糖尿病创面大肠杆菌谷氧还蛋白GrxB的原核表达纯化及多克隆抗体制备  

Prokaryotic expression and purification of glutaredoxin GrxB from diabetic wound derived Escherichia coli and preparation of polyclonal antibody

作　　者：白丽民 张筱薇 李笑眉 周圣杰 应杰 徐刚 Bai Limin;Zhang Xiaowei;Li Xiaomei;Zhou Shengjie;Ying Jie;Xu Gang(Graduate School of Dalian Medical University,Dalian 116044,China;Clinical College of Yangzhou University/Department of Burns and Plastic Surgery,Northern Jiangsu People’s Hospital,Yangzhou 225001,China)

机构地区：[1]大连医科大学研究生院,大连116044 [2]扬州大学临床医学院/扬州大学附属苏北人民医院烧伤整形科,扬州225001

出　　处：《中华实验外科杂志》2023年第1期43-46,共4页Chinese Journal of Experimental Surgery

摘　　要：目的原核表达糖尿病创面来源大肠杆菌谷氧还蛋白(Grx)GrxB,制备GrxB多克隆抗体。方法根据美国国家生物技术信息中心(NCBI)公布的大肠杆菌K12标准株grxB基因序列(Gene ID:946926)设计扩增引物,以本科室前期分离到的一株糖尿病创面大肠杆菌基因组(煮沸裂解法获得)为模板,通过聚合酶链反应(PCR)扩增出grxB全长663 bp,使用EcoRⅠ和XhoⅠ双酶切后连接至表达载体pET-30a。将重组质粒pET-30a-grxB转化至大肠杆菌BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导表达GrxB蛋白,使用N^(2+)镍离子层析柱对蛋白进行纯化。纯化后的重组GrxB蛋白免疫BALB/c小鼠,完成免疫程序后使用蛋白质印迹法(Western blot)和间接酶联免疫吸附试验(ELISA)对多克隆抗体的特异性和效价进行测定。两组间比较采用t检验。结果经测序,扩增出的grxB序列与K12标准株的完全一致,并成功表达出可溶性重组GrxB蛋白,大小为31.2 kDa,与预测大小一致。血清51200倍稀释后,免疫组效价显著高于磷酸盐缓冲液(PBS)组(1.146±0.066比0.12±0.011,t=15.000,P<0.05),免疫组血清与重组GrxB蛋白和大肠杆菌全菌均具有良好的特异性反应。结论成功表达可溶性重组GrxB蛋白,并获得高滴度多克隆抗体。Objective To detect the prokaryotic expression of glutaredoxins(Grx)B(GrxB)from diabetic wound-derived Escherichia coli(E.coli),and to prepare GrxB polyclonal antibody.Methods The amplification primers were designed according to the grxB gene sequence of E.coli K12 strain(Gene ID:946926)published by NCBI.Using the genome of a diabetic wound E.coli strain isolated in the early stage of our department as a template,the full-length grxB open reading frame(ORF)of 663 bp was amplified by polymerase chain reaction(PCR),which was digested with EcoRⅠ and XhoⅠ and then ligated into the expression vector pET-30a.The recombinant plasmid pET-30a-grxB was transformed into E.coli BL21(DE3),induced by IPTG,and the GrxB protein was expressed in large quantities and was purified by N^(2+)nickel ion chromatography.The purified GrxB protein was used to immunize BALB/c mice,and the specificity of this polyclonal antibody was assessed by Western blotting and indirect enzyme-linked immunosorbent assay(ELISA)after the immunization program was completed.T-test was used for comparison between the two groups,and the difference was statistically significant with P<0.05.Results After sequencing,the amplified grxB sequence was completely consistent with that of the E.coli K12 strain,and the soluble recombinant GrxB protein was successfully expressed with a predicted size of 31.2 kDa.After 51200-fold dilution,the titer of the immune group was significantly higher than that of the phosphate buffer saline(PBS)group(1.146±0.066 vs.0.120±0.011,t=15.000,P<0.05),and immunized serum had a good specific reaction with recombinant GrxB protein and E.coli strain.Conclusion The soluble recombinant GrxB protein was successfully expressed and high titer polyclonal antibody was obtained.It lays a material foundation for further research on the biological function of E.coli GrxB.

关 键 词：大肠杆菌 谷氧还蛋白 蛋白纯化 多克隆抗体 

分 类 号：R587.2[医药卫生—内分泌]