摘要
【目的】验证牛支原体P48蛋白在细胞中的位置,并为后续功能的研究奠定基础.【方法】运用Overlap PCR扩增获得点突变后的牛支原体武威分离株P48基因,并将其克隆至pGEM-T Easy,将测序正确的质粒克隆至表达载体pET-28a中,构建原核表达质粒pET-P48,表达质粒转化大肠杆菌BL21(DE3),并在IPTG诱导下成功获得融合蛋白表达产物rMbP48,大小约为51ku.重组蛋白纯化后免疫新西兰大白兔制备多克隆抗体.在分离牛支原体膜蛋白的基础上应用Western-blot及间接ELISA对P48蛋白在细胞中的分布进行分析.【结果】P48蛋白主要存在于牛支原体的膜分离相.全菌免疫荧光试验进一步证实脂蛋白P48位于牛支原体的膜表面.【结论】牛支原体P48蛋白是存在于牛支原体表面的一种具有免疫原性的脂蛋白.本研究为进一步研究P48蛋白的生物学特性及建立高效的牛支原体诊断方法奠定了基础.
[Objective] To verify the location of Mycoplasma boris P48 protein in the cell, and to lay the foundation for the follow-up study its function. [Method] The P48 gene from M. boris Wuwei strain was amplified by Overlap PCR. Sequencing and analyzing were accomplished after P48 gene was cloned into pGEM-T Easy. Then the gene was cloned into prokaryotic expression vector pET-28a （q-） and the recom- binant expression plasmid pET-P48 was transformed into E. coli BL21 （DE3）. After induction by IPTG, the recombinant protein rMbP48 was expressed,which the relative molecular weight was about 51ku. Then the rabbit was immunized with purified recombinant protein and the polyclonal antibodies against rMbF48 were prepared. Subsequently,the subcellular localization of P48 was accomplished using Western-blot and ELISA on the basis of the separation of the membrane proteins. [Result] The results showed that P48 pro-tein mainly distributed in the cytomembrane. That was further confirmed by whole cell immunofluorescence test. [Conclusion] Mr. boris P48 protein is a lipoprotein that has immunogenic and present in the surface of M. boris,which laid the foundation for the future study of the biological characteristics and establish an ef- ficient diagnostic methods of P48 protein of M. boris
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2016年第2期1-7,共7页
Journal of Gansu Agricultural University
基金
甘肃省自然科学基金项目"新生犊牛传染性肺炎病原的分离
鉴定及快速诊断方法研究"(1308RTZA235)
关键词
牛支原体
P48
基因克隆
原核表达
亚细胞定位
Mycoplasma boris
P48
gene clone
prokaryotic expression
subcellular localization
作者简介
胡国明（1989-），男，硕士研究生，研究方向为传染病与病原分子生物学．E-mail：hgm900915@126．com
通信作者：包世俊，男，副教授，硕士生导师，研究方向为动物传染病与流行病学、动物病原分子生物学及基因工程．E-mail：bsjdy@126．com
