麻鸭白细胞介素18成熟蛋白基因的克隆、表达及生物学活性

根据GenBank发表的鸭IL-18 cDNA基因序列，设计、合成一对引物，直接从成年麻鸭脾淋巴细胞提取总RNA，经反转录-聚合酶链反应（RT-PCR）扩增麻鸭IL-18成熟蛋白基因，扩增产物进行了T-A克隆、测序，获得了麻鸭IL-18成熟蛋白基因全序列，其大小为513 bp，编码一条由170个氨基酸残基组成的多肽。将该基因片段亚克隆到原核表达载体pQE30，构建重组表达质粒pQE30-mDuIL18，转化大肠杆菌M15，并用IPTG 诱导。重组菌菌体裂解物经SDS-PAGE可检测到相对分子质量为19.76 Ku的重组蛋白，Western blotting证实该重组蛋白可与兔抗鸡IL-18血清发生特异性反应。表达产物以包涵体形式存在，包涵体经变性、复性处理后进行鸭T淋巴细胞转化试验表明，重组蛋白具有明显促进鸭T细胞转化的生物学活性。用mDuIL-18（150 ng or 200 ng /鸭）和AIV 疫苗免疫鸭2周后，HI抗体平均滴度达到7.5–7.7 log 2 ，而只用AIV 疫苗免疫和用mDuIL-18（100 ng /鸭）和AIV 疫苗免疫的HI抗体平均滴度仅达到6.3–6.6 log 2，表明mDuIL-18提高AIV灭活油乳苗诱导的免疫应答。这为研究鸭IL-18结构和生物学应用，以及研制新型免疫佐剂和免疫调节剂奠定基础。

Cloning , Expression of Duck Interleukin-18 Mature Protein Gene and Biological Activity

One pair of primers was designed and synthesized according to duck IL-18 gene sequences published in GenBank．Duck IL-18 mature protein gene was amplified by RT-PCR from total RNA extracted directionally from ma duck splenocyte ,then was cloned and sequenced．The result suggested that the nucleotide sequence of duck IL-18 mature protein gene be consisted of 513 bp in length, encoding 170 amino acid residues．A prokaryotic expression plasmid of mDuIL-18 , pQE30-mDuIL18 , was obtained by subcloning the encoding region of the DuIL-18 mature peptide into pQE30. pQE30-mDuIL18 was transformed E.coli M15 ,and induced by IPTG. The expression of p IL-18 mature protein gene was identified by SDS-PAGE and Western-blotting. The results revealed it had a molecular weight of 19755,and could be specifically recognized by the rabbit sera to chicken IL-18. The expressed products exist at the form of inclusion body. After being degenerated and then renaturated, the activity of the inclusion bodies were detected by by MTT. In ducks injected intramuscularly with rduIL-18 protein (150 ng or 200 ng per duck, respectively) and AIV vaccine 2 weeks after immunization, the average titers of HI antibodies to AIV reached 7.5–7.7 log 2, while the average titers of HI antibody to AIV were 6.3–6.6 log 2 in ducks only vaccinated with AIV vaccine or with 100 ng rduIL-18 and AIV vaccine. The results clearly showed that 150 ng rduIL-18/duck strengthened in vivo immune responses induced by the inactivated oil emulsion AIV vaccine.Development of recombinant pQE30-mDuIL18 paved the way for future study of biological function of expressed product and development of recombinant fowlpox viruses(rFPV) coexpressing duck IL-18 and protective antigen gene.