中华蜜蜂Apidaecin的重组表达及其抗菌活性

doi:10.3864/j.issn.0578-1752.2019.04.016

Abstract

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

CHEN WenFeng,WANG HongFang,LIU ZhenGuo,ZHANG WeiXing,CHI XuePeng,XU BaoHua. Recombinant Expression and Antimicrobial Activity of Apidaecin in Apis cerana cerana[J].Scientia Agricultura Sinica, 2019, 52(4): 767-776.

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References 34

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基因名称 Gene name	引物序列 Primer sequence (5′-3′)	引物用途 Description	登录号 GenBank accession number
API-F	TGTGGGTTGAATAACTATTGATAA	cDNA sequence primer, forward	NM_001011613.1
API-R	GTTATTTCACGTGCTTCATATTC	cDNA sequence primer, reverse	NM_001011613.1
GAPDH-F	GGTTGTCTCCTGCGACTTCA	Standard control primer, forward	NM_001289726.1
GAPDH-R	TGGTCCAGGGTTTCTTACTCC	Standard control primer, reverse	NM_001289726.1
IL10-F	ACTGCTATGCTGCCTGCTCT	Real-time PCR primer, forward	NM_010548.2
IL10-R	GACTGGGAAGTGGGTGCAGT	Real-time PCR primer, reverse	NM_010548.2
TNF-α-F	CCAGCCGATGGGTTGTACCT	Real-time PCR primer, forward	NM_001278601.1
TNF-α-R	CAAATCGGCTGACGGTGTGG	Real-time PCR primer, reverse	NM_001278601.1
IL6-F	TGGGACTGATGCTGGTGACA	Real-time PCR primer, forward	NM_031168.2
IL6-R	ACAGGTCTGTTGGGAGTGGT	Real-time PCR primer, reverse	NM_031168.2
ZO-2-F	CCACCTCGCACGCATCACAG	Real-time PCR primer, forward	XM_017318137.1
ZO-2-R	TGGTCCTTCACCTCTGAGCACTAC	Real-time PCR primer, reverse	XM_017318137.1
Claudin-1-F	TCGGCTCCATCGTCAGCACTG	Real-time PCR primer, forward	NM_016674.4
Claudin-1-R	AGATGGCCTGAGCGGTCACG	Real-time PCR primer, reverse	NM_016674.4
IFN-γ-F	GGCTCTGGAGGCTGGAGGAAG	Real-time PCR primer, forward	NM_008337.4
IFN-γ-R	TGATAGGCGGTGAGGCTACAAGG	Real-time PCR primer, reverse	NM_008337.4

分组 Group	步骤1 Step 1 （第1—6天腹腔注射 Intraperitoneal injection at 1st-6th days）	步骤2 Step 2 （第6天腹腔注射后1 h再注射 Repeated intraperitoneal injection was performed 1 h after the first injection at 6th day）
A：正常对照组 Normal control group	0.5 mL生理盐水0.5 mL Saline	0.5 mL生理盐水0.5 mL Saline
B：pHT43空载体 pHT43 Empty carrier	0.5 mL pHT43	0.5 mL生理盐水 0.5 mL Saline
C：染菌对照组Infection control group	0.5 mL生理盐水0.5 mL Saline	0.5 mL菌悬液0.5 mL Bacteria suspension
D：pHT43空载体染菌组 pHT43 Empty carrier infection group	0.5 mL pHT43	0.5 mL菌悬液0.5 mL Bacteria suspension
E：20 mg·L^-1重组Apidaecin染菌组 20 mg·L^-1 Recombinant Apidaecin infection group	0.5 mL重组Apidaecin 0.5 mL Recombinant Apidaecin	0.5mL菌悬液0.5 mL Bacteria suspension
F：150 mg·L^-1头孢噻肟染菌组 150 mg·L^-1 Cefotaxime infection group	0.5 mL头孢噻肟溶液 0.5 mL Cefotaxime solution	0.5 mL菌悬液0.5 mL Bacteria suspension

Recombinant Expression and Antimicrobial Activity of Apidaecin in Apis cerana cerana

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