[1]蔡远凤,贾城壹,王光丽.霍乱弧菌溶血素共调节蛋白（Hcp）的原核表达、纯化及多克隆抗体制备[J].现代检验医学杂志,2024,39(05):189-192+204.[doi:10.3969/j.issn.1671-7414.2024.05.035]
　CAI Yuanfeng,JIA Chengyi,WANG Guangli.Prokaryotic Expression, Purification and Polyclonal Antibody Preparation of Hemolysin Co-Regulatory Protein (Hcp) from Vibrio Cholerae[J].Journal of Modern Laboratory Medicine,2024,39(05):189-192+204.[doi:10.3969/j.issn.1671-7414.2024.05.035]
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霍乱弧菌溶血素共调节蛋白（Hcp）的原核表达、纯化及多克隆抗体制备()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年05期
页码:
189-192+204
栏目:
论著
出版日期:
2024-09-15

文章信息/Info

Title:
Prokaryotic Expression, Purification and Polyclonal Antibody Preparation of Hemolysin Co-Regulatory Protein (Hcp) from Vibrio Cholerae
文章编号:
1671-7414（2024）05-189-05
作者:
蔡远凤1; 贾城壹2; 王光丽3
（1. 绥阳县中医医院检验科，贵州绥阳 563399；2. 遵义市第一人民医院输血科，贵州遵义563099；3. 遵义医科大学附属医院医学检验科，贵州遵义 563000）
Author(s):
CAI Yuanfeng1; JIA Chengyi2; WANG Guangli3
（1. Department of Clinical Laboratory，Suiyang County Hospital of Traditional Chinese Medicine，Guizhou Suiyang 563359, China；2. Department of Blood Transfusion, the First People’s Hospital of Zunyi, Guizhou Zunyi 563099, China；3. Department of Medical Labo
关键词:
非O1/ 非O139群霍乱弧菌; 溶血素共调节蛋白; 原核表达; 多克隆抗体
分类号:
R378.3；R392-33
DOI:
10.3969/j.issn.1671-7414.2024.05.035
文献标志码:
A
摘要:
目的 原核表达、纯化霍乱弧菌溶血素共调节蛋白（hemolysin coregulatory protein，Hcp），并制备其多克隆抗体。方法 PCR 扩增霍乱弧菌Hcp 基因并克隆入pET28a 载体中构建重组表达载体；将重组载体pET28a-hcp 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Hcp 蛋白行Ni-NTA 柱纯化，纯化的Hcp 蛋白免疫BALB /c 小鼠以制备多克隆抗体，并用间接酶联免疫吸附试验（ELISA）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Hcp 蛋白的特异性识别。结果 重组载体pET28a-hcp 的酶切片段与预期相符，测序结果与GenBank 数据库中hcp 基因序列一致，成功构建pET28a-hcp 重组质粒，重组质粒经异丙基-β-D- 硫代半乳糖苷（IPTG）诱导表达相对分子量为28kD 的目的蛋白；经Ni-NTA 柱纯化后获得较纯的Hcp 蛋白，免疫小鼠可获得效价为1∶512 000 的抗Hcp 多克隆抗体（anti-Hcp）；Western blot 鉴定结果显示anti-Hcp 具有识别霍乱弧菌Hcp 蛋白的特异性。结论 成功获得可溶形式表达的Hcp 蛋白，免疫小鼠后获得高效价的抗Hcp 多克隆抗体，为后续研究Hcp 蛋白在非O1/ 非O139 群霍乱弧菌T6SS 致病过程中的作用奠定了基础。
Abstract:
Objective To explore prokaryotic expression, purification of hemolysin coregulatory protein （Hcp） of Vibrio cholerae, and preparation of its polyclonal antibodies. Methods PCR was used to amplify Vibrio cholerae Hcp gene and clone it into pET28a vector to construct recombinant expression vector. The recombinant vector pET28a-hcp was transformed into E.coil BL21 (DE3) for expression condition optimization and expression form identification. The soluble Hcp protein was purified by Ni-NTA column. The purified Hcp protein was used to immunize BALB/c mice to prepare polyclonal antibodies. The antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA) to evaluate its immunogenicity. Western blot was used to analyze the specific recognition of antibodies to Hcp protein in Vibrio cholerae. Results The enzyme fragment digested by recombinant vector pET28a-hcp was consistent with the expected, the sequencing results were consistent with the Hcp gene sequence in the GenBank database, and the pET28a-hcp recombinant plasmid was successfully constructed. The recombinant plasmid was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) to express the target protein with a relative molecular weight of 28 kD. The pure Hcp protein was obtained after purification by Ni-NTA column, and then Hcp polyclonal antibody (anti-Hcp) with a titer of 1∶512 000 could be obtained from immunized mice. Western blot results showed that anti-Hcp had specificity in recognizing Hcp protein in Vibrio cholerae. Conclusion The soluble expression of Hcp protein is successfully obtained, and high-titer polyclonal antibodies against Hcp are obtained after immunization of mice, which may lay a foundation for subsequent studies on the role of Hcp protein in the pathogenesis of T6SS in non-O1/non-O139 V. cholerae.

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备注/Memo

备注/Memo:
基金项目：贵州省科技厅基础研究计划项目（NO：黔科合基础-ZK[2021] 一般470）。
作者简介：蔡远凤（1980-），女，本科，副主任技师，研究方向：医学检验及相关研究，E-mail:49854699@qq.com。
通讯作者：王光丽（1996-），女，硕士研究生，检验技师，研究方向：细菌致病分子机制，E-mail：1947952722@qq.com。
更新日期/Last Update: 2024-09-15