Pvax1

The 1,191 bp ROP1 coding sequence (GenBank ID: M71274) was amplified by PCR from genomic DNA of T. gondii tachyzoites using gene-specific forward (5′-CGCGGATCCACGATGGAGCAAAGGCTGCCAA-3′) and reverse (5′-CGGAATTCTTATTGCGATCCATCATCCTGC-3′) primers, introducing Bam HI and Eco R1 restriction sites (as underlined). The PCR product was cloned into the respective restriction sites of pVAX1 (Invitrogen, USA) and pVAX1-GFP vectors. The constructs were verified by sequencing in both directions to ensure fidelity (GenScript Inc., Piscataway, NJ, USA).

Vectors consisted of pEGFP-N1 (BD Biosciences, Franklin Lakes, NJ, USA), pVAX1/rhBMP-2 (Fig 1A) and control vector was pVAX1 (Invitrogen) without insert. The pBMP-2 construct contained the full-length human recombinant BMP-2 cDNA. Plasmid DNA was isolated, purified and cleared from endotoxins (EndoFree Plasmid Maxi kit, Qiagen K.K., Tokyo, Japan).

The complete ROP38 gene sequence of ME49 strain in ToxoDB database (TGME49_242110) was used to design specific primers ROP38U1 (5′-CGGGGTACCATGAAAAATACTCTGTTGTCA-3′) and ROP38D1 (5′- TGCTCTAGATCAAAATTGATGCGTTCTTAT-3′), in which Kpn I and Xba I recognition sites were introduced in both primers and underlined. The ORF of TgROP38 gene was amplified by PCR using genomic DNA (gDNA) from T. gondii RH strain as template. The PCR product was purified by using TIANquick Midi Purification Kit (TIANGEN, Beijing, China). The purified PCR product and vector pVAX I (Invitrogen, Carlsbad, California, USA) were then cleaved by Kpn I and Xba I. The TgROP38 fragment was then inserted into vector pVAX I using T4 ligase enzyme, and formed pVAX-ROP38. The concentration of the recombinant plasmids was determined using a spectrophotometer at OD₂₆₀ and OD₂₈₀. The plasmids were diluted with PBS to a final concentration of 1 μg/μl and stored at -20°C.

p3M2e was synthesized by GenScript Co., Ltd. Each M2e sequence was linked with a GSG4 linker (Gly–Ser–Gly–Gly–Gly–Gly). The eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) was used to construct the DNA vaccine. For p-p3M2e, p3M2e was digested with EcoRI and XhoI and then cloned into pVAX1 to create p-p3M2e. To construct p-tPA-p3M2e, the tPA signal sequence was amplified by PCR using the following primers: 5′-CCCAAGCTTATGGATGCAATGAAGAG AGGGCTCTGCTGTGTGCTGCTGCTG-3′ and 5′-CCGGAATTCGCTGGGCGAA ACGAAGACTGCTCCACACAGCAGCAGCACACAGCAGAG-3′. The PCR product was then digested with HindIII and EcoRI, followed by ligation into p-p3M2e to create p-tPA-p3M2e. The single-copy M2e plasmids (p-tPA-pM2e and p-pM2e) were constructed using similar methods. The plasmids were propagated in Escherichia coli DH5α bacteria and purified using NucleoBond® Xtra (MACHEREY-NAGEL GmbH and Co. KG).
The M2e peptide SLLTEVETPIRNEWGCRCNGSSD was synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (>95% purity).

The method of constructing MTB ag85a/b chimeric DNA was described previously (29 (link)). Briefly, the DNA encoding amino acids 125-282 in the Ag85B protein of MTB H₃₇Rv were amplified by PCR with specific oligonucleotide primers containing the site of endonuclease enzyme Acc I. PCR product was purified and then inserted into the endonuclease enzyme Acc I site of the ag85a gene that was cloned into eukaryotic expression vector pVAX1 (Invitrogen Life Technologies Corporation, Carlsbad, CA, USA). The length of gene sequence encoding for strong immunogen Ag85A was 888 bp, and the Ag85B was 474 bp. MTB ag85a/b chimeric DNA vaccine was thus constructed by inserting the sequence encoding amino acids 125-282 of Ag85B protein into nucleotide 430-435 (endonuclease enzyme Acc I site) of Ag85A DNA vaccine. In this study, the MTB ag85a/b chimeric DNA was produced and purified by Guangzhou Baiyunhan Baidi Biological Medicine Co.Ltd (Guangdong province, China).

Eukaryotic expression plasmid pVAX1 was purchased from Invitrogen (USA). The full length ORF2 gene of PCV2 HeB-1 strain was amplified by using the primers 5′-ATCGCTAGCGCCGCCACCATGACGTATCCAA-3′ and 5′-CCCAAGCTTTCACTTAGGGTTAAGT-3′, cut with Nhe I/Hind III and ligated into pVAX 1 yielding pVAX1-ORF2. The ORF2-C3d-P28.3 fusion protein was designed by cloning three tandem repeats of the porcine homologue of C3d-P28 (HM026945.1) in frame at the 3′ end of the ORF2 gene. Linkers composed of two repeats of four glycines and a serine [(G4S)2] were fused at the junctures of ORF2 and C3d-P28 and between each C3d-P28 repeat. The ORF2-C3d-P28.3 gene with Nhe I/Hind III in 5′ and 3′ ends was synthesized commercially (Takara, Japan) and was ligated into pVAX1 yielding pVAX1-ORF2-C3d-P28.3.
Escherichia coli strain DH5a was used as the host for all plasmids. Plasmids were purified from cultures of E. coli using an EndoFree Plasmid Mega kit (QIAGEN). Plasmids were verified by appropriate restriction enzyme digestion and gel electrophoresis. The purity of DNA preparations was verified by optical density reading at 260 and 280 nm.