摘要
通过引物定点突变PCR法,扩增了猪肺炎支原体(Mhp)168株黏附因子P97基因抗原决定簇R1区基因片段,并将该基因片段插入表达载体pET-32 a(+)中构建重组质粒,再将该重组质粒转入大肠杆菌BL21(DE3)。DNA序列分析结果表明所构建的重组质粒含有正确的目的基因。经0.1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)37℃诱导4 h,重组质粒表达的目的蛋白量可达到总蛋白的23%。W estern b lotting和ELISA试验结果证明所表达的重组蛋白具有猪肺炎支原体抗原性。
In this study the R1 region of P97 gene of Mycoplasma hyopneumoniae （Mhp） strain 168 was amplified by PCR using a pair of insite-mutation primers. The PCR product was inserted into expression plasmid pET-32a（ ＋ ） . The recombinant plasmid was transformed into competent cells of BL21 （ DE3 ） and it contained correct object fragment by sequencing. It was induced to express by 0. 1 mmol/L isopropyl-β-D-thiogalactoside （IPTG） at 37℃ for 4 hours. The dose of the recombinant protein was nearly 23% of the total product. The expression product of interest and its biological activities were characterized with western blotting and ELISA analyses.
出处
《江苏农业学报》
CSCD
北大核心
2005年第3期207-211,共5页
Jiangsu Journal of Agricultural Sciences
基金
国家科技攻关计划子专题(2002BA514A-16-6)
江苏省自然科学基金(BK2004169)
江苏省农业三项工程项目[SX(2003)049]
关键词
猪肺炎支原体
P97基因
克隆
表达
Mycoplasma hyopneumoniae
P97 gene
cloning
expression
