Noti enzymes

DEFB118 gene was obtained by complete gene synthesis (Beijing Cycle-Tech Biotechnology Co., Ltd., China). The company provides a cloned strain of DH5α-PMD19-DEFB118 containing the target gene DEFB118. And sequencing was identified in Shenggong Biological Engineering Co., Ltd. Sequencing results were compared by software DNAMAN. The plasmid PMD19-DEFB118 was extracted by using Plasmid Mini Kit I (Omega, America) according to the manufacturer's recommendations. The plasmid pMD19-DEFB118 and expression vector pET32a (+) were double-digested with EcoRI and NotI enzymes (Takara, Japan) at 37°C for 4 h. After purification by agarose gel electrophoresis, the isolated DNA fragments were ligated by T4 DNA ligase (Takara, Japan). And then, the ligated product was transformed into E. coli DH5α cells using the heat shock method and plated on LB agar containing kanamycin (50 μg/mL). The positive colonies were randomly picked, then confirmed by restriction enzyme digestion and sequenced by Sangon Biotech (Shanghai, China).

PNKL gene was obtained by complete gene synthesis (Beijing Cycle-Tech Biotechnology Co., Ltd., Beijing, China). The company provides a cloned strain of DH5α-PMD19-PNKL containing the target gene porcine NK-lysin (PNKL). Sequencing identification was. Performed at ShengGong Biological Engineering Co., Ltd. Sequencing results were compared by software DNAMAN. The plasmid PMD19-PNKL was extracted by using Plasmid Mini Kit I (Omega, Norcross, GA, USA) according to the manufacturer’s recommendations. The plasmid pMD19-PNKL and expression vector pET32a (+) were double-digested with EcoRI and NotI Enzymes (Takara, Otsu, Japan) at 37 °C for 4 h. After purification by agarose gel electrophoresis, the isolated DNA fragments were ligated by T4 DNA ligase (Takara, Otsu, Japan). Then, the ligated product was transformed into E. coli DH5α cells using the heat shock method and plated on LB agar containing kanamycin (50 µg/mL). The positive colonies were randomly picked, then confirmed by restriction enzyme digestion and sequenced by Sangon Biotech (Shanghai, China).