Cells and viruses

The 293T, Vero E6 cells and Baby hamster kidney cell (BHK) cells were purchased from ATCC (ATCC number: CRL-1586) and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 100 U of penicillin, and 100 μg of streptomycin per ml at 37 with 5% CO2. HTNV strain 84Fli, isolated from liver of a fatal fetus in China, were grown in Vero E6 cells as previously described.

Construction of plasmids for DNA vaccination

Plasmids expressing HTNV strain 84Fli N protein (pcDNA3/S) and glycoproteins (Gn and Gc, pcDNA3/M) were constructed previously [22] with pcDNA3 vector (Invitrogen, Karlsruhe, Germany). A plasmid, pCTLA-4-C expressing eCTLA-4-antigen fusion protein, was a kind gift from Prof. Mengji Lu. pCTLA-4-C was constructed on pcDNA3 vector background with antigen fragment inserted downstream of eCTLA-4 between EcoRV and Xhol (BioLabs, USA) restriction sites. The S and M fragments respectively encoding N protein and glycoproteins (Gn and Gc) were amplified by RT-PCR with the following primers containing restriction enzyme sites (EcoRV and Xhol): S forward: 5′-GGA TAT CAT GGC AAC TAT GGA GGA A-3′; S reverse: 5′-GCA CTC GAG TTA TAG TTT TAA AGG CTC TTG GTT GG-3′, M forward: 5′-GGA TAT CAT GGG GGT ATG GAA GTG GCT AGT A-3′; M reverse: 5′-GCA CTC GAG CTA TGA CTT TTT ATG CTT TCT TAC AGG-3′. The amplified fragments of S and M were digested with EcoRV and Xhol and then respectively inserted into the corresponding site of pCTLA-4-C predigested with EcoRV and Xhol to generate pcDNA3/eCTLA4-S and pcDNA3/eCTLA4-M. Insertion of correct nucleotide sequence was verified by sequencing. DNA plasmids were prepared with the Giga plasmid purification kit (QIAGEN, Germany), and then dissolved in phosphate-buffered saline (PBS) in a final concentration of 1 mg/ml.

The S and eCTLA4-S fragments were also further cloned into pET30a vector (Merck, Darmstadt, Germany) respectively to generate pET30a/S and pET30a/eCTLA-S for the identification of expression of HTNV N and eCTLA4-N fusion protein in prokaryotic system induced by isopropyl-beta-D-thiogalactoside (IPTG).

Expression and Identification of eCTLA4-HTNV N and eCTLA4-GP fusion proteins

Prokaryotic expression of HTNV N and eCTLA4-NP fusion protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described previously [23]. 293T and BHK cell lines were used for transfection experiment. Transient transfection was performed by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacture’s instructions. Transient expression of fusion protein, eCTLA4-N and eCTLA4-GP (Gn and Gc) was verified by western-blot or immuno-fluorescence assay (IFA) respectively as described previously.

Immunization of mice with HTNV recombinant DNA plasmid

Female 6-8 weeks old C57BL/6C mice (H-2Kb) were housed in the facility of Chinese Academy of Medical Sciences Breeding Laboratories under specific-pathogen-free conditions. Mice were pretreated by intramuscular injection of 100 μl 0.25% bupivacaine in quadriceps with 50 μl in each side. 24 hours later, groups of mice were injected intramuscularly (i.m.) three times at one week interval with 100 μg of DNA plasmids in the presence or absence of 10 μg of CpG1826 motifs (Sangon Biotech, Shanghai, China). DNA plasmids expressing HTNV N (or eCTLA4-N) and HTNV GP (or eCTLA4-GP) were mixed equally with 50 μg of each. A group of mice was injected i.m. with either 100 μg of pcDNA3 vector or 100 μl PBS alone as a negative control. Sera of 5 mice per group for serological assay were collected before each immunization and one week after third immunization, and for cellular immune response assay were collected one week after second immunization.