猪白细胞介素-18基因克隆、序列分析及其真核表达载体的构建

采集6月龄河南良杂猪的脾脏，分离淋巴细胞后直接提取总RNA，进行反转录-聚合酶链反应（RT-PCR）扩增，扩增产物进行T-A克隆、测序，获得了河南良杂猪IL-18全基因序列，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。与GenBank上已发表序列ABO10003进行比较，核苷酸同源性为99.8%，在第550位处 (以ATG为1计)由A→G，存在有意义突变。与GenBank下载读取的ABO10003、AF176949、AY262109、NM1997序列进行比较分析，氨基酸同源性分别为99%、98.5%和99.8%、99%。将该基因片段克隆到真核表达载体pcDNA3.1，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，为核酸疫苗的研究应用奠定了基础。

Gene Cloning and Eukaryotic Expressist Vector Constructing of Porcine lnterleukin-18

Interleukin18 (IL-18) is a novel potent inducer of IFN-γproduction. 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 was cloned and sequenced. The result suggested that the full length of porcine IL-18 gene is consisted of 579bp,and encodes 192 amino acids. The homology of the nucleotides compared to ABO10003 is 99.8%,and at 550 base the nucleotide 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 pcDNA3.1 and transformed into host E.colistrain JM109 for identification.The result showed that the recombinant plasmid of pcDNA-ILl8m was constructed correctly,which paved the way of nucleotide vaccine.