关键词: 中华蜜蜂, 抗菌肽, 重组表达, 大肠杆菌, 抗菌活性

Abstract:

【Objective】In order to study the recombinant expression of antimicrobial peptide Apidaecin of Apis cerana cerana in Bacillus subtilis, verify whether the purified recombinant antimicrobial peptide Apidaecin has antibacterial activity in vivo and in vitro or not, and to provide a theoretical basis for the development of new antimicrobial peptide preparations with safe antibacterial and immunomodulatory functions.【Method】The experiment used Apidaecin in Apis mellifera ligustica as templates to clone the antimicrobial peptide Apidaecin in A. c. cerana, and his-pHT43/Apidaecin expression vector was successfully constructed. In addition, the competent cells were successfully prepared according to the method provided by MoBiTec. The cells were prepared and used on demand to ensure the activity. Protein was isolated and purified using 6×His-Tagged Protein Purification Kit (soluble protein), and the Easy II Protein Quantitative Kit (BCA) was used for concentration determination. The purified recombinant antimicrobial peptide Apidaecin acted on Escherichia coli K88. The disk diffusion test and minimum inhibitory concentration (MIC) were conducted in vitro. In vivo, mice were used as experimental animal models, mice in the experimental group were injected with recombinant antibacterial peptide Apidaecin by intraperitoneal injection, mice in the negative control group were injected with normal saline of the same volume, and the positive control group was injected with the same volume of cefotaxime, then artificially infected with E. coli K88. After 24 hours of infection, the mice were dissected, intestinal samples and blood samples were obtained, and the protective effect of Apidaecin on the mice infected with E. coli K88 was discussed from the perspective of intestinal barrier and immune function. Serum immunoglobulin (IgA, IgG, IgM) and intestinal sIgA levels were determined by using ELISA Kit in the experiment. In addition, the expression levels of intestinal tightly-connected protein genes (claudin-1, ZO-2) and intestinal cytokines (proinflammatory cytokines TNF-α, IFN-γ, IL6 and anti-inflammatory factor IL10) were determined by qRT-PCR. 【Result】The Apidaecin in A. c. cerana was cloned, containing 183 bp, encoding 60 amino acids, including signal peptide sequence of Apidaecin, a basic RR dipeptide and amino acid sequence of Apidaecin (containing 8 highly conserved amino acid sequences). The protein encoded is named AccApidaecin, its molecular weight is 15.6 kD and isoelectric point is 5.33. About 20 mg antimicrobial peptides could be purified from 1 L supernatant at the concentration of IPTG was 3 mmol·L ^-1, induction temperature was 30℃, and inducing time was 12 h. The recombinant antibacterial peptide Apidaecin showed obvious antibacterial ring in vitro compared with the negative control, and the measured MIC was 10 mg·L ^-1. In mice, there were significant differences in immunoglobulin between the bacterial test group injected with recombinant antimicrobial peptide Apidaecin by intraperitoneal injection and the bacterial test group injected with saline (P<0.05), which indicated that recombinant Apidaecin could effectively alleviate the increase of immunoglobulin content in mice caused by E.coli K88. The gene expression level of intestinal related proteins in mice was also significantly different (P<0.05), which indicated that the recombinant Apidaecin could effectively protect the intestinal tract of mice infected with E. coli K88.【Conclusion】The antimicrobial peptide Apidaecin in A. c. cerana was successfully expressed in B. subtilis. The purified antibacterial peptide Apidaecin in A. c. cerana has a good antibacterial effect on E. coli K88 in vitro. In addition, it can be injected into mice through abdominal cavity to improve the immune function of mice and effectively resist the infection of E. coli K88 to mice.

Key words: Apis cerana cerana, antimicrobial peptide, recombinant expression, Escherichia coli, antimicrobial activity