猪白细胞介素18全基因的克隆、真核表达质粒的构建及其在猪肾细胞中的表达  

Cloning and construction of eukaryotic expressing plasmid of porcine interleukin18 gene and its expression in porcine kidney(PK15)

作　　者：郑兰兰[1,2] 崔保安[1,2] 陈红英[1,2] 陈瑞亮[1] 方忠意[1,2] 李小康[1,2] 赵丽[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《中国兽医学报》2009年第1期30-35,共6页Chinese Journal of Veterinary Science

基　　金：国家"十五"食品安全重大攻关专项(2001BA804A30-11);河南省重大科技攻关项目(0223013800)

摘　　要：通过RT-PCR方法自猪脾脏淋巴细胞中扩增mpIL-18基因,序列测定表明,plL-18全基因核苷酸长度为579bp,编码192个氨基酸。将其克隆到真核表达载体pcDNA3.1中,构建重组质粒pcDNA-ILl8m,所获重组质粒经过酶切、测序鉴定,证实含有目的片段,且连接、构建正确。阳性克隆鉴定后,在脂质体作用下转染猪肾细胞(PK15),通过提取RNA检测到了mpIL-18在PK15细胞中的表达。One pair of primers were designed according to porcine IL-18 gene sequence published in GenBank. Porcine IL 18 gene was amplified by RT-PCR from porcine splenocytes total RNA extracts and then cloned and sequenced. The result suggested that the full length of porcine IL- 8 gene is consisted of 579 bp,and encodes 192 amino acids. The homology of the nucleotides compared to ABO10003 is 99.8% ,and at 550 base the nueleotide changed from A to G. The homology of the amino acids compared to ABO10003,AF176949,AY262109,NM1997 are 99%, 98.5%,99.8% and 99%. Then,got pIL-18 gene,and subcloned it into the eukaryotic expressing plasmid vector peD- NA3.1 and transformed into host E. coli strain JM109 for identification. The result showed that the recombinant plasmid of pcDNA-IL18m was constructed correctly. Meanwhile, the PK15 cells were transfected with pcDNA3. 1 （＋）-IL18 construct by cationic liposome 48 hours later, and the mRNA of the target gene was determined by RT- PCR. It was demonstrated that the accuracy of plasmid pcDNA3.1（＋ ）-IL18 construct was confirmed by a series of molecular biology techniques transfection of the PK15 cells with plasmid pcDNA3.1 （＋）-IL18 caused a transient expression of the IL18 protein. It is concluded that the construction and expression of plasmid pcDNA3.1-IL18 would provide the possibility to investigate the immunogenicity of the recombinant plasmid and to prepare a new type of nucleotide vaccine.

关 键 词：猪 IL-18基因 克隆 转染 表达 

分 类 号：Q78[生物学—分子生物学]