Identification and analysis of toxins in novel Bacillus thuringiensis strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera (Lep.: Noctuidae).

Despite the successful application of toxins from Bacillus thuringiensis as biological control agents against pests, pests are showing resistance against an increasing number of Bacillus thuringiensis toxins due to evolution; thus, new toxins with higher toxicity and broad-spectrum activity against insects are being increasingly identified. To find new toxins, whole genome sequencing of the novel B. thuringiensis strain Bt S3076-1 was performed, and ten predicted toxic genes were identified in this study, including six cry genes, two tpp genes, one cyt gene and one vip gene, among which six were novel toxins. Subsequently, SDS‒PAGE analysis showed that the major proteins at the spore maturation stage were approximately 120 kDa, 70 kDa, 67 kDa, 60 kDa and 40 kDa, while active proteins after trypsin digestion (approximately 70 kDa and 40 kDa) exhibited LC50 values of 149.64 µg/g and 441.47 µg/g against Spodoptera frugiperda and Helicoverpa armigera larvae, respectively. Furthermore, pathological observation results showed that the peritrophic membrane of Spodoptera frugiperda and Helicoverpa armigera larvae was degraded. These findings will provide an experimental reference for further research on the insecticidal activity, toxicity spectrum and synergism of these toxins in Bt S3076-1.

[Preparation of monoclonal antibody against recombinant human interferon-ß1a].

OBJECTIVE: To prepare the monoclonal antibody (mAb) against recombinant human interferon-ß1a (rhIFN-ß1a). METHODS: BALB/c mice were immunized with purified rhIFN-ß1a. The spleen lymphocyte cells from the one whose blood specific antibody titer exceeded 1:10 000 were fused with Sp2/0 cells. The fused cells went through HAT selection, serial dilution, and positive selection until stable hybridoma cell lines were obtained. The equilibrium dissociation constant (K(D)) of antibodies was calculated based on ELISA result. The cell line with the best KD value was cultured in a stirred-tank bioreactor with a fed-batch strategy to produce secreted anti-rhIFN-ß1a monoclonal antibody. The mAb released into the cell culture supernatant were purified by ultrafiltration and protein-G affinity chromatography. RESULTS: Thirteen hybridoma cell lines which stably produced anti-rhIFN-ß1a antibody were retrieved from standard hybridoma fusion and selection procedures. One of the 13 cell lines, 1E8 with the KD value of 4.6 × 10⁻9 mol/L was cultured and antibody titer in the cell culture supernatant reached 1:5000. The mAb with 90% purity was recovered from the two-steps purification. CONCLUSION: The anti-rhIFN-ß1a mAb with high purity has been successfully obtained.