摘要
为获得单增李斯特菌溶血素(Listeriolysiono,LLO)蛋白及其多克隆抗体。根据单增李斯特菌溶血素蛋白的基因Hly序列设计了一对特异性引物,扩增出Hly基因,克隆入原核表达载体p ET-32a,并转化于大肠杆菌BL21中,在诱导剂IPTG的作用下重组LLO蛋白成功表达。SDS-PAGE结果表明重组蛋白分子量约为69.3 ku,且主要以包涵体形式表达LLO蛋白。将重组蛋白纯化后免疫新西兰大白兔,获得兔抗LLO抗体。Western-blot检测到一条约69.3 ku的特异性条带,表明该抗体具有较强的特异性。单增李斯特菌溶血素基因Hly在大肠杆菌中已成功表达,且制备的多克隆抗体可用于LLO蛋白的检测。
To obtain LLO protein and its polyclonal antibody of Listeria monocytogenes.Hly gene from Listeria monocytogenes was amplificated by PCR using the primers designed according to the Hly gene sequence in Gen Bank.Purified PCR products were digested with nucleic acid restriction enzyme,and then inserted into p ET-32 a vector to construct recombinant expression plasmid.The positive recombinant plasmid was transformed into BL21 E. coli-comp ETent cells upon induction with isopropyl-β-D-thiogalactopyranoside（ IPTG）. The expression form of LLO protein was analyzed about 69.3 ku by SDS-PAGE analysis.A New Zealand white rabbit was vaccinated with purified LLO protein to obtain rabbit anti-LLO antibody. Western-blot showed that a69.3 ku HLY protein band of specificity appeared in the purified expression products,which showed that the antibody had strong specificity.LLO protein and its Polyclonal antibody were successfully prepared,which provides material basis for indenfitication of LLO protein.
作者
洪伟鸣
宋亮
左伟勇
HONG Wei-ming SONG Liang ZUO Wei-yong(Jiangsu Key Laboratory of Veterinary Biotechnologic Drug, Jiangsu Agri-animal Husbandry Vocational College,Taizhou, Jiangsu 225300, Chin)
出处
《江西农业大学学报》
CAS
CSCD
北大核心
2017年第1期175-181,共7页
Acta Agriculturae Universitatis Jiangxiensis
基金
国家自然科学基金(31302096)
江苏省自然科学基金(BK2011536)
江苏省"333人才工程"
江苏省"六大人才高峰"项目~~
关键词
单增李斯特菌
LLO蛋白
原核表达
多克隆抗体
Listeria monocytogenes
LLO protein
prokaryotic expression
polyclonal antibody
