猪白细胞介素12 p35、p40亚基基因的克隆和序列分析

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。

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

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.