Viccillin

The plasmids encoding each ribosomal protein were purified using the ASKA library [36 (link)]. Then, the plasmids were introduced into E. coli BL21 (DE3) lacZ::kmr. The cells were grown at 37 °C to OD₆₀₀ = 0.5–0.6 in 5 mL LB medium containing 100 μg/ml ampicillin (Viccillin^® for injection, Meiji Seika Pharma), incubated at 37 °C for 3 h with 0.1 or 1 mM IPTG (Nacalai tesque), and then centrifuged at 3,000 rpm for 5 min. Then, the pellets of the cells were suspended in 200 μL of lysis buffer of 2 M urea, 50 mM ammonium bicarbonate, and with a pH of 8.0 and transferred into 1.5 mL microtubes in an ice-water bath. The cells were disrupted using a sonicator (BIORUPTOR UCD-250, Cosmo Bio, Tokyo, Japan) for 10 min at 4 °C (Level 5, a 30-s sonication with 30-s interval). Then, the lysates were centrifuged at 14,000 g for 10 min at 4 °C, and the supernatants were collected. The ribosomal proteins were quantified using the LCMS-8060 (Shimadzu, Kyoto, Japan) as described below.

Animals were pre-anesthetized with an intramuscular injection of 0.04 mg/kg of medetomidine (Domitor; Nippon Zenyaku Kogyo, Koriyama, Japan), 0.40 mg/kg of midazolam (Dormicam 10 mg; Astellas Pharma, Tokyo, Japan), and 0.40 mg/kg of butorphanol (Vetorphale; Meiji Seika Pharma, Tokyo, Japan). They were also administered 15 mg/kg ampicillin (Viccillin; Meiji Seika Pharma, Co., Ltd.) and hydrated subcutaneously with 2 mL/head of fluid (KN No.1 injection; Otsuka Pharmaceutical, Tokyo, Japan). Thereafter, animals were anesthetized by inhalation with 1.0–3.0% isoflurane (Forane; Abbott Japan, Tokyo, Japan) via a ventilation mask. Anesthetization management was performed by spontaneous respiration during the operation, monitoring the heart rate and the arterial oxygen saturation. After oocyte collection or embryo transfer, 0.20 mg/kg atipamezole (Antisedan; Nippon Zenyaku Kogyo) was administered intramuscularly into the animals. For postoperative analgesia and infection control, 1.2 mg/kg ketprofen and 15 mg/kg ampicillin were administered once daily for 3 consecutive days following the operations.