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

doi:10.13560/j.cnki.biotech.bull.1985.2021-1299

摘要/Abstract

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。

关键词: 霍乱弧菌, HlyA蛋白, 原核表达, 多克隆抗体

Abstract:

It is aimed to express and purify hemolysin HlyA protein of Vibrio cholerae in prokaryotic cells,and to prepare and identify the polyclonal antibody of mouse anti-HlyA. The hlyAgene was amplified from the genome Vibrio cholerae via PCR and cloned into the expression plasmid pET28a,pET32a and pCold TF to construct the recombinant plasmid pET28a-hlyA,pET32a-hlyA and pCold TF-hlyA, and they were transformed into E. coli BL21（DE3）. The conditions used to induce expression were optimized and the expressive forms were identified. The soluble form of hlyA protein was purified by Ni-NTA column. The purified HlyA protein was used as antigen for immunization of BALB/c mice to prepare polyclonal antibody. The antibody titer was determined to test the immunogenicity by indirect ELISA. The antibody specific recognition of HlyA protein in Vibrio cholerae was analyzed by Western blot and verified by mass spectrometry. The hemolytic activity of the purified hlyA protein and the neutralizing activity of the antibody were analyzed. The HlyA protein was expressed as inclusion bodies in the pET28a-hlyA and pET32a-hlyA vectors,and as soluble form in the pCold TF-hlyA vector. HlyA was purified by affinity chromatography on an Ni-NTA agarose column to get higher purity. The recombinant HlyA protein could not lyse rabbit red blood cells,but immunized mice can obtain polyclonal antibody with higher titer. The results of Western blot and mass spectrometry showed that the HlyA polyclonal antibody can specifically recognize the HlyA protein in Vibrio cholerae,and the antibody can effectively inhibit the hemolytic activity of Vibrio cholerae. The soluble form of HlyA protein and the high titer anti-HlyA polyclonal antibody were successfully obtained,which laid the foundation for the follow-up study of the role of HlyA protein in the pathogenic process of V. cholerae.

Key words: Vibrio cholerae, HlyA protein, prokaryotic expression, polyclonal antibody

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/表 9

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6 HlyA蛋白免疫前后小鼠血清抗体效价分析

Fig.6 Analysis of serum antibody titer in mice before and after immunization with HlyA protein

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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