Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection

Muhammad, Azharuddin; Toufeeq, Shahzad; Yu, Hai-Zhong; Wang, Jie; Zhang, Shang-Zhi; Li, Bing; Li, Zhen; Yang, Li-Ang; Hu, Pei; Ma, Yan; Xu, Jia-Ping

Primers with restricted enzymes sites of EcoR I and Xho l were designed, the purpose of which was the amplification of Bmp38 and BmS6K domains for the recombinant protein expressions (Table 1) . Using the pMD 19T vector, the PCR products (purified) were cloned as per previous protocol (Yu et al. 2017a ). Positive colonies were randomly selected for sequencing DNA and for the confirmation of amplified sequence. Next, the plasmids were extracted, digested, purified, and ligated into the pET-28a vector (Novagen). The resulting recombinant plasmids pET-28a-Bmp38 and BmS6K were confirmed by DNA sequencing and then transformed into Escherichia coli BL21 (DE3; Novagen) competent cells. After this activity, addition of isopropyl β-D-thiogalactoside was done and kept in an incubator for time period of 4 h at 37°C. The cells were harvested by centrifugation at 5,800 × g for 10 min. For cell pellets suspension, binding buffer (20 mM tris-HCl, 500 mM NaCl, 5 mM imidazole at pH 7.9) was used. This activity was then disrupted by sonication on ice. The procedure for recombinant proteins purification was used after centrifugation at 12,000 × g for time period of 20 min at temperature of 4°C by using a Ni-NTA Fast Start Kit (Qiagen, Inc., Valencia, CA) as per protocol of manufacturer. The quality and identity of purified proteins was evaluated. For this purpose, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed by using anti-His primary antibody (Transgene Biotech, Beijing, China), and antigenantibody complexes were detected with a horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Transgene Biotech; 1:5,000 dilution).