生物技术通报 ›› 2022, Vol. 38 ›› Issue (7): 269-277.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1299
王光丽1,2(), 范婵1,2, 王辉1,2, 卢惠芳1,2, 夏灵尹1,2, 黄健1,2, 闵迅1,2()
收稿日期:
2021-10-14
出版日期:
2022-07-26
发布日期:
2022-08-09
作者简介:
王光丽,女,硕士研究生,研究方向:细菌致病机制;E-mail: 基金资助:
WANG Guang-li1,2(), FAN Chan1,2, WANG Hui1,2, LU Hui-fang1,2, XIA Ling-yin1,2, HUANG Jian1,2, MIN Xun1,2()
Received:
2021-10-14
Published:
2022-07-26
Online:
2022-08-09
摘要:
原核表达、纯化霍乱弧菌HlyA蛋白,制备并鉴定其多克隆抗体。PCR扩增霍乱弧菌hlyA基因并克隆入pET28a、pET32a和 pCold TF载体中构建重组表达载体;将重组载体pET28a-hlyA、pET32a-hlyA和pCold TF-hlyA转化E. coil BL21(DE3)中,进行表达条件优化及表达形式鉴定。获取可溶性形式的HlyA蛋白行Ni-NTA柱纯化,纯化的HlyA蛋白免疫BALB /c小鼠以制备多克隆抗体,并用间接ELISA法检测抗体效价,以评估其免疫原性。再以Western blot法分析抗体对霍乱弧菌中HlyA蛋白的特异性识别,并行质谱验证。分析纯化的HlyA蛋白的溶血活性及其抗体的中和活性。pET28a-hlyA、pET32a-hlyA载体只能诱导出包涵体表达的HlyA蛋白,pCold TF-hlyA载体诱导出可溶性表达的HlyA蛋白。经Ni-NTA柱纯化后获得较纯的HlyA蛋白,该蛋白不能裂解兔红细胞,但免疫小鼠可获得较高效价的多克隆抗体;Western blot和质谱鉴定均显示HlyA多克隆抗体能特异性识别霍乱弧菌中的HlyA蛋白,且该抗体可有效抑制霍乱弧菌分泌上清液的溶血活性。成功获得可溶表达的HlyA蛋白,免疫小鼠后获得高效价的抗HlyA多克隆抗体,为后续研究HlyA蛋白在霍乱弧菌致病过程中的作用奠定了基础。
王光丽, 范婵, 王辉, 卢惠芳, 夏灵尹, 黄健, 闵迅. 霍乱弧菌溶血素HlyA的原核表达、纯化及多克隆抗体制备与鉴定[J]. 生物技术通报, 2022, 38(7): 269-277.
WANG Guang-li, FAN Chan, WANG Hui, LU Hui-fang, XIA Ling-yin, HUANG Jian, MIN Xun. Prokaryotic Expression,Purification,Identification,and Polyclonal Antibody Preparation of Vibrio cholerae Hemolysin HlyA[J]. Biotechnology Bulletin, 2022, 38(7): 269-277.
图1 pET28a-hlyA、pET32a-hlyA和pCold TF-hlyA重组质粒的构建 M:DNA marker DL5000 bp;A:hlyA基因PCR扩增电泳图(1:hlyA片段);B-D:分别是pET28a-hlyA、pET32a-hlyA和pCold TF-hlyA重组质粒菌液PCR鉴定电泳图(1:阳性对照;2-4:阳性克隆菌)
Fig.1 Construction of recombinant plasmids pET28a-hlyA,pET32 a-hlyA and pCold TF-hlyA. M:DNA marker DL5000 bp;A:electrophoresis results of hlyA gene PCR amplification(1:hlyA fragment). B-D:Electrophoresis results of pET28a-hlyA,pET32a-hlyA and pCold TF-hlyArecombinant plasmid bacterial liquid PCR,respectively(1:positive control;2-4:positive cloning bacteria)
图2 pET28a-hlyA在E. coli BL21(DE3)中诱导表达的SDS-PAGE分析 M:180 kD 蛋白marker;A:1:未诱导 pET28a-hlyA-E. coliBL21(DE3)全菌;2-3:10℃,120 r/min,0.05 和0.1 mmol/L IPTG 诱导16 h的全菌;4:15℃,100 r/min,0.05 mmol/L IPTG 诱导12 h的全菌;5和7:诱导菌裂解后沉淀;6和8:诱导菌裂解后上清;B:1:未诱导pET28a-hlyA-E. coliBL21(DE3)全菌;2:23℃,100 r/min,0.05 mmol/L IPTG诱导10 h全菌裂解后沉淀;3:23℃诱导菌裂解后上清
Fig.2 SDS-PAGE analysis of pET28a-hlyA expression pro-ducts in E. coli BL21(DE3)induced with IPTG M:180 kD protein marker;A:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA;2-3:induced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA at 10℃,120 r/min by 0.05 mmol/L and 0.1 mmol/L IPTG for 16 h;4:induced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA at 15℃,100 r/min by 0.05 mmol/L IPTG for 12 h;5 and 7:lysate pellet of induced bacteria;6 and 8:lysate supernatant of induced bacteria. B:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET28a-hlyA;2:lysate pellet of induced bacteria at 23℃ by 0.05 mmol/L IPTG for 10 h;3:lysate supernatant of induced bacteria at 23℃
图3 pET32a-hlyA在E. coli BL21(DE3)中诱导表达的SDS-PAGE分析 M:250 kD 蛋白marker;A:1:未诱导pET32a-hlyA-E. coli BL21(DE3)全菌;2和3:15℃,100 r/min,IPTG分别0.05和0.1 mmol/L诱导16 h全菌;4和5:25℃,120 r/min IPTG分别0.05和0.1 mmol/L诱导10 h全菌;6-9:诱导菌裂解后上清;10-13:诱导菌裂解后沉淀;B:1:未诱导pET32a-hlyA-E. coli BL21(DE3)全菌;2和5:20℃,100 r/min,IPTG分别0.05和0.1 mmol/L诱导12 h全菌;3和6:诱导菌裂解后沉淀;4和7:诱导菌裂解后上清
Fig.3 SDS-PAGE analysis of pET32a-hlyA expression pr-oducts in E. coli BL21(DE3)induced with IPTG M:250 kD protein marker. A:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA;2-3:induced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA at 15℃,100 r/min by 0.05 and 0.1 mmol/L IPTG for 16 h;4 and 5:induced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA at 25℃,120 r/min by 0.05 and 0.1 mmol/L IPTG for 10 h;6-9:lysate supernatant of induced bacteria;10-13:lysate pellet of induced bacteria. B:1:uninduced E. coli BL21(DE3)with recombinant plasmid pET32a-hlyA;2 and 5:induced E. coliBL21(DE3)with recombinant plasmid pET32a-hlyA at 20℃,100 r/min by 0.05 and 0.1 mmol/L IPTG for 12 h;3 and 6:lysate pellet of induced bacteria;4 and 7:lysate supernatant of induced bacteria
图4 pCold TF-hlyA在E. coliBL21中诱导表达和HlyA蛋白纯化的SDS-PAGE分析 M:250 kD蛋白质marker;1:未诱导pCold TF -hlyA- E.coli BL21全菌;2:诱导全菌;3:诱导菌裂解后上清;4-9:分别为含10、20、30、40、300、500 mmol /L 咪唑的1×Washing Buffer经Ni柱后的洗涤液
Fig.4 SDS-PAGE analysis of pCold TF-hlyA expression products in E. coli BL21 induced with IPTG and the purified HlyA protein M:250 kD protein marker;1:uninduced E. coli BL21 with recombinant plasmid pCold TF -hlyA;2:induced E. coli BL21 with recombinant plasmid pCold TF -hlyA;3:lysate supernatant of induced bacteria;4-9:1×Washing Buffer contain of 10,20,30,40,300,500 mmol/L imidazole through Ni-chelating affinity chromatography
图5 重组表达的HlyA蛋白溶血活性测定 1:阳性对照(1% Trion-X);2:阴性对照(PBS);3:0 μg HlyA 蛋白与2%兔红细胞共孵育;4:4 μg HlyA 蛋白与2%兔红细胞共孵育;5:8 μg HlyA 蛋白与2%兔红细胞共孵育
Fig.5 Hemolytic activity determination of recombinant expressive protein HlyA 1:Positive control(1% Trion-X);2:negative control(PBS);3:0 μg HlyA protein incubateed with 2% rabbit red blood cells;4:4 μg HlyA protein incubateed with 2% rabbit red blood cells;5:8 μg HlyA protein co-incubated with 2% rabbit red blood cells
图7 Western blot分析抗HlyA多克隆抗体的特异性 1:HN375菌上清;2:HN375△hlyA菌上清
Fig.7 Analysis specificity of anti-HlyA polyclonal antibody by Western blot 1:supernatant of HN375 bacterial;2:supernatant of HN375△hlyA bacterial
图8 HlyA蛋白的质量图谱 展示一条与HlyA蛋白氨基酸序列匹配肽段序列SASFTVDWDHPVFTGGRP-VNLQLASFNNR的质谱峰图
Fig.8 Mass spectrometric profiles of HlyA protein A mass spectrum of the peptide sequence SASFTVDWDHPVFTGGRPVNLQLAS-FNNR that matches the amino acid sequence of the HlyA protein is shown
图9 抗HlyA多克隆抗体中和活性检测 A:抗HlyA多克隆抗体对HN375△tagH菌株溶血活性抑制效应分析;B:抗HlyA多克隆抗体对HN375菌株和两株临床菌株溶血活性抑制效应分析。***:表示两组之间数据存在显著性差异(P < 0.001)
Fig. 9 Detection of Anti-HlyA polyclonal antibody neutrali-zation activity A:Analysis of the inhibitory effect of anti-HlyA polyclonal antibody on the hemolytic activity of HN375△tagH strain;B:Analysis of the inhibitory effect of anti-HlyA polyclonal antibody on the hemolytic activity of HN375 strain and two clinical strains. *** indicates that there is a significant difference in the data between two groups(P < 0.001)
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