摘要
目的本研究以猪鼻支原体表面的可变脂蛋白(vlp)家族为研究对象,鉴定其各成员对宿主细胞的黏附能力。方法根据已公布的猪鼻支原体vlp家族7种成员的基因序列设计引物,构建重组表达质粒pET-32a(+)/vlp,筛选获得阳性克隆。重组工程菌经IPTG诱导表达目的蛋白,经镍柱亲和层析获得7种纯化的vlp重组蛋白。利用间接免疫荧光法及微孔板定量法分别对vlp各个成员的黏附功能进行鉴定。结果成功构建了7种重组表达质粒pET-32a(+)/vlp,获得阳性重组工程菌。经IPTG诱导及镍柱亲和层析纯化,获得了较纯的7种vlp重组蛋白。间接免疫荧光及微孔板黏附定量试验检测结果显示,重组蛋白vlpA、vlpB、vlpC、vlpE、vlpG可黏附PK细胞,而重组vlpD和vlpF蛋白黏附能力不明显。结论本研究成功构建了猪鼻支原体7种vlp的重组蛋白,并对其黏附功能进行了初步鉴定,证明部分vlp属于猪鼻支原体的黏附因子。
Mycoplasma hyorhinis（M.hyorhinis）is a zoonotic pathogen,which is highly prevalent in pig farms,causing a variety of chronic inflammations,and is also related to various human cancers.However,the adhesion molecules and pathogenic mechanism of M.hyorhinis are not clear yet.In this study,we performed several experiments to identify the function of the variable lipoprotein（Vlp）family in the adherence to the host cell.According to the gene sequences of the vlp family,seven genes of Vlp had been cloned into the prokaryotic expression vector pET-32a（＋）.The recombinant Vlp（rVlp）proteins were induced by IPTG,expressed in E.coli,and purified by affinity chromatography.The adhesion of Vlp to host cell was identified by indirect immunofluorescence assay and the microtiter plate adherence assay.Result showed that seven kinds of recombinant expression plasmid pET-32a（＋）/vlp were constructed successfully,and the purified rVlp proteins were obtained.The results of adherence assays showed that the purified vlpA,vlpB,vlpC,vlpE and vlpG proteins were able to bind to PK cell,while the adhesion ability of the purified vlpD and vlpF proteins were not obvious.This study successfully constructed seven kinds of rVlp proteins,and the data indicated that some of Vlp was the adhesins of M.hyorhinis.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2016年第4期338-343,共6页
Chinese Journal of Zoonoses
基金
国家自然科学基金项目(No.31300155)
江苏省自然科学基金项目(No.BK20130702)
江苏省农业科技自主创新资金项目(No.cx(14)5039)联合资助~~
关键词
猪鼻支原体
可变脂蛋白
细胞黏附
Mycoplasma hyorhinis
variable lipoprotein
cell adhesion
