Clonexpress 2 kit

The fiber-1 and different fiber-1 truncations were amplified using corresponding primers listed in Table 1 and cloned into linear pcDNA3.1 using ClonExpressTM II kit (Vazyme, Nanjing, China) to generate pcDNA3.1-Fiber-1 and other fiber-1 truncations. These recombinant plasmids were expressed in LMH cells and the epitopes recognized by the mAbs were identified using IFA. To evaluate the conservation of the identified B cell epitopes in Fiber-1, the sequences of Fiber-1 among different DAdV-3 and other representive DAdV isolates deposited in Genbank were aligned using DNASTAR Megalign software.

Viral RNAs of XZ299 and H9N2 mutant were extracted and cDNA was synthesized as previously described [22 (link)]. The HA (GenBank accession No: MN227199) and NA(Neuraminidase) (GenBank accession No: MN227201) genes from XZ299 and the HA gene from H9N2 mutant were amplified and cloned into the linear influenza vector pDP2002 by the ExnaseTM II, provided by ClonExpressTM II kit (Vazyme Biotech Co., Ltd., Nanjing, China), as previously described [23 (link)]. Two recombinant viruses designated as rgPR8-H9 166N and rgPR8-H9 166D were rescued by transfection in the cocultured 293T and MDCK cells as previously described [23 (link)]. Briefly, 1 μg of the HA plasmid derived from XZ299 and H9N2 mutant respectively, and 1 μg of NA, NP, PB1, PB2, PA, MP, and NS plasmid derived from PR8 (A/Puerto Rico/8/34 (H1N1)) each were first mixed in 250 μL of Opti-MEM medium and then mixed with 16 μL of TransIT^®-LT1 Transfection Reagent (Mirus Bio LLC, Madison, WI, USA). The mixture was incubated at room temperature for 45 min, and then 1 mL of Opti-MEM medium was added. The mixture was then inoculated onto the co-cultured 293T and MDCK cells. After 12 h post-transfection, the medium was changed with 2 mL of fresh Opti-MEM medium with 1μg/mL TPCK-Trypsin. At day 4 post-transfection, the rescued viruses in the supernatant of the transfected cells were collected and titrated in MDCK cells.

The full-length ORFs of OcFLS genes were amplified from their corresponding pEASY-Blunt derived plasmids and then inserted into EcoR I and Hind III sites of pET-28a (+) (Novagen, Madison, USA) using the ClonExpress II kit according to the manufacturer’s instructions (Vazyme, Nanjing, China), respectively. The resultant pET28a-OcFLS2 was transformed into E. coli Transetta (DE3) (TransGen Co. Ltd., Beijing, China) for the recombinant expression. The other plasmid pET28a-OcFLS1 was co-transformed into E. coli BL21(DE3) strain with a chaperone plasmid pGro7 (Takara Biotechnology Co., Ltd., Dalian, China) to improve soluble expression as previously reported [42 , 51 (link)]. These recombinant cells were cultivated until the optical density at 600 nm (OD₆₀₀) reached 0.6 and was induced at 20 °C overnight by the addition of isopropyl-D-thiogalactopyranoside (IPTG) to reach a final concentration of 0.3 mM. The soluble expressions of OcFLS genes in E. coli were verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the bacterially produced OcFLS proteins were purified and quantified as previously described [52 , 53 (link)].