【作者】 王爽；

【导师】 朱道银；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2008， 硕士

【摘要】 目的:构建鼠Lcn2蛋白的原核表达质粒,并在E.coli中高效表达和纯化重组的Lcn2蛋白,并了解其生物学活性。方法:采用RT-PCR法从小鼠巨噬细胞中扩增出Lcn2的基因片段,将其插入原核表达质粒pET32a(+),构建重组质粒pET32a(+)-lcn2。重组质粒经酶切和DNA测序鉴定后,将pET32a(+)-lcn2转化E.coli BL21(DE3)plysS,用IPTG诱导目的基因表达。His-tag in-gel stain鉴定后,采用Ni-NTA树脂亲和纯化目的蛋白。然后采取逐步降低尿素浓度的方法使重组蛋白复性。然后将重组蛋白分别添加到LB培养基中,利用倾注法接种E.coli BL21(DE3),同时另取一个普通琼脂平板做对照,观察重组Lcn2蛋白对大肠杆菌生长的影响。以E.coli BL21(DE3)、乙型链球菌和金黄色葡萄球菌为研究对象,将含重组Lcn2蛋白的纸片分别添加到涂有上述三种细菌的培养基中,用药物敏感性试验纸片法来判断其蛋白对细菌生长有无抑制作用。结果:经核苷酸序列测定和双酶切鉴定结果表明,成功构建了重组质粒pET32a(+)-lcn2。在大肠杆菌BL21(DE3)plysS中用IPTG诱导表达后,获得了Lcn2蛋白。重组蛋白经SDS-PAGE分析分子量约为21KD。细菌生长试验表明,在含有重组蛋白的培养基上生长的菌落明显小于对照,但菌落数量没有显著差异。在接种大肠杆菌、乙型链球菌的培养基上出现明显抑菌环,金黄色葡萄球菌的培养基上未见抑菌环。结论:成功构建了重组表达质粒pET32a(+)-lcn2,并获得了具有生物学活性的重组蛋白。细菌生长试验证实其对革兰氏阳性菌和革兰氏阴性菌生长都有一定的抑制作用,但对金黄色葡萄球菌无效。细菌生长试验结果提示蛋白Lcn2是一种有用的抑菌剂。更多还原

【Abstract】 Objective: To construct prokaryotic expression plasmids carrying murine macrophage lcn2 gene, express the recombinant proteins in E.coli BL21(DE3)plysS and observe its biological activity.Methods: The gene from genome of murine macrophage lcn2 was amplified by RT-PCR and inserted into prokaryotic expression vector PET32a(+) to obtain recombinant plasmid pET32a(+)-lcn2. After identified by restriction digestion and DNA sequencing, the constructed recombinant plasmids was transformed into E.coli BL21(DE3)plysS. Then E.coli was induced with IPTG.and the product was analyzed by SDS-PAGE and His-tag in-gel Stain.Then the expressed protein was purified.The recominant protein Lcn2 was added into the LB medium ,and E.coliBL21(DE3) was subcultured on the medium by pouring.A common LB medium without Lcn2 as the control was operated by the same way. Finally , E.coliBL21(DE3),Streptococcus hemolyticus, Staphylococcu saureus were respecrively subcultured on the corresponding medium, the bacteriostatic activity of recombinat Lcn2 was observed by disk diffusion antimicrobial suceptibility test.Result: Restriction endonuclease digestion and DNA sequence analysis indicated that the prokaryotic expression vector was constracted successful.The expressed protein induced by IPTG in E.coliBL21(DE3)plysS had relative molecular mass of about 21 kDa.Bacteria growth test demonstrated that the colonise in medium with recominant protein was smaller than that in control LB medium,but the bacterial numbers is not different.Except for the medium with Staphylococcus aureus,bacteriostatic circles was observed on medium with E.coli and Streptococcus respecrively.Conclusion: The recominant expression plasmid pET32a(+)-lcn2 was constracted successful, and the recominant protein Lcn2 was successfully prepared.Bacteria growth test demonstrated that the recombinant Lcn2 could inhibit the growth of gram-positive bacterias and gram-negative bacteria , but it had no effect for Staphylococcus aureus.These results indicated Lcn2 might be a useful bacteriostatic agent.更多还原