比较在台式生物反应器中使用五种大肠杆菌菌株以获得由针对 VEGF 的抗体与 MAP30 毒素偶联而成的重组免疫毒素的最大产量。

Comparison of Five Escherichia coli Strains to Achieve the Maximum Yield of a Recombinant Immunotoxin Consisting of an Antibody against VEGF Conjugated with MAP30 Toxin in a Benchtop Bioreactor.

机构信息

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71468-64685, Shiraz, Iran.

Depatment of Microbiology, Faculty of Basic Sciences, Jahrom Branch, Islamic Azad University, Jahrom, Iran.

出版信息

Recent Pat Biotechnol. 2024;18(1):52-62. doi: 10.2174/1872208317666230316111554.

DOI:10.2174/1872208317666230316111554

PMID:38205774

Abstract

BACKGROUND

Cancer is among the leading causes of death worldwide, imposing high costs on the health systems of all societies. Extensive biological studies are required to discover appropriate therapies. has long been regarded as one of the main biotechnological bio-factories to produce recombinant protein-based therapeutics. In the present study, five strains of were compared to achieve the maximum production of a previously designed recombinant immunotoxin-carrying MAP30 toxin against VEGF-overexpressed cancer cells in a benchtop bioreactor.

METHODS

The recombinant immunotoxin coding gene sequence was extracted from the NCBI database. The host used to produce the recombinant immunotoxin were five strains of BL21 (DE3), DH5α, SHuffleT7, XL1-Blue, and Rosetta-gami (DE3). CaCl method was used for bacterial transformation. Bacterial growth measurements were performed using optical density measurements at 600 nm. The immunotoxin production was measured using SDS-PAGE analysis. The best-producing strain was cultivated in a 10-L benchtop stirred tank bioreactor. Recent patents on this field were also studied.

RESULTS

The results demonstrated that the BL21 (DE3) strain had the highest expression of recombinant protein in comparison to other strains. Moreover, the cell growth of BL21 (DE3) and SHuffleT7 strains before transformation in the LB medium, were significantly higher in comparison to other strains. Additionally, the transformation of Rosettagami was associated with decreased cell proliferation. The transformation of the XL1-Blue strain did not effect cell growth. Analysis of the growth kinetics demonstrated appropriate proliferation of the transformed BL21 (DE3) cells in the laboratory benchtop bioreactor.

CONCLUSIONS

Based on the results of this study, the BL21 (DE3) strain could be used as a suitable host for the production of the recombinant immunotoxin against VEGF in stirred tank bioreactor, which can be employed for the treatment of tumors. Yet, its precise mechanism must be explored in extensive studies.

摘要

背景

癌症是全球主要死因之一，给所有社会的卫生系统带来了巨大的负担。为了发现合适的治疗方法，需要进行广泛的生物学研究。一直被认为是生产基于重组蛋白的治疗性生物制药的主要生物技术生物工厂之一。在本研究中，比较了五种菌株，以在台式生物反应器中达到对 VEGF 过表达癌细胞的先前设计的携带 MAP30 毒素的重组免疫毒素的最大产量。

方法

从 NCBI 数据库中提取重组免疫毒素编码基因序列。用于生产重组免疫毒素的宿主是 BL21（DE3）、DH5α、ShuffleT7、XL1-Blue 和 Rosetta-gami（DE3）的五种菌株。采用氯化钙法进行细菌转化。通过在 600nm 处测量光密度来测量细菌生长。使用 SDS-PAGE 分析测量免疫毒素的产生。在 10L 台式搅拌罐生物反应器中培养最佳生产菌株。还研究了该领域的最新专利。

结果

结果表明，与其他菌株相比，BL21（DE3）菌株的重组蛋白表达量最高。此外，LB 培养基中 BL21（DE3）和 ShuffleT7 菌株在转化前的细胞生长明显高于其他菌株。此外，Rosettagami 的转化与细胞增殖减少有关。XL1-Blue 菌株的转化对细胞生长没有影响。生长动力学分析表明，转化后的 BL21（DE3）细胞在实验室台式生物反应器中能够适当增殖。