金黄色葡萄球菌膜蛋白nEbpS原核表达系统的构建及抗体的制备

【作者】 李传芬；

【导师】 李云龙； 胡成进；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2008， 硕士

【摘要】 金黄色葡萄球菌(金葡菌)是临床常见致病菌之一。目前研究发现,该菌主要是通过自身的弹性纤维结合蛋白(Elastin binding proteins of S. aureus,EbpS)侵染宿主。EbpS是两次跨膜蛋白,全基因长为1458bp,编码486AA,分子量为117kDa。并且N端的59个氨基酸(amino acid,AA)是金葡菌和宿主弹性蛋白结合的主要位点。本课题在获得金黄色葡萄球菌全基因组的基础上,首先根据Genbank中ATCC25923标准菌株基因组序列及EbpS蛋白的基因序列,设计出针对EbpS蛋白N端(N terminal of Elastin Binding Proteins of S. aureus,nEbpS)的引物,利用PCR方法采用高保真酶从提取的标准菌株基因组中扩增出nebps基因片段,片段长度为684bp,末端加A,双酶切后将目的片段基因按照正确的读码框克隆到T克隆载体pMD19-T中,得到含nebps基因片段的pMD19-T(+)重组子,转化大肠杆菌DH5α感受态细胞,并对经氨苄青霉素初步筛选的阳性重组子进行双酶切鉴定和测序鉴定。将双酶切鉴定正确并且测序结果与Genbank上nEbpS基因序列完全一致的阳性重组质粒双酶切处理,再与同样经双酶切处理的表达载体pQE30连接,得到重组表达载体pQE30(+)/nebps,转化大肠杆菌表达菌M15,37℃,1mM IPTG,200rpm诱导重组蛋白表达。经SDS PAGE和Western blotting检测后表明分子量约53kDa的融合蛋白表达,与预期分子量大小(53.33kDa)一致,并且表达蛋白大部分以可溶形式存在。利用目的蛋白的多聚组氨酸标签进行镊柱亲和层析纯化融合蛋白,经Western blotting鉴定,融合蛋白与His单抗能够结合。纯化出的蛋白经PBS透析后超滤管浓缩,进行SDS-PAGE电泳检测纯度后,考马斯亮蓝染色法蛋白定量,采用100mg/ml的浓度与弗氏佐剂乳化制备抗原,免疫新西兰兔子,收获抗血清,通过与金葡菌菌体的凝集反应进行鉴定。实验结果说明,重组质粒pQE30(+)/nebps能够成功地在大肠杆菌表达菌M15中表达出目的蛋白,所获得的融合蛋白与预期大小相同。利用亲和层析纯化后的蛋白制备抗原,免疫动物获得抗血清。更多还原

【Abstract】 Staphylococcus aureus is a common bacteria . Mainly through its own elastic fiber surface protein binding protein (Elastin Binding Proteins of S. aureus, nEbpS) infected host. nEbpS owns 1458 bp gene ,which encodes a protein of 486 AA, molecular weight of 25 kDa.The N-terminal 59 AA are the binding sites between Staphylococcus aureus and the main elastic protein.A pair of primers were designed and synthesized according to the sequence of nEbpS gene on the basis of the sequence of Staphylococcus aureus genome from Genbank. And the whole of N-terminal of Staphylococcus aureus gene was amplified by high-fidelity PCR technique from the Staphylococcus aureus genome. Add adenosine to the terminal. Then the recombinant clone plasmids pMD19-T(+)/nebps was constructed after double restriction endonucleases digestion and the host strain DH5αwas transformed. The selected clones were verified by PCR,double restriction endonucleases and DNA sequencing. DNA sequencing results showed that the sequence of DNA fragments inserted into the vector was identical to the one that had been reported before from Genebank. Then the recombinant express plasmids pQE30(+)/nebps was constructed in the similar way as the clone vector. After verifying by PCR and double restriction endonucleases digestion, the plasmid was transformed into expression strain M15 to express nEbpS protein with 37℃and 1mM IPTG induced. The expressed protein with molecular weight of 53kDa was detected by SDS-PAGE electrophoresis and Western blotting. The protein nEbps fused with Trx-His tag. The molecular weight was identical to prospective one(53.33 kDa), and most protein was soluble.The expressed fuse protein was purified by the method of affinity chromatography according to the manual of the producer. Then the purified protein nEbps fused with His tag was detected by Western blotting. The purified product showed good reactivity to anti-His tag antibody. The purified protein was concentrated by ultrafiltration and quantitated by Coomassie brilliant blue to 100 mg / ml concentration.Then misced antigen with complete Freund’s adjuvant emulsion to immune animals, tnen harvest antiserum.The antibody was detected by agglutination reaction whih Staphylococcus aureus. The experimental results show that, the fragment of 684bp nEbpS gene was successfully expressed in M15 contained the recombined plasmid pQE30(+)/nebps. The molecular weight of the expressed protein was identical to the anticipant one. Then the expressed nEbpS protein was purified by affinity chromatography and prepared as antigen for animal immunization. And the antiserum was confirmed to own antibody by agglutination reaction.更多还原