Pgex 4t 1 vector

For cell surface binding assays, an N-terminal fragment of CpTIPH containing ITGA and VCBS domains (amino acids 57 to 503) was expressed as a recombinant GST-fusion protein (marked as rCpTIPH-NF in Figure 1). Corresponding DNA fragment (1341-bp) was amplified by PCR from total genomic DNA isolated from C. parvum oocysts using a TIANamp Stool DNA Kit (TIANGEN, Beijing, China) and primers 5′-GGA TCC TTT AAT TTC CCT GA-3′ and 5′-GAA TTC ATT GTT TTT CTG AGA ATG CT-3′ (restriction sites underlined). Thermal cycling used the following conditions: 95 °C for 5 min, followed by 35 cycles at 94 °C for 45 s, 55 °C for 45 s and 72 °C for 90 s, and a final extension at 72 °C for 10 min.
The PCR products were purified using a Gel/PCR Extraction Kit (BIOMIGA, San Diego, CA, USA) and cloned into a pGEX-4T-1 vector (Invitrogen) for expression as a glutathione-S-transferase (GST)-fusion protein in the BL21(DE3) strain of Escherichia coli (TIANGEN, Beijing, China). Recombinant protein was purified by an affinity chromatography using glutathione-sepharose 4B following manufacturer’s protocol (GE Healthcare, Stockholm, Sweden). The purity and molecular weight were evaluated by SDS-PAGE, followed by Coomassie Blue staining. This GST-fusion protein containing 447 aa N-terminal fragment without signal peptide was designated as GST-CpTIPH-NF.

Primers were designed according to the sequences of the tle1^AH and vgrG genes of A. hydrophila NJ-35 in GenBank (accession number NZ_CP006870). Tle1^AH was cloned into the pGEX-4T-1 vector (Invitrogen) for expression with an N-terminal glutathione-S-transferase (GST) tag. VgrG/hcp was cloned into the pET-28a vector (Invitrogen) with a His tag. The GST-Tle1^AH and His-VgrG/Hcp proteins were expressed in BL21 (DE3) cells (CWBIO, Beijing, China). The transformed cells were cultured in LB medium at 37 °C to an OD₆₀₀ of 0.8, at which time the fusion protein expression was at the highest level and most was expressed as soluble protein not as an inclusion body based on our preliminary experiment. Protein expression was induced with 100 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 °C for 20 h. The cultures were harvested by centrifugation at 8000 × g, resuspended in 1 × PBS and lysed by sonication. The lysate was centrifuged at 12 000 × g for 15 min at 4 °C to remove precipitate, and then the supernatant was loaded on a Ni²⁺-NTA column (GE Healthcare, Shanghai, China) to purify the proteins. The eluted proteins were collected and dialyzed for pull-down assays.

The PPRV N protein cDNA (GenBank accession no. HQ197753.1) was cloned into the mammalian expression vector p3 ×Flag-CMV10 vector (Sigma) with the EcoRI and KpnI restriction enzymes to generate the p3 ×Flag-N plasmid. The pHA-NCL plasmid encoding the NCL protein with a HA tag was obtained by cloning NCL cDNA (GenBank accession no.XM_012163051.2) into a pCMV-HA vector (Clontech). For prokaryotic expression of the glutathione S-transferase (GST)-tagged N protein, DNA encoding the N protein was subcloned into the pGEX-4T-1 vector (Invitrogen) with the EcoRI and NotI restriction endonucleases. Truncated mutants of NCL and PPRV N were generated from pHA-NCL and p3 ×Flag-N by conventional PCR (the primers will be made available upon request). All constructs were confirmed by sequencing (Sangon Biotech).