基于合成基因重组蛋白的 B 型肉毒神经毒素免疫检测系统的研制。

Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein.

机构信息

Biotechnology Division, Defence Research Development & Establishment, Gwalior, India.

出版信息

Indian J Med Res. 2011 Jul;134(1):33-9.

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3171914/

Abstract

BACKGROUND & OBJECTIVES: Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridium botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach.

METHODS

The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated.

RESULTS

The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2,048,000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes.

INTERPRETATION & CONCLUSIONS: There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples.

摘要

背景与目的

肉毒神经毒素（A-G）是世界上毒性最强的物质之一，由专性厌氧细菌肉毒梭菌产生。在这七种血清型中，A、B、E 和 F 对人类具有重要意义。在印度，已经报道了肉毒梭菌的流行情况以及肉毒中毒的爆发情况。由于其极高的毒性，它被归类为生物战剂 A 类。到目前为止，印度还没有商业检测系统来检测肉毒中毒。本研究旨在使用合成基因方法开发针对肉毒神经毒素血清型 B 的免疫检测系统。

方法

使用 PCR 重叠引物合成肉毒神经毒素 B 型的截断片段，从氨基酸 1-450 开始；构建的基因克隆到 pQE30UA 载体中，并转化到大肠杆菌 SG13009 中。使用不同浓度的异丙基硫代半乳糖苷（IPTG）诱导优化重组蛋白的表达，然后使用抗 His 抗体进行 Western blot 分析确认表达。在变性条件下使用 Ni-NTA 亲和层析纯化重组蛋白。使用明矾佐剂在 BALB/c 小鼠中产生针对重组蛋白的抗体，并测试其与其他肉毒梭菌血清型以及密切相关的梭菌的交叉反应性。开发了一种用于检测肉毒神经毒素的 ELISA 试验，并估计了最低检测限。

结果

在 IPTG 浓度为 0.5 mM 时，诱导后 4.3 小时达到最大产量表达出重组蛋白。使用 Ni-NTA 亲和层析将重组蛋白纯化至均一水平。用 1:2048000 的效价在小鼠中产生了多克隆抗体。开发的抗体具有高度特异性，检测重组蛋白的灵敏度约为 15 ng/ml，并且与其他血清型没有交叉反应性。