Neomycin sulfate

RB-Rev, variant RB-Rev, and HIV-1 RRE RNA were custom-synthesized and HPLC-purified by Sangon Biotech Co., Ltd. (Shanghai, China). ICR 191 dihydrochloride was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA), while neomycin sulfate was purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). Proflavin and arginine were purchased from Macklin Biotechnology Co., Ltd. (Shanghai, China). Additionally, RB was purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China). Tryptophan was obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). The remaining reagents were all of the analytical grade, commercially available, and employed without further purification.
RNase-free tubes and tips were purchased from Thermo Science & Technologies Co., Ltd. (Waltham, MA, USA), and diethylpyrocarbonate (DEPC) water was obtained from Phygene Biotechnology Co., Ltd. (Fuzhou, China). The RNA solutions were prepared using DEPC water to prevent nuclease contamination.

To prove intestinal flora regulation on mucosal structure, 12 D280 ZN Hens were divided into two groups, one control and one multiple‐antibiotic feeding group. In the control group, hens were fed with a basal diet and free access to water. However, in the multiple‐antibiotics group, hens were supplied with water containing Ampicillin Sodium (1 g l⁻¹ water, MB1378; Meilunbio, Dalian, China), Neomycin Sulfate (1 g l⁻¹ water, MB1716; Meilunbio), Metronidazole (1 g l⁻¹ water, MB2200; Meilunbio, Dalian, China) and Vancomycin HCl (0.5 g l⁻¹ water, MB1260; Meilunbio) for five consecutive days. On the sixth day, all the hens were killed after anaesthesia. The gut lumen content was then collected sterilely, further used for Colony‐Forming Units (CFUs) counting and 16S rRNA gene sequencing. Meanwhile, the intestinal tissue and intestinal crypt were collected for morphology, mRNA transcription and protein expression assays.