禽巴氏杆菌C48-1外膜蛋白H基因与鸡IL-18基因真核共表达载体的构建及表达

根据鸡IL-18和真核质粒pcDNA3的序列，设计1对特异性引物，以重组质粒pcDNA3／IL-18为模板，扩增出具有完整真核表达结构的CMV-cIL-18-BGHpA片段。将此片段非定向插入重组表达质粒pcDNA3／ompH中，经酶切分析、PCR鉴定及序列测定证实成功构建了共表达禽巴氏杆菌C48-1外膜蛋白H基因和鸡IL-18基因的重组表达质粒。将共表达质粒转染Cos7细胞，经RT—PCR、SDS—PAGE及Western blotting检测，证明了外膜蛋白H基因及鸡IL-18基因均获得了瞬时表达，为研究禽巴氏杆菌C48-1外膜蛋白H的免疫原性和鸡IL-18在基因疫苗中的免疫调节作用奠定了基础。

Construction of Eukaryotic Expression Vector and Expression of Outer Membrane Protein H Gene of Avian Pasteurlla Multocida C48-1 and Chicken Interkine-18 Gene

A pair of primers was designed according to the cIL-18 and pcDNA3 nucleotide sequence. With the primers ,the whole segment of CMV-cIL-18-BGHpA was amplified by PCR from recombinant plasmid pcDNA3/cIL-18 which contains the full length cIL-18 gene,and then inserted into recombinant plasmid pcDNA3/ompH to generate a co expression plasmid pcD- NA3/ompH clL-18. The recombinant plasmid was transferred on the Cos7 cells by Polybrene. RT-PCR identification indicated that both ompH and cIL-18 genes had already inserted into Cos7 cell chromosome. SDS-PAGE and Western blotting analysis demonstrated that both ompH and cIL 18 gene expressed instantaneously in Cos7 cell. These results lay the foundation for research on development of immunogenicity of C48-1 ompH and immunoaccommodation of cIL-18 in DNA vaccine.