猪白细胞介素12 p35、p40亚基基因的克隆和序列分析

Cloning and sequence analysis genes of porcine interleukin-12 p35 and p40 subunit

目的:克隆猪白细胞介素(IL)-12p35、p40亚基基因,为制备猪囊尾蚴的复合基因疫苗奠定基础。方法:分别分离成年猪的外周血单个核细胞和脾细胞,培养10h后用IFN-γ(100ng/ml)刺激增殖12h,随后加入细菌脂多糖(1μg/ml)刺激培养3h,分别收集细胞并提取细胞总RNA,用RT-PCR方法扩增猪IL-12p35、p40cDNA编码基因,克隆至pMD18-T载体,经PCR和双酶切鉴定后进行序列测定。结果:猪IL-12p35、p40cDNAPCR产物电泳结果证明所克隆的基因分别为616bp和1011bp,基因测序结果与GenBank报道的基因序列各有一个碱基差异,但编码氨基酸无差异,证实分别为猪IL-12p35、p40cDNA编码基因。结论:成功克隆了猪IL-12p35、p40cDNA编码基因。

Objective：To clone genes of encoding porcine interleukin-12 p35 and p40 subunits,for preparing the compound nucleir acid vaccine against the porcine bladder worm.Methods：Peripheral blood mononuclear cells and splenic lymphocytes of porcine were isolated and cultured for 10 hours,and stimulated with 100 ng/ml IFN-γ for 12 hours,followed by stimulated with 1 μg/ml lipopolysaccharide for 3 hours.Total RNA from these stimulated porcine lymphocytes were extracted,cDNA of genes for porcine IL-12 p35 and p40 subunit were ampliphied by RT-PCR assay and inserted into pMD18-T vector.The cloned genes were sequenced.Results：The PCR products of porcine IL-12 p35 and p40 subunits were 616 bp and 1 011 bp,respectively,verified by electrophoresis,sequence analysis for cloned genes in pMD18-T vector showed one base was different in each but none affecting amino acids coding,confirmed to be genes of porcine IL-12 p35 and p40 subunit.Conclusions：cDNA encoding porcine interleukin-12 p35 and p40 subunit have been successfully cloned into cloning vector pMD18-T.