Rani Sarita, Ponmariappan S, Sharma Arti, Kamboj D V, Jain A K
Biotechnology Division, Defence Research Development & Establishment, Jhansi Road, Gwalior- 474 002, M.P., India.
2SOS in Zoology, Jiwaji University, Gwalior - 474 002, M.P., India.
Protein Pept Lett. 2017;24(6):495-502. doi: 10.2174/0929866524666170301115727.
Clostridium botulinum is an obligate anaerobic, Gram positive bacterium that secretes extremely toxic substances known as botulinum neurotoxins (BoNTs) that cause serious paralytic illness called botulism. Based upon the serological properties, these neurotoxin have been classified into seven serotypes designated from A to G. Due to extreme toxicity of BoNTs, these neurotoxins have been designated as category A biowarfare agents. There is no commercial neutralizing antibody available for the treatment of botulism. Hence there is an urgent need to develop therapeutic intervention for prevention and cure of botulism within short period. BoNT antiserum injection is still the effective treatment.
In the present study, the recombinant light chain of BoNT/E was successfully purified in soluble form. The purified rBoNT/E LC was used for the generation of polyclonal antibody in rabbit. In order to find out the neutralizing capacity of generated antisera, rabbit antiserum was incubated with 20 LD50 of botulinum neurotoxin type E for 1 hour at 37°C and then injected intraperitoneally (IP) into mice. Further in another set of experiments antiserum was administered in different ways that included administration of - antiserum and BoNT/E toxin simultaneously without preincubation, one after another at the same and different time points for its therapeutic ability. To find out cross neutralization capacity, rBoNT/E LC antiserum was pre-incubated with 5 LD50 of BoNT/A, BoNT/B, BoNT/F and then injected (IP) into mice. In all the cases mice were observed continuously for 96 hours.
The results clearly indicate that developed polyclonal rabbit antiserum showed serotype specific neutralization of BoNT/E toxin only but not of BoNT/A, BoNT/B and BoNT/F.
The developed antibodies will be used for preventive and therapeutic intervention of type 'E' botulism.
肉毒梭菌是一种专性厌氧的革兰氏阳性菌，可分泌极具毒性的物质，即肉毒神经毒素（BoNTs），这些毒素会引发一种严重的麻痹性疾病——肉毒中毒。根据血清学特性，这些神经毒素已被分为七种血清型，分别命名为A至G。由于肉毒神经毒素具有极高的毒性，它们被指定为A类生物战剂。目前尚无用于治疗肉毒中毒的商业化中和抗体。因此，迫切需要在短时间内开发出预防和治疗肉毒中毒的治疗性干预措施。肉毒神经毒素抗血清注射仍是有效的治疗方法。
在本研究中，成功以可溶性形式纯化了肉毒神经毒素E型的重组轻链。纯化的重组肉毒神经毒素E型轻链用于在兔体内产生多克隆抗体。为了确定所产生抗血清的中和能力，将兔抗血清与20个半数致死剂量（LD50）的E型肉毒神经毒素在37°C下孵育1小时，然后腹腔注射到小鼠体内。在另一组实验中，以不同方式给予抗血清，包括在不预先孵育的情况下同时给予抗血清和肉毒神经毒素E型，以及在相同和不同时间点先后给予，以研究其治疗能力。为了确定交叉中和能力，将重组肉毒神经毒素E型轻链抗血清与5个LD50的A、B、F型肉毒神经毒素预先孵育，然后腹腔注射到小鼠体内。在所有情况下，持续观察小鼠96小时。
结果清楚地表明，所产生的兔多克隆抗血清仅对E型肉毒神经毒素表现出血清型特异性中和作用，而对A、B、F型肉毒神经毒素无中和作用。
所开发的抗体将用于E型肉毒中毒的预防和治疗干预。
