幽门螺杆菌空泡毒素相关蛋白A重组蛋白的构建、克隆及表达（你提供的原文不完整，推测补充了“Helicobacter pylori vacuolating cytotoxin-associated protein A”后的完整内容，以便你参考准确译文）

Construction, Cloning, and Expression of CagA Recombinant Protein of .

作者信息

Shapouri Moghaddam Abbas, Mansouri Shamseddin, Neshani Alireza, Firoozeh Farzaneh, Matinpur Azade, Khaledi Azad, Ghazalibina Mehran

机构信息

Department of Immunology, BuAli Research Institute, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Antimicrobial Resistance Research Center, Department of Microbiology, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran.

出版信息

Avicenna J Med Biotechnol. 2020 Apr-Jun;12(2):135-138.

PMID:32431799

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7229452/

Abstract

BACKGROUND

This study aimed to assess construction and expression of CagA recombinant protein of in BL21.

METHODS

Bioinformatics was used in designing the desired gene by Gene Runner. Next, the construct was subcloned to pET21b vector and this process was confirmed by Polymerase Chain Reaction (PCR), enzyme digestion and sequencing techniques. Then, it was cloned in the Escherichia coli BL21 as an expression host. Expression of protein was verified using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting technique. For purification of the protein, the Ni-NTA column was used. Protein concentration was determined by the Bicinchoninic Acid Protein Assay Kit (Parstoos). Finally, Western blotting was performed using CagA antibodies and normal human serum for determining immunogenicity feature with human antiserum.

RESULTS

According to the results of the present study, CagA construct was cloned into the pET21b vector and after confirmation and cloning in host expression, recombinant protein with the size of 38 was successfully expressed and purified. The recombinant CagA protein showed immunogenicity characteristics with human antiserum.

CONCLUSION

In conclusion, only 5'-end of recombinant protein CagA with high immunogenicity effects was successfully constructed, cloned and expressed. Also, CagA recombinant protein showed good immunogenicity activity with human antiserum.

摘要

背景

本研究旨在评估空肠弯曲菌CagA重组蛋白在大肠杆菌BL21中的构建与表达。

方法

运用生物信息学通过Gene Runner软件设计所需基因。接着，将构建体亚克隆至pET21b载体，此过程通过聚合酶链反应（PCR）、酶切及测序技术得以证实。随后，将其克隆至大肠杆菌BL21作为表达宿主。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹技术验证蛋白表达。为纯化该蛋白，使用镍-亚氨基二乙酸（Ni-NTA）柱。通过二辛可宁酸蛋白测定试剂盒（帕斯托斯）测定蛋白浓度。最后，使用CagA抗体和正常人血清进行蛋白质免疫印迹，以确定该蛋白与人抗血清的免疫原性特征。