内江猪IL-15基因的表达与免疫增强作用研究

Expression of Neijiang porcine interleukin 15 gene and study on immunologic enhancement of the expressed IL-15

参照GenBank中猪IL-15基因的mRNA序列设计1对特异性引物,从经由刀豆蛋白A诱导培养的内江猪外周血淋巴细胞扩增猪IL-15基因;构建其成熟肽原核表达载体pMAL-pIL-15,在Rosetta(DE3)pLysS中表达;构建其真核表达载体pCI-pIL-15,与pCI-gD(PRV)联合免疫雌性BALB/c小鼠以研究其免疫增强作用。测序结果显示,内江猪IL-15基因与发表的猪IL-15基因的核苷酸同源性为97%～99%,含489 bp的完整开放阅读框。SDS-PAGE分析显示,表达的融合蛋白约为52.5 ku,约占菌体总蛋白的27%。Western-blot检测显示,所表达的融合蛋白为麦芽糖结合蛋白的融合蛋白。MTT法试验证实,该融合表达产物经初步纯化、透析复性后,可明显增强淋巴细胞增殖;小鼠免疫试验显示,pCI-pIL-15能够促进小鼠脾CD4+、CD8+T细胞增殖,加强PRV-gD基因疫苗特异性中和抗体的产生。

A pair of specific primers was designed according to the mRNA sequences of porcine IL-15 gene in GenBank. A Neijiang porcine IL-15 gene was cloned from the PBMC stimulated by Con A. The pro-karyotic expression vector pMAL plL 15 was constructed,and expressed in Rosetta（DE3） pLysS. The eukaryotie expression vector pCI-pIL-15 was constructed. The female BALB/c mice were immunized by the combination of pCI-pIL-15 with the pCI-gD（PRV）. The cloned Neijiang porcine IL-15 gene fragment had a complete open reading frame of 489 bp in size. Sequence analysis indicated that the nueleotide sequence of the gene fragment shared 97% to 99% homology with relevant sequences reported previously. SDS-PAGE analysis showed that the target fusion protein was about 52.5 ku in molecular weight and approximately made up 27% of the total bacterium proteins. Western-blot analysis indicated that it was the target fusion protein of maltose-binding protein（MBP）. After the fusion protein was purified roughly and renatured, MTT assay confirmed that it could enhance the proliferation of PBMC. The immunization test to BALB/c mice showed that the pCI plL 15 could promote the proliferation of CD4^＋ and CD8^＋ T lymphocyte cells and enhanced the neutralization antibody level of the PRV-gD gene vaccine.