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

中国生物工程杂志

2007, Vol. 27

Issue (1): 41-46

研究报告

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

郑兰兰¹,崔保安²,陈红英³,贾云飞⁴,陈瑞亮³,方忠意³,李小康³,赵丽⁵

1. 河南农业大学
2. 河南农业大学河南省动物性食品安全重点实验室
3. 信阳师范学院生命科学学院
4. 河南农业大学经济与管理学院
5.

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

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摘要： 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。

Abstract: 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.

收稿日期: 2006-08-17 出版日期: 2010-05-10

通讯作者: 崔保安 E-mail: baoancui@henau.edu.cn

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	郑兰兰
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	贾云飞
	陈瑞亮
	方忠意
	李小康
	赵丽

引用本文:

郑兰兰崔保安陈红英贾云飞陈瑞亮方忠意李小康赵丽. 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[J]. 中国生物工程杂志, 2007, 27(1): 41-46.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I1/41

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