Preparation of Recombinant Botulinum Neurotoxin Serotype B Based on Sortase A-Mediated Enzymatic Ligation

ZHAO Mingming; WANG Zhibo; YU Qi; CHAI Chuang; LIANG Haoran; WANG Hanqi; REN Honglin

doi:10.15933/j.cnki.1004-3268.2026.06.013

Abstract：To establish a safe and efficient preparation technology system of botulinum neurotoxin serotype B(BoNT/B),BoNT/B was divided into light chain and heavy chain N-terminal domain(LHN)and heavy chain C-terminal domain(HC)by prokaryotic expression method.The LPETG pentapeptide sequence was introduced into the C-terminus of the LHN protein and the glycine(Gly)was introduced into the N-terminus of the HC protein.The prokaryotic expression vectors of pET-32a-LHN and pET-28a-SUMO-HC were constructed accordingly,respectively.The recombinant LHN and HC proteins were induced and purified.Using the sortase A(SrtA)-mediated transpeptidation reaction,the in vitro covalent coupling of LHN and HC was achieved,and recombinant BoNT/B was prepared.To evaluate the toxicity of recombinant BoNT/B,eighteen female KM mice were randomly divided equally into 6 groups(n=3).Mice in each group were intraperitoneally injected with diluted recombinant BoNT/B at mass concentrations of 200,100,10,1 and 0.1 µg/mL,respectively.Gelatin phosphate buffer was set as the negative control,and the morbidity and mortality of mice were continuously observed within 96 hours.The results showed that following expression,purification,and tag removal,specific protein bands of LHN and HC were observed at approximately 98.67 ku and 52.15 ku,respectively,via sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Under the catalysis of SrtA,the two fragments were successfully covalently ligated to generate recombinant BoNT/B with a molecular weight of approximately 150.82 ku.The toxicity test in mice showed that the recombinant BoNT/B had obvious neurotoxicity.The recombinant BoNT/B with mass concentration≥1 µg/mL could cause typical botulinum poisoning symptoms and death in mice.There was no death in mice in the group with mass concentration<1 µg/mL and the control group.In conclusion,the pET-32a-LHN and pET-28a-SUMO-HC expression vectors were successfully constructed and efficiently expressed in the Escherichia coli prokaryotic expression system.The purified LHN and HC domain proteins were capable of conjugating to form recombinant BoNT/B under SrtA catalysis.The mouse challenge test confirmed that the prepared recombinant BoNT/B exhibited neurotoxicity.