KHẢO SÁT ĐIỀU KIỆN NUÔI CẤY BIỂU HIỆN CHUỖI NHẸ ĐỘC TỐ THẦN KINH TYPE HUYẾT THANH B TÁI TỔ HỢP CỦA VI KHUẨN Clostridium botulinum TRONG TẾ BÀO Escherichia coli BL21 (DE3)

Tóm tắt

Botulinum neurotoxin (BoNT) is a protein known to cause poisoning through the gastrointestinal tract. Despite the low incidence rate of the disease, it exhibits a high mortality rate (5-10%) if not promptly diagnosed and treated. Most human poisoning cases are attributed to toxin types A, B, and E (with types A and B being the most toxic), while incidences involving other toxin types are infrequent. The BoNT protein is composed of two chains: the heavy chain (HC, approximately 100 kDa) and the light chain (LC, approximately 50 kDa), with the latter being the toxic domain of BoNT. Research into creating BoNT toxin light chains establishes the scientific basis for developing preventive vaccines and immune diagnostic kits.

To generate the botulinum neurotoxin type B light chain (BoNT/B-LC), a modified gene encoding the light chain from positions 1-450 amino acids was synthesized. This gene was then inserted into the plasmid pET-32a(+) and expressed in Escherichia coli BL21 (DE3). To improve recombinant protein expression productivity, the study optimized the process for expressing recombinant BoNT/B protein at the laboratory flask scale. The E. coli strain BL21 carrying the BoNT/B-LC gene was cultured at 37°C in LB medium with an IPTG concentration of 500 µM and sampled at 12 hours. The optimized process increased recombinant TrxBoNT/B-LC protein content from approximately 268 mg/L to 380 mg/L of culture medium, a 1.7-fold increase compared to the previous optimization.