摘要
目的构建携带eap基因的原核表达载体,诱导表达具有活性的重组EAP融合蛋白。方法PCR法扩增金黄色葡萄球菌基因组DNA,回收、纯化的扩增产物与pMD18-T载体相连接得重组质粒pMD18-T-EAP,转化E.coliBL21(DE3)感受态细胞,酶切鉴定;未酶切组作为对照组重组质粒pMD18-T-EAP和pET28a(+)表达载体分别用NdeI和XhoI限制性内切酶双酶切、连接,转化E.coliBL21(DE3)感受态细胞,酶切鉴定;空载体作为对照组。用不同浓度(终浓度1、2、4、8mmol/L)和不同诱导时间(1、2、3、4、5、6h)的异丙基-β-D-硫代半乳糖苷(IPTG)对阳性重组菌进行表达优化,分别取E.coli上清液和沉淀做电泳分析。应用MagneHis-蛋白纯化系统纯化重组EAP融合蛋白,并通过薄层扫描测定蛋白质的浓度。结果所获eap基因与GeneBank的基因序列同源性>99%;氨基酸同源性达100%。重组质粒经IPTG诱导,阳性重组菌转化子均有表达;当吸光度(A)值等于0.6～0.8时,相对分子质量约70000处出现目的蛋白条带。破碎的重组菌pET28a-EAP上清液中目的蛋白条带较清楚,沉淀中几乎看不到。终浓度1mmol/L为最佳蛋白表达工作浓度。IPTG诱导1h重组EAP融合蛋白有一定量的表达,随着时间的延长,表达量增加不明显,3h时的表达量达最高,之后,蛋白表达量变化不明显。表达的重组EAP融合蛋白含量占全菌体蛋白的29.6%。结论成功地克隆和表达了金黄色葡萄球菌重组EAP融合蛋白,为进一步研究以EAP蛋白作为免疫原预防和治疗由金黄色葡萄球菌引起的疾病奠定基础。
Objective To construct a prokaryotic expression vector carrying eap gene, and to express recombinant EAP fusion protein with activities. Methods Genomic DNA of Staphylococcus aureus （S. aureus） was used as template to amplified eap gene by PCR. The purified eap gene fragments were introduced into pMD18-T vectors, transformed into E. coli BL21（DE3） pLysS competent cells, and then identified by restriction endonuclease digestion. Non-digested cells were set as a control group. Then, pMD18-T-EAP plasmids as well as pET28a positive vectors were double digested with Nde I and Xho I, then, the purified eap gene were ligated with linear pET28a positive vector, transformed into E. coli. BL21（DE3） pLysS competent cells, and identified by restriction enzyme digestion. Blank vector was set as a control, as for protein expression, the IPTG concentrations （1, 2, 4, or 8 mmol/L） induced time（1, 2, 3, 4, 5, or 6 hours） were optimized, respectively. The supernatant and sediment of induced E. coli cells were then analyzed by SDS-PAGE. MagneHisTM was employed to purify the recombinant protein and the concentration of the protein was determined by thin-layer scanning. Results The eap gene shared 99% nucleotide homology and 100% amino acid homology to GenBank sequences. EAP protein was expressed in E. coli BL21（DE3） pLysS host cells induced with IPTG Electrophoresis showed a protein band at 70 000 when A value was 0.6-0.8. The protein existed in the supernatant, but could be hardly observed in the sediment. Expression of the protein was optimal at a concentration of 1 mmol/L. Expression could be detected after 1-hour induction by IPTG. The level of the expression did not increase with time and reached its peak at 3 hours and then maintained the level during the experiment. The concentration of recombinant EAP fusion protein accounted for 29.6% of all the proteins expressed in host cells. Conclusion eap gene from S. aureus can be cloned and expressed successfully, which make it possible to prevent and treat S.
出处
《中国医药生物技术》
CSCD
2008年第3期210-213,共4页
Chinese Medicinal Biotechnology
关键词
葡萄球菌
金黄色
克隆
分子
基因
表达
胞外黏着蛋白
Staphylococcus aureus
Cloning, molecular
Gene expression
Extracellular adherence protein
