与抗原相连时可以明显提高抗原分子的免疫原性。CR2/CD21在其功能发挥过程中起到重要作用, P29为编码C3d与CR2结合区域的基因。从健康AA肉鸡肝脏中RT-PCR克隆C3d cDNA, 设计引物克隆P29至pUC19载体, 利用同裂酶 BamH I和Bgl II构建pUC-P29.n。酶切获得P29.n, 将其克隆至真核表达载体pCDNA3.1(+)。最后RT-PCR扩增新城疫F基因, 定向克隆至真核表达载体pCDNA-P29.n中P29.n的上游, 构建完成新城疫F基因疫苗。3周龄SPF鸡进行基因免疫, 结果pCDNA-F-P29.4、pCDNA-F-P29.6较pCDNA-F都能够提高HI抗体水平及保护力, 虽然HI抗体水平不及灭活苗, 但是能够抵抗致死量病毒的攻击, 并且pCDNA-F-P29.6效果更好。目前发表的关于C3d的佐剂作用的文章多是关于鼠C3d, 相应的抗原不能够自然感染鼠类, 关于鸡C3d的报道较少。研究结果为进一步开发和利用鸡C3d奠定了基础。

关键词:鸡C3d; pUC- P29.n; HI; F基因疫苗

Abstract:

After cloning the C3d cDNA of AA broilers using the liver mRNA source, a pair of primers were designed to subclone the P29 gene to the pUC19 plasmid. Several tandems of P29 were constructed in the pUC19 plasmid using a pair of isoschizomers-BamH I and Bgl II. The pUC- P29.n was igested to get the gene of P29.n that was then cloned to pCDNA3.1 (+) plasmid. After this, the F Gene of Newcastle Disease Virus was cloned through RT-PCR and inserted into the upstream of the P29.n that was in the pCDNA-P29.n, and the DNA vaccines containing F gene against NDV with C3d-P29 as molecular adjuvant were constructed. Several groups of Specefic Pathogen Free chickens were injected with these recombinant plasmids. The pCDNA-F-P29.4 and pCDNA-F-P29.6 group had higher HI antibody titers than the pCDNA-F group. The pCDNA-F-P29.4 and pCDNA-F-P29.6 group’s HI antibody titers did not achieve titers as high as the inactive vaccine group. However, they all provided protection against the lethal F48E9 virus challenge.

Key words:C3d: pUC- P29.n; HI; F DNA Vaccine