Cloning, expression and monoclonal antibody of porcine interferon-regulated antiviral gene

Background IRAV (interferon-regulated antiviral gene) was identified with antiviral activity as a novel interferon-stimulated gene. IRAV is upregulated in response to type I and type II IFNs and a number of virus. However, the antiviral activity of IRAV to virus infection is poorly understood. Methods To obtain the antibody against IRAV, we cloned the full-length IRAV complementary DNA (cDNA) from porcine kidney cells firstly. And then, the porcine IRAV protein was expressed in Escherichia coli BL21 (DE3), purified and immunized to female BALB/c mice. Finally, we determined the specificity of the MAbs by indirect ELISA, Western blotting and IFA. Results Five strains of hybridoma cells named 2B10, 2G12, 2H1,5A8 and 2C5 secreting anti-IRAV MAbs were obtained. By western blot analysis and indirect immunofluorescence assay, the MAbs were identified with the specific reaction with the overexpressed porcine IRAV protein in PK15 cells. Conclusions The MAbs against porcine IRAV, identified by western blot and IFA, provid a valuable tool to study the biological function of IRAV in the future.


Cloning and sequence analysis of porcine IRAV
Total RNA of swine was isolated from porcine spleen tissue using the TRIzol (Invitrogen). The SuperScript III Reverse Transcriptase was used to synthesize cDNA fragments from extracted total RNA with oligo(dT) primer according to the manufacturer's instructions. Based on the porcine mRNA sequence (GenBank ID nos. NM_001244321), the primers (Table 1) were designed to clone the partial IRAV gene by reverse transcriptase polymerase chain reaction (RT-PCR). Rapid amplification of cDNA ends (RACE) was also performed using porcine IRAV-specific primers (Table 1)  The open reading frame (ORF) of the porcine IRAV was analyzed by Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/) on NCBI. Phylogenetic tree based on the predicted amino acid sequence of IRAV was constructed in the MEGA 4 program with 1,000 bootstrap replications using the Neighbor-joining method and the P distance algorithm of correction.

Expression vector construction and subcellular localization
The ORF of porcine IRAV was amplified from the cDNAs obtained from the porcine spleen tissue using the primers designed based on the eukaryotic expression vector p3×FLAG CMV 7.1 (Terminal FLAG tag; Sigma-Aldrich) to produce the pFLAG-pIRAV (Table 1), and the prokaryotic expression vector pCold-I to produce the p-Cold-pIRAV (Table 1).
PK-15 cells were plated in 6-well culture plates and transfected at 70-80% confluency with pFLAG-pIRAV in Gibco OPti-MEM cell culture medium (Life Technologies) using FuGENE HD transfection reagent (Promega) according to the manufacturer's instructions. After 48 h transfection, the cells were washed and fixed with paraformaldehyde. After staining with DAPI, the cells were observed under a confocal immunofluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Purification of rpIRAV protein and immunization procedure of mice
The recombinant plasmid p-Cold-pIRAV was expressed in Escherichia coli BL21 (DE3) at 16℃ for 16 hours by the addition of 0.5 mM of isopropyl-β-D-thiogalactoside (IPTG). The induced rpIRAV protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The recombinant protein was purified by Nickel Magnetic Beads (Biotool, Shanghai, China) after centrifugation and ultrasonication.
With the equal amount of Freund's complete adjuvant, the purified rpIRAV protein was mixed and emulsified to immune 6-week-old female BALB/c mice through subcutaneous injection. After 2 weeks, each mouse was subcutaneously injected using the same dose of antigen emulsified in Freund's incomplete adjuvant at a 1:1 (v/v) ratio. The immunization was repeated twice at 2-week intervals.
Before cell fusion, booster immunization was given 3-4 days in advance. After that, mice were euthanized by cervical dislocation and their spleen were removed aseptically based on animal welfare law of China.

Indirect enzyme-linked immunosorbent assay
Indirect ELISA was used to choose the serum and cell culture containing the highest titer of anti-rpIRAV antibodies. The ELISA Plates were plated with 200 ng/well rpIRAV protein diluted with 100 µL carbonate bicarbonate buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 [pH 9.6]) and coated at 4℃ overnight.
Then the plates were blocked with 5% skimmed milk in phosphate buffer with 0.05% Tween-20 (PBST) at 37℃ for 1 hour. Followed by washing for three times, the plates were incubated at 37℃ containing 100 µL diluted cell culture supernatant or antibodies. An hour later, the plates were incubated with HRP-conjugated goat anti-mouse IgG with 1:20,000 dilution in PBST at 37℃ for 1 hour after washing thrice with PBST. And then, away from light, the plates were incubated with 100 µL/well of TMB liquid for 15 minutes at room temperature. Being stopped by 50 µL/well 2 M H 2 SO 4 , these plates were read at OD450 value to screen the positive hybridoma cells compared with negative control coating with His-tag protein.

Preparation of anti-rpIRAV protein-specific monoclonal antibody
By indirect ELISA, we determined the mice whose serum contains the highest titer of anti-rpIRAV antibodies. Their spleen cells were fused with SP2/0 myeloma cells under the action of 50% PEG as fusion agents. The hybridoma cells were cultured in hypoxanthine-aminopterin-thymidine (HAT) screening culture medium with 20% FBS in 96-well plates at 37℃ in a humidified 5% CO 2 incubator.
When the cells covered between a third and a half of the bottoms of 96-well plates, we used indirect ELISA to filter the positive hybridomas. The positive hybridoma cells were injected into pristinetreated BALB/c mice after cloning four times by limiting dilution to generate abundant ascetic fluid containing the MAb.

Western blot analysis
Western blot was used to confirm the specificity of the MAbs (18). The monoclonal antibody cell lysate was transferred to the nitrocellulose (NC) membrane after collection and separation through 10% SDS-PAGE. The membrane was blocked for 1 hour at room temperature with 5% skimmed milk on a shaking table in TBST (TBS with 0.1% Polysorbate-20). And then, the NC membrane was incubated with the anti-FLAG antibody, anti-β-actin antibody and anti-IRAV MAbs for 1 hour at room temperature and washed for three times with TBST. After that, the HRP-conjugated goat anti-mouse IgG (1:6000 dilution in TBST) was added on the NC membrane for 1 hour at room temperature. Washing the NC membrane as the method above, we developed color using the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA)

Indirect immunofluorescence assay
Immunofluorescence assays (IFAs) were performed as described previously (19). PK-15 cells were plated in a six-well plate. After 80%confluency, the cells were fixed by paraformaldehyde for 30 minutes at room temperature. Then washing by PBS for three times, cells were blocked with 10%

Subcellular localization
The ORF of porcine IRAV was amplified from the cDNAs, and cloned into p3 × FLAG CMV 7.1 vector to produce the pFLAG-pIRAV. PK-15 cells in 6-well culture plates was transfected at 70-80% confluency with the pFLAG-pIRAV using FuGENE HD transfection reagent (Promega). After 48 h transfection, the cells were washed and fixed with paraformaldehyde, followed by incubating with Anti-FLAG antibody and secondary antibody (Green). Cell nucleus were stained by DAPI (Blue) (20; 21). The fluorescence signals were visualized by confocal immunofluorescence microscopy. IRAV fusion proteins were detected to be distributed predominantly in the cytoplasm of the PK-15 cells (Fig. 2).

Expression and purification of rpIRAV protein
The p-Cold-pIRAV was produced by cloning and inserting porcine IRAV ORF to the prokaryotic expression vector pCold-I, which was confirmed by sequencing (19). The recombinant plasmid p-Cold-pIRAV expressed in Escherichia coli BL21 (DE3) migrated at 35 kDa on SDS-PAGE, which was consistent with the expected molecular weight (Fig. 3). The optimized studies indicated that the highest expression of the recombinant protein was found with conditions of 16℃ and 0.2 mM IPTG for 16 h incubation. In the solubility study, the induced protein was found mainly in the supernatant in E. coli efficiently, and was easily purified by using His-binding Nickel Magnetic Beads (Biotool, Shanghai, China) after centrifugation and ultrasonication (Fig. 3) (19).

Generation of MAbs against porcine IRAV
Five groups of BALB/c mice were immunized with the purified rpIRAV protein to prepare MAbs. Indirect ELISA was used to choose the serum from immunized mice containing the highest titer of anti-rpIRAV antibodies (18). Before cell fusion, booster immunization was given 3-4 days in advance. The spleen cells of mice were fused with SP2/0 myeloma cells to generate hybridoma cell lines expressing MAbs against rpIRAV. After subcloning by limiting dilution and screening for four times, five cell lines secreting positive MAbs were obtained and named 2B10, 2G12, 2H1, 5A8 and 2C5 (Fig. 4A).

Reactivity of MAbs against rpIRAV
To obtain the ascites containing MAbs against rpIRAV, the MAbs 2B10, 2G12, 2H1, 5A8 and 2C5 cell lines were injected to mice (19). After a week, the ascites was extracted from mice, and purified by MAb ProteinG Spin Columns (Thermo Fisher Scientific, Rockford, IL). Followed by overexpressing porcine IRAV protein in PK-15 cells, the specificity of the MAbs was identified by Western blot analysis and Immunofluorescence assays (IFAs). Western blot analysis showed that the MAbs 2B10, 2G12, 2H1, 5A8 and 2C5 against porcine IRAV protein (Fig. 4A), and IFAs indicated that the MAbs 5A8 against porcine IRAV protein (Fig. 4B).

Discussion
IRAV is an novel IFN-stimulated gene (ISG) with antiviral activity against DENV and HCV (3; 4; 7). The published microarray data shows that IRAV is upregulated in response to type I and type II IFNs There are several porcine IRAV-like sequences on GenBank, but the sequences do not contain gene annotation. In the present study, porcine IRAV gene and the termini of it was cloned by RT-PCR and RACE from the IRAV mRNA of porcine spleen tissues, which contains an ORF (858 bp) encoding a polypeptide of 285 amino acids that shares the highest level of AA sequence identity (96.2%) with the whale IRAV. Phylogenetic analyses indicated that porcine IRAV protein clustered with the IRAV protein of whale, canine, bovine and sheep (21). Subcellular localization data of porcine IRAV showed that it localized to the cytoplasm.
For the porcine IRAV protein shares low AA sequence identity with human (92.8%), mouse (92.1%) and Macaca mulatta (91.8%) IRAV proteins, it is difficult to detected the endogenous porcine protein using the antibodies of human, mouse and Macaca mulatta IRAV proteins. So, we expressed porcine IRAV gene bacterially to generate the MAbs against porcine IRAV. A high level of antibody induced by the purified rIRAV protein was detected using indirect ELISA in immunized mice. By hybridoma technique, MAbs against porcine IRAV named 2B10, 2G12, 2H1,5A8 and 2C5 were generated. This antibody, identified by Western blot and IFA, provided a valuable tool for further investigation of the antiviral activity of IRAV.
In our current study, the recombinant porcine IRAV protein was expressed and purified for preparing  Table   Table 1. Primers used in the present study.