淋病奈瑟菌NGO2105蛋白660~1468肽段的表达纯化及多克隆抗体的制备与鉴定  

Expression and purification of a peptide fragment of 660-1468 amino acids in Neisseria gonorrhoeae NGO2105 protein as well as preparation and identification of its polyclonal antibody

作　　者：夏灵尹 卢琴 王小素 黄美容 闵迅 黄健 Xia Lingyin;Lu Qin;Wang Xiaosu;Huang Meirong;Min Xun;Huang Jian(Department of Laboratory Medicine,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou,China;Department of Blood Transfusion,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou,China)

机构地区：[1]遵义医科大学附属医院检验科,遵义563000 [2]遵义医科大学附属医院输血科,遵义563000

出　　处：《中华皮肤科杂志》2023年第3期216-221,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81760358、82260330)。

摘　　要：目的原核表达淋病奈瑟菌NGO2105蛋白的660~1468肽段,制备并鉴定其多克隆抗体。方法将pCold TF-NGO2105_(660-1468 aa)重组质粒转化至E.coli BL21(DE3)菌中进行蛋白表达。包涵体蛋白经变性复性处理后纯化目的蛋白。目的蛋白免疫BALB/c小鼠制备多克隆抗血清;采用ELISA法检测抗体效价,Western印迹分析抗体对淋病奈瑟菌中NGO2105蛋白的特异性,流式细胞仪分析抗血清与淋病奈瑟菌的亲和力,黏附抑制实验评估抗NGO2105_(660-1468 aa)抗体对淋病奈瑟菌对人宫颈上皮细胞ME-180细胞黏附作用的抑制能力。不同处理组间比较采用t检验。结果NGO2105_(660-1468 aa)蛋白以包涵体的形式呈现,通过变复性和纯化后得到了可溶性NGO2105_(660-1468 aa)蛋白,以该蛋白免疫小鼠后抗血清的效价为5.12×10^(6),流式细胞仪分析结果显示,该抗体能较好与淋病奈瑟菌的NGO2105_(660-1468 aa)片段结合。黏附抑制实验结果提示,相比未处理组的黏附率(100%),抗NGO2105_(660-1468 aa)抗体在20和40倍稀释时均能显著抑制淋病奈瑟菌的黏附作用(黏附率=52.9%、79.2%;t=8.40、5.29;P<0.001、=0.006),呈现一定的浓度梯度依赖。结论成功表达及纯化出NGO2105_(660-1468 aa)肽段蛋白,制备出高效价的多克隆抗体,该抗体与淋病奈瑟菌有较好的亲和力且有黏附抑制能力。Objective To prokaryotically express a peptide fragment of 660-1468 amino acids in Neisseria gonorrhoeae NGO2105 protein,and to prepare and identify its polyclonal antibody.Methods The pCold TF-NGO2105_(660-1468 aa) recombinant plasmid was transformed into the bacterium Escherichia coli BL21(DE3)for protein expression.After the inclusion body protein was denatured and renatured,the target protein was purified.Then,BALB/c mice were immunized with the target protein to prepare a polyclonal antiserum;the antibody potency was evaluated by enzyme-linked immunosorbent assay,the specificity of the antibody against NGO2105 protein in Neisseria gonorrhoeae was analyzed by Western blot analysis,the affinity of the antiserum with Neisseria gonorrhoeae was analyzed by flow cytometry,and adhesion inhibition assay was performed to evaluate the inhibitory effect of anti-NGO2105_(660-1468 aa) antibody on the adhesion of Neisseria gonorrhoeae to human cervical epithelial ME-180 cells.Comparisons between different groups were performed by using t test.Results The NGO2105_(660-1468 aa) protein was expressed as the inclusion body,and the soluble target protein was obtained by denaturation,renaturation,and purification.After immunization of mice with the target protein,the antiserum titer was 5.12×10^(6),and flow cytometry showed that the antibody bound well to the Neisseria gonorrhoeae NGO2105_(660-1468 aa).Adhesion inhibition assay showed that the anti-NGO2105_(660-1468 aa) antibody significantly inhibited the adhesion of Neisseria gonorrhoeae to ME-180 cells,and the inhibitory effect was concentration-dependent to some extent,with the adhesion rates of Neisseria gonorrhoeae treated with 20-and 40-fold dilutions of the anti-NGO2105_(660-1468 aa) antibody being 52.9%and 79.2%respectively,significantly lower than the adhesion rate in the untreated group(100%,t=8.40,5.29,P<0.001,=0.006,respectively).Conclusion The NGO2105_(660-1468 aa) protein was successfully expressed and purified,and a highly potent polyclonal antibody was prepare

关 键 词：淋病奈瑟球菌 NGO2105蛋白 多克隆抗体 抗体亲和力 细菌黏附 免疫接种 疫苗 

分 类 号：R759.2[医药卫生—皮肤病学与性病学]