Duong Ngoc-Diem, Nguyen-Phuoc Khai-Hoan, Do Kim-Yen Thi, Nguyen Nguyet-Thu Thi, Tran Thuoc Linh, Tran-Van Hieu
University of Science, 227 Nguyen Van Cu Street, Ward 4, District 5, Ho Chi Minh City, Vietnam.
Vietnam National University, Ho Chi Minh City, Vietnam.
J Genet Eng Biotechnol. 2021 May 11;19(1):70. doi: 10.1186/s43141-021-00172-9.
Acute hepatopancreatic necrosis disease (AHPND) is caused by toxin-producing strains of Vibrio parahaemolyticus which contain deadly binary toxins PirA and PirB encoded in pVA1 plasmid. The polyclonal antibodies against PirB protein could be used to develop immunochromatographic test strip for in-field diagnosis of AHPND.
In this study, PirB gene was amplified, cloned, and expressed in E. coli. The expressed protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot probed with 6xHis antibodies. Then, the recombinant PirB (rPirB) was purified using Ni-Sepharose column. Rabbits were immunized with the purified rPirB, and produced antibodies were analyzed using Ouchterlony double immunodiffusion. The antibody titration and antibody purification were performed by ELISA and affinity chromatography, respectively. Finally, antibody specificity and sensitivity were evaluated by dot blotting. The present study showed a high titer of polyclonal antibodies in rabbit serum after immunization and the titer increased steadily during the immunization schedule. The highest titer of antibody reached up to 2,560,000 with LOD of 0.1 ng/mL. The purified antibodies showed no cross-reactivity with proteins from other Vibrio species, and the detection threshold ranged from 6.25 to 12.5 ng toxin/dot.
This study highlights the production of high titer and specific polyclonal antibodies as an initial material towards the development of lateral-flow strip test.
急性肝胰腺坏死病（AHPND）由产毒素的副溶血性弧菌菌株引起，这些菌株含有在pVA1质粒中编码的致命二元毒素PirA和PirB。抗PirB蛋白的多克隆抗体可用于开发免疫层析试纸条，用于现场诊断AHPND。
在本研究中，扩增、克隆了PirB基因，并在大肠杆菌中表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测表达的蛋白，并用6xHis抗体进行Western印迹检测。然后，使用Ni-Sepharose柱纯化重组PirB（rPirB）。用纯化的rPirB免疫兔子，并使用双向免疫扩散法分析产生的抗体。分别通过ELISA和亲和层析进行抗体滴定和抗体纯化。最后，通过斑点印迹评估抗体的特异性和敏感性。本研究表明，免疫后兔血清中多克隆抗体滴度较高，且在免疫过程中滴度稳步上升。抗体的最高滴度达到2560000，检测限为0.1 ng/mL。纯化的抗体与其他弧菌属的蛋白无交叉反应，检测阈值为6.25至12.5 ng毒素/点。
本研究强调了产生高滴度和特异性的多克隆抗体作为开发侧向流试纸条检测的初始材料。
