BACKGROUND
porcine reproductive and respiratory syndrome  has been recognized as one of the most serious infectious diseases of swine since its first appearance in north america in  <dig>  <cit> .
the disease is characterized by severe reproductive failure and severe pneumonia in neonatal pigs and is caused by prrs virus , a member of the arteriviridae family, order nidoviridales  <cit> .
pigs mount a rapid antibody response to infection by prrsv, but the antibodies are mainly directed to the n- and m-proteins and are non-neutralizing  <cit> .
the primary neutralization epitope of some north american prrsvs is located in the middle of the glycoprotein  <dig>  ectodomain  <cit> .
a truncated gp <dig> without the signal peptide sequence or the predicted transmembrane regions  is able to elicit protective antibodies capable of detecting prrsv-infected cells and of distinguishing this virus from others  <cit> .
currently, killed-virus and modified-live prrsv vaccines are used to control prrs.
however, both of these types of vaccines have inherent drawbacks and the development of novel prrsv vaccines is urgently needed  <cit> .
recent advances in innate immunity research have indicated that pathogen-associated molecular patterns  are promising molecular adjuvants for subunit vaccines  <cit> .
flagellin, the structural component of the flagellar filament in various locomotive bacteria, is a ligand for toll-like receptor  <dig>  in host cells  <cit> .
an increasing number of studies have demonstrated the effectiveness of flagellin as an adjuvant  <cit> , and flagellin is an effective inducer of innate immune effectors, such as cytokines and nitric oxide, thereby stimulating the activation of adaptive immune responses  <cit> .
in the present study, we cloned a truncated rgp <dig> gene and constructed the fusion protein rgp5-fljb using a prokaryotic system.
we then determined the tlr-5-specific bioactivity of fusion protein rgp5-fljb by detecting the expression levels of the cytokine interleukin  <dig>  in hek293-mtlr <dig> cells by sandwich enzyme-linked immunosorbent assay .
last, we assessed the immunogenicity of rgp <dig> and the adjuvant properties of fljb in a mouse immunization assay.
elisa-based detection of gp5-specific and prrsv-specific antibodies suggested that fljb could enhance the immunogenicity of gp <dig> and induce a robust humoral immune response, thus providing more effective antibodies against prrsv.
RESULTS
construction of expression plasmids bearing rgp <dig> and rgp5-fljb
a truncated rgp <dig> gene lacking the signal peptide sequence and transmembrane regions was amplified using a linker-based overlap-pcr strategy.
the gene of interest was inserted into the expression plasmids pcoldi and pgex-6p- <dig> to add a his or gst tag, respectively.
recombinant plasmid pcold-rgp5-fljb was constructed by using enzymes to digest the plasmid pcold-rgp <dig> and inserting the pcr product fljb .
the dna sequencing results indicated that the sequences of the inserts were identical to the template sequences.fig.
<dig> schematic representation for the construction of rgp <dig> and rgp5-fljb.
rgp <dig> fragment with the deletion of its signal peptide  and transmembrane regions  was amplified by overlap-pcr, and inserted into the bamhi and ecori digested expression vector pcoldi or pgex-6p- <dig> to create pcold-rgp <dig> or pgex-6p-1-rgp <dig> respectively.
flagellin fljb gene  was amplified from the genomic dna of attenuated salmonella typhimurium sl <dig> strain and cloned into the ecori and sali sites of pcold-rgp <dig>  resulting in a recombinant plasmid pcold-rgp5-fljb.
“l” represents the linker sequence ggggs
expression and purification of rgp <dig> and rgp5-fljb proteins
the expression of his-rgp <dig>  gst-rgp <dig>  and rgp5-fljb was induced by addition of iptg to the culture medium.
sds-page results indicated that the molecular weights of the tagged proteins were about 15 kda for his-rgp <dig>  40 kda for gst-rgp <dig>  and 67 kda for rgp5-fljb, as expected .
his-rgp <dig> and rgp5-fljb were purified via a ni-nta affinity column and verified by sds-page analysis .fig.
<dig> sds-page analysis of recombinant bacteria of bl <dig>  , bl <dig>  and bl <dig>  .
lanes: m, molecular weight markers;  <dig>  product of bl <dig> induced by iptg;  <dig>  lysate supernatant of bacteria bearing his-rgp <dig> induced by iptg;  <dig>  inclusion bodies of bacteria bearing his-rgp <dig> induced by iptg;  <dig>  product of bacteria bearing his-rgp <dig> not induced.
lanes: m, molecular weight markers;  <dig>  product of bl <dig> induced by iptg;  <dig>  lysate supernatant of bacteria bearing gst-rgp <dig> induced by iptg;  <dig>  inclusion bodies of bacteria bearing gst-rgp <dig> induced by iptg.
lanes: m, molecular weight markers;  <dig>  product of bl <dig> induced by iptg;  <dig>  product of bacteria bearing rgp5-fljb not induced;  <dig>  lysate supernatant of bacteria bearing rgp5-fljb induced by iptg;  <dig>  inclusion bodies of bacteria bearing rgp5-fljb induced by iptg
fig.
<dig> sds-page analysis of purified his-rgp <dig> and rgp5-fljb proteins.
the fused his-rgp <dig>  and rgp5-fljb  were purified via ni-nta affinity columns.
lanes: m, molecular weight markers;  <dig> and  <dig>  purified his-rgp <dig> or rgp5-fljb proteins
immunoblotting
to analyze the immunoreactivity of the rgp <dig> and fusion protein rgp5-fljb, polyclonal antibodies against prrsv or fljb were used as the primary antibodies in a western blotting assay.
the results showed that the bacterially-expressed rgp <dig> and rgp5-fljb were able to react with the prrsv-specific antibody .
additionally, western blotting with anti-fljb antibodies confirmed the presence of a highly purified 67 kda protein corresponding to rgp5-fljb .fig.
<dig> western blotting analysis of his-rgp <dig> and rgp5-fljb proteins.
analysis of his-rgp <dig> and rgp5-fljb with an anti-prrsv polyclonal antibody.
lanes: m, molecular weight markers;  <dig>  1 × sds-loading buffer;  <dig>  purified his-rgp <dig> protein;  <dig>  purified rgp5-fljb protein.
analysis of rgp5-fljb with an anti-fljb polyclonal antibody.
lanes: m, molecular weight markers;  <dig>  lysate supernatant of bl <dig> induced by iptg;  <dig>  inclusion bodies of bl <dig> induced by iptg;  <dig>  product of bl <dig> induced by iptg
tlr- <dig> activation by purified fusion protein rgp5-fljb
after removal of endotoxin from the purified proteins rgp <dig> and rgp5-fljb, very low endotoxin levels, less than  <dig>  eu/μg, were detected in the protein preparations, indicating that lps contamination in the prepared proteins was negligible.
to verify the activation of innate immune signaling by fusion protein rgp5-fljb, we conducted a tlr-5-specific bioactivity assay.
stimulation with rgp5-fljb at several different protein concentrations elicited a robust production of il- <dig> that was significantly higher than that elicited by stimulation with similar concentrations of rgp <dig> alone .
the observed up-regulation of chemokine il- <dig> following stimulation with rgp5-fljb suggested that the prepared endotoxin-free rgp5-fljb was able to activate the innate immune response in vitro.fig.
<dig> tlr5-specific activity of recombinant fusion proteins.
the tlr5-specific activity of recombinant flagellin fusion protein was examined on the hek293-mtlr <dig> cell line expressing mouse tlr <dig>  cells were treated with endotoxin-free recombinant proteins rgp <dig> or rgp5-fljb at the concentration of  <dig> and 100 ng/ml for 5 h. for positive controls, hek293-mtlr <dig> cells were treated with the tlr <dig> agonist flagellin.
supernatants were collected and expression levels of il- <dig> were then evaluated by elisa.
error bars indicate standard deviations of the means.
statistical significance was determined at p <  <dig>   or p <  <dig>
effects of rgp5-fljb treatment on gp5-specific and prrsv-specific serum antibodies
to examine the antibody response to the recombinant protein in an animal model, we administered the endotoxin-free fusion protein rgp5-fljb via an intraperitoneal injection.
the results showed that immunization with the fusion protein induced a significantly enhanced gp5-specific and prrsv-specific igg response after three immunizations compared with immunization using rgp <dig> alone .
the titer in the rgp5-fljb immunization group was elevated almost four-fold after the third immunization compared with the titer after the second immunization.
co-administration of the rgp <dig> with r <dig> or alum also yielded a higher igg response than immunization with rgp <dig> alone .fig.
<dig> immunization schedule and gp5-specific igg antibody titers in serum.
c3h/hej mice were randomly divided into five groups  and immunized intraperitoneally either with rgp <dig>  rgp5-fljb, rgp5 + r <dig>  rgp5 + aluminium adjuvant, or pbs, respectively.
these mice were immunized three times on days  <dig>   <dig>  and  <dig> at a dose of 50 μg rgp <dig>  50 μg rgp5-fljb, 10 μg r <dig> or isochoric aluminium adjuvant in 200 μl.
blood was collected from eye sockets on days  <dig>   <dig>   <dig> and  <dig> for analysis of anti-gp <dig> igg titers by elisa.
gp5-specific igg antibody titers in serum at day  <dig> after the boost and third immunization.
igg <dig> and igg2a levels in serum at day  <dig> after the third immunization.
data reflects the mean ± sd by using the student t test at p <  <dig>  , p <  <dig>   or p <  <dig>
fig.
<dig> prrsv-specific igg antibody titers in serum at day  <dig> after the third immunization.
c3h/hej mice were randomly divided into five groups  and immunized intraperitoneally either with rgp <dig>  rgp5-fljb, rgp5 + r <dig>  rgp5 + aluminium adjuvant, or pbs, respectively.
these mice were immunized three times on days  <dig>   <dig>  and  <dig> at a dose of 50 μg rgp <dig>  50 μg rgp5-fljb, 10 μg r <dig> or isochoric aluminium adjuvant in 200 μl.
blood was collected from eye sockets on days  <dig> for analysis of anti-prrsv igg titers by elisa.
data reflects the mean ± sd by using the student t test at p <  <dig>   or p <  <dig>
immune subtype induced by fusion protein rgp5-fljb
we investigated the subtype of the immune response after three immunizations with fusion protein rgp5-fljb by detecting serum igg <dig> and igg2a via indirect elisa.
co-administration of rgp <dig> with r <dig> or alum induced a robust th <dig> immune response.
the igg1/igg2a ratio in the group of mice immunized with rgp <dig> alone was  <dig> , whereas in the group of mice immunized with rgp <dig> together with r <dig> or alum this ratio was  <dig>  or  <dig> , respectively.
the igg1/igg2a ratio in the rgp5-fljb immunization group was significantly higher  than that in the rgp <dig> alone immunization group, and reached a level equivalent to the igg1/igg2a ratio in the rgp5 + alum immunization group , suggesting a strong th <dig> immune response.
the longevity of gp5-specific serum igg
to monitor the longevity of the gp5-specific serum igg, immunized animals were bled at 12-day intervals for 36 days after the third immunization and serum igg levels were determined by indirect elisa.
the elisa plates were coated with gst-tagged gp <dig> antigen.
we analyzed the time course of the levels of gp5-specific antibodies induced by rgp <dig> alone, rgp5-fljb, or rgp5 + r <dig> .
the igg titer in the rgp5-fljb group reached its peak  on day  <dig> post-third immunization, and then gradually fell.
the rgp5-fljb immunization induced an approximately 7– <dig> fold increase in the gp5-specific igg titers compared with the rgp <dig> alone immunization over the different tested time points, and the igg titer of the rgp5-fljb group was still significantly higher than that of the rgp <dig> alone group on day  <dig> post-third immunization.
these results suggest that rgp5-fljb immunization produces both a higher and longer-lasting anti-gp <dig> igg titer than immunization with rgp <dig> alone.fig.
<dig> the longevity of gp5-specific serum igg titers.
mice were bled for 36 days at 12 days intervals after 3rd immunization, and serum igg levels were determined by indirect elisa.
the elisa plates were coated with gst-taged gp <dig> antigen.
the time course of gp5-specific antibodies induced by rgp <dig> alone, rgp5-fljb or rgp5 + r <dig> was analyzed.
data reflects the mean ± sd by using the student t test at p <  <dig>  , p <  <dig>   or p <  <dig>
discussion
prrsv infection has been recognized as a severe threat to the pig industry.
a hallmark of the swine antibody response against prrsv is the abundant nonneutralizing antibodies detected early in infection followed by a low neutralizing antibody titer that appears at least 3 weeks after infection  <cit> .
the viral glycoprotein can induce a potent humoral immune response, and the production of anti-gp <dig> antibody is associated with the disappearance of viremia  <cit> .
the escherichia coli -based prokaryotic expression system is a powerful host cell system for expressing heterologous genes  <cit> .
many glycoproteins from cells or viruses produced in this system have shown biological activities  <cit> .
therefore, we aimed to express a truncated rgp <dig> gene  <cit>  without the signal peptide sequence and transmembrane regions in e. coli.
the fused his-rgp <dig> was purified via a ni-nta affinity column by its his-tag .
the immunoreactivity of rgp <dig> was determined by western blotting, and the results show that the heterologously-expressed protein reacts with an anti-prrsv polyclonal antibody , indicating that the recombinant protein retained the biological activity of the wild-type protein.
tlrs recognize distinctive ligands, play key roles in innate immunity, and can contribute to the development of appropriate adaptive immune responses  <cit> .
recently, several tlr ligands have been widely studied as adjuvants for immunotherapy and vaccination  <cit> .
among the defined tlr agonists, flagellin is the only protein with defined genetic codes  <cit> .
to investigate the adjuvant activity of fusion protein rgp5-fljb, phase ii flagellin fljb was amplified from the s. typhimurium sl <dig> strain and the recombinant plasmid pcold-rgp5-fljb was constructed.
the fusion protein preparation had a high degree of purity, as only one protein band was observed .
flagellins from gram-negative bacteria undergo a direct interaction with the leucine-rich regions in tlr- <dig> and activate a range of inflammatory cells via a tlr-5-dependent signaling pathway  <cit> .
we conducted a tlr-5-specific bioactivity assay and the results showed that stimulation with fusion protein rgp5-fljb induced a significantly higher expression of il- <dig> than stimulation with rgp <dig> alone, at several different protein concentrations , indicating that the innate immune response was activated by flagellin fljb.
recently, an ever increasing number of studies have described the adjuvant property of flagellin in the context of a broad range of recombinant vaccines  <cit> .
we tested the ability of rgp5-fljb to act as an adjuvant by administering the fusion protein rgp5-fljb to mice, and found that immunization with the fusion protein induced a significantly-enhanced gp5-specific and prrsv-specific igg response compared with immunization with rgp <dig> alone , indicating that the polyclonal antibodies against rgp <dig> were able to react with prrsv and fljb is an efficacious adjuvant for the induction of antigen-specific igg production.
imiquimod, a synthetic tlr- <dig> agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccines  <cit> .
in this study, the gp5-specific igg titer following rgp5 + r <dig> co-administration was significantly upregulated after the third immunization  and persisted for almost 5 weeks .
the robust igg1/igg2a ratio post-third immunization in the rgp5-fljb group, similar to that of the rgp5 + r848/alum co-administration groups, indicated that a strong gp5-specific th <dig> response was induced by the fusion protein rgp5-fljb .
the intranasal administration of other antigens in combination with flagellin has been shown to significantly increase the antigen-specific iga titer, not only in the mucosal compartment but also in the serum  <cit> .
additionally, previous studies found that immunization via the intranasal but not the subcutaneous route elicited sporozoite neutralizing antibodies capable of inhibiting > 90 % of sporozoite invasion  <cit> .
these findings suggest that a comparison of different administration routes of flagellin is needed in future studies.
CONCLUSIONS
we designed a truncated rgp <dig> gene and prepared his-rgp <dig>  gst-rgp <dig>  and rgp5-fljb proteins with a prokaryotic expression system.
the immunoreactivites of his-rgp <dig> and fusion protein rgp5-fljb were confirmed by western blotting.
after the removal of endotoxin in our rgp <dig> and rgp5-fljb preparations, we verified the activation of an innate immune response via a tlr-5-specific bioactivity assay and investigated the adjuvant activity of fljb via administration of rgp5-fljb protein to mice.
our findings confirm that fljb could serve as an excellent immunoadjuvant for the production of gp5-specific and prrsv-specific igg antibodies and the induction of a robust humoral immune response.

 porcine reproductive and respiratory syndrome has been recognized as one of the most serious infectious diseases of swine since its first appearance in north america in. the disease is characterized by severe reproductive failure and severe pneumonia in neonatal pigs and is caused by prrs virus, a member of the arteriviridae family, order nidoviridales. pigs mount a rapid antibody response to infection by prrsv, but the antibodies are mainly directed to the n- and m-proteins and are non-neutralizing. the primary neutralization epitope of some north american prrsvs is located in the middle of the glycoprotein ectodomain. a truncated gp without the signal peptide sequence or the predicted transmembrane regions is able to elicit protective antibodies capable of detecting prrsv-infected cells and of distinguishing this virus from others. currently, killed-virus and modified-live prrsv vaccines are used to control prrs. however, both of these types of vaccines have inherent drawbacks and the development of novel prrsv vaccines is urgently needed. recent advances in innate immunity research have indicated that pathogen-associated molecular patterns are promising molecular adjuvants for subunit vaccines. flagellin, the structural component of the flagellar filament in various locomotive bacteria, is a ligand for toll-like receptor in host cells. an increasing number of studies have demonstrated the effectiveness of flagellin as an adjuvant, and flagellin is an effective inducer of innate immune effectors, such as cytokines and nitric oxide, thereby stimulating the activation of adaptive immune responses. in the present study, we cloned a truncated rgp gene and constructed the fusion protein rgp5-fljb using a prokaryotic system. we then determined the tlr-5-specific bioactivity of fusion protein rgp5-fljb by detecting the expression levels of the cytokine interleukin in hek293-mtlr cells by sandwich enzyme-linked immunosorbent assay. last, we assessed the immunogenicity of rgp and the adjuvant properties of fljb in a mouse immunization assay. elisa-based detection of gp5-specific and prrsv-specific antibodies suggested that fljb could enhance the immunogenicity of gp and induce a robust humoral immune response, thus providing more effective antibodies against prrsv.  construction of expression plasmids bearing rgp and rgp5-fljb a truncated rgp gene lacking the signal peptide sequence and transmembrane regions was amplified using a linker-based overlap-pcr strategy. the gene of interest was inserted into the expression plasmids pcoldi and pgex-6p- to add a his or gst tag, respectively. recombinant plasmid pcold-rgp5-fljb was constructed by using enzymes to digest the plasmid pcold-rgp and inserting the pcr product fljb. the dna sequencing results indicated that the sequences of the inserts were identical to the template sequences.fig. schematic representation for the construction of rgp and rgp5-fljb. rgp fragment with the deletion of its signal peptide and transmembrane regions was amplified by overlap-pcr, and inserted into the bamhi and ecori digested expression vector pcoldi or pgex-6p- to create pcold-rgp or pgex-6p-1-rgp respectively. flagellin fljb gene was amplified from the genomic dna of attenuated salmonella typhimurium sl strain and cloned into the ecori and sali sites of pcold-rgp resulting in a recombinant plasmid pcold-rgp5-fljb. “l” represents the linker sequence ggggs expression and purification of rgp and rgp5-fljb proteins the expression of his-rgp gst-rgp and rgp5-fljb was induced by addition of iptg to the culture medium. sds-page results indicated that the molecular weights of the tagged proteins were about 15 kda for his-rgp 40 kda for gst-rgp and 67 kda for rgp5-fljb, as expected. his-rgp and rgp5-fljb were purified via a ni-nta affinity column and verified by sds-page analysis.fig. sds-page analysis of recombinant bacteria of bl, bl and bl. lanes: m, molecular weight markers; product of bl induced by iptg; lysate supernatant of bacteria bearing his-rgp induced by iptg; inclusion bodies of bacteria bearing his-rgp induced by iptg; product of bacteria bearing his-rgp not induced. lanes: m, molecular weight markers; product of bl induced by iptg; lysate supernatant of bacteria bearing gst-rgp induced by iptg; inclusion bodies of bacteria bearing gst-rgp induced by iptg. lanes: m, molecular weight markers; product of bl induced by iptg; product of bacteria bearing rgp5-fljb not induced; lysate supernatant of bacteria bearing rgp5-fljb induced by iptg; inclusion bodies of bacteria bearing rgp5-fljb induced by iptg fig. sds-page analysis of purified his-rgp and rgp5-fljb proteins. the fused his-rgp and rgp5-fljb were purified via ni-nta affinity columns. lanes: m, molecular weight markers; and purified his-rgp or rgp5-fljb proteins immunoblotting to analyze the immunoreactivity of the rgp and fusion protein rgp5-fljb, polyclonal antibodies against prrsv or fljb were used as the primary antibodies in a western blotting assay. the results showed that the bacterially-expressed rgp and rgp5-fljb were able to react with the prrsv-specific antibody. additionally, western blotting with anti-fljb antibodies confirmed the presence of a highly purified 67 kda protein corresponding to rgp5-fljb.fig. western blotting analysis of his-rgp and rgp5-fljb proteins. analysis of his-rgp and rgp5-fljb with an anti-prrsv polyclonal antibody. lanes: m, molecular weight markers; 1 × sds-loading buffer; purified his-rgp protein; purified rgp5-fljb protein. analysis of rgp5-fljb with an anti-fljb polyclonal antibody. lanes: m, molecular weight markers; lysate supernatant of bl induced by iptg; inclusion bodies of bl induced by iptg; product of bl induced by iptg tlr- activation by purified fusion protein rgp5-fljb after removal of endotoxin from the purified proteins rgp and rgp5-fljb, very low endotoxin levels, less than eu/μg, were detected in the protein preparations, indicating that lps contamination in the prepared proteins was negligible. to verify the activation of innate immune signaling by fusion protein rgp5-fljb, we conducted a tlr-5-specific bioactivity assay. stimulation with rgp5-fljb at several different protein concentrations elicited a robust production of il- that was significantly higher than that elicited by stimulation with similar concentrations of rgp alone. the observed up-regulation of chemokine il- following stimulation with rgp5-fljb suggested that the prepared endotoxin-free rgp5-fljb was able to activate the innate immune response in vitro.fig. tlr5-specific activity of recombinant fusion proteins. the tlr5-specific activity of recombinant flagellin fusion protein was examined on the hek293-mtlr cell line expressing mouse tlr cells were treated with endotoxin-free recombinant proteins rgp or rgp5-fljb at the concentration of and 100 ng/ml for 5 h. for positive controls, hek293-mtlr cells were treated with the tlr agonist flagellin. supernatants were collected and expression levels of il- were then evaluated by elisa. error bars indicate standard deviations of the means. statistical significance was determined at p <  or p <  effects of rgp5-fljb treatment on gp5-specific and prrsv-specific serum antibodies to examine the antibody response to the recombinant protein in an animal model, we administered the endotoxin-free fusion protein rgp5-fljb via an intraperitoneal injection. the results showed that immunization with the fusion protein induced a significantly enhanced gp5-specific and prrsv-specific igg response after three immunizations compared with immunization using rgp alone. the titer in the rgp5-fljb immunization group was elevated almost four-fold after the third immunization compared with the titer after the second immunization. co-administration of the rgp with r or alum also yielded a higher igg response than immunization with rgp alone.fig. immunization schedule and gp5-specific igg antibody titers in serum. c3h/hej mice were randomly divided into five groups and immunized intraperitoneally either with rgp rgp5-fljb, rgp5 + r rgp5 + aluminium adjuvant, or pbs, respectively. these mice were immunized three times on days and at a dose of 50 μg rgp 50 μg rgp5-fljb, 10 μg r or isochoric aluminium adjuvant in 200 μl. blood was collected from eye sockets on days and for analysis of anti-gp igg titers by elisa. gp5-specific igg antibody titers in serum at day after the boost and third immunization. igg and igg2a levels in serum at day after the third immunization. data reflects the mean ± sd by using the student t test at p < , p <  or p <  fig. prrsv-specific igg antibody titers in serum at day after the third immunization. c3h/hej mice were randomly divided into five groups and immunized intraperitoneally either with rgp rgp5-fljb, rgp5 + r rgp5 + aluminium adjuvant, or pbs, respectively. these mice were immunized three times on days and at a dose of 50 μg rgp 50 μg rgp5-fljb, 10 μg r or isochoric aluminium adjuvant in 200 μl. blood was collected from eye sockets on days for analysis of anti-prrsv igg titers by elisa. data reflects the mean ± sd by using the student t test at p <  or p <  immune subtype induced by fusion protein rgp5-fljb we investigated the subtype of the immune response after three immunizations with fusion protein rgp5-fljb by detecting serum igg and igg2a via indirect elisa. co-administration of rgp with r or alum induced a robust th immune response. the igg1/igg2a ratio in the group of mice immunized with rgp alone was, whereas in the group of mice immunized with rgp together with r or alum this ratio was or, respectively. the igg1/igg2a ratio in the rgp5-fljb immunization group was significantly higher than that in the rgp alone immunization group, and reached a level equivalent to the igg1/igg2a ratio in the rgp5 + alum immunization group, suggesting a strong th immune response. the longevity of gp5-specific serum igg to monitor the longevity of the gp5-specific serum igg, immunized animals were bled at 12-day intervals for 36 days after the third immunization and serum igg levels were determined by indirect elisa. the elisa plates were coated with gst-tagged gp antigen. we analyzed the time course of the levels of gp5-specific antibodies induced by rgp alone, rgp5-fljb, or rgp5 + r. the igg titer in the rgp5-fljb group reached its peak on day post-third immunization, and then gradually fell. the rgp5-fljb immunization induced an approximately 7– fold increase in the gp5-specific igg titers compared with the rgp alone immunization over the different tested time points, and the igg titer of the rgp5-fljb group was still significantly higher than that of the rgp alone group on day post-third immunization. these results suggest that rgp5-fljb immunization produces both a higher and longer-lasting anti-gp igg titer than immunization with rgp alone.fig. the longevity of gp5-specific serum igg titers. mice were bled for 36 days at 12 days intervals after 3rd immunization, and serum igg levels were determined by indirect elisa. the elisa plates were coated with gst-taged gp antigen. the time course of gp5-specific antibodies induced by rgp alone, rgp5-fljb or rgp5 + r was analyzed. data reflects the mean ± sd by using the student t test at p < , p <  or p <  discussion prrsv infection has been recognized as a severe threat to the pig industry. a hallmark of the swine antibody response against prrsv is the abundant nonneutralizing antibodies detected early in infection followed by a low neutralizing antibody titer that appears at least 3 weeks after infection. the viral glycoprotein can induce a potent humoral immune response, and the production of anti-gp antibody is associated with the disappearance of viremia. the escherichia coli -based prokaryotic expression system is a powerful host cell system for expressing heterologous genes. many glycoproteins from cells or viruses produced in this system have shown biological activities. therefore, we aimed to express a truncated rgp gene without the signal peptide sequence and transmembrane regions in e. coli. the fused his-rgp was purified via a ni-nta affinity column by its his-tag. the immunoreactivity of rgp was determined by western blotting, and the results show that the heterologously-expressed protein reacts with an anti-prrsv polyclonal antibody, indicating that the recombinant protein retained the biological activity of the wild-type protein. tlrs recognize distinctive ligands, play key roles in innate immunity, and can contribute to the development of appropriate adaptive immune responses. recently, several tlr ligands have been widely studied as adjuvants for immunotherapy and vaccination. among the defined tlr agonists, flagellin is the only protein with defined genetic codes. to investigate the adjuvant activity of fusion protein rgp5-fljb, phase ii flagellin fljb was amplified from the s. typhimurium sl strain and the recombinant plasmid pcold-rgp5-fljb was constructed. the fusion protein preparation had a high degree of purity, as only one protein band was observed. flagellins from gram-negative bacteria undergo a direct interaction with the leucine-rich regions in tlr- and activate a range of inflammatory cells via a tlr-5-dependent signaling pathway. we conducted a tlr-5-specific bioactivity assay and the results showed that stimulation with fusion protein rgp5-fljb induced a significantly higher expression of il- than stimulation with rgp alone, at several different protein concentrations, indicating that the innate immune response was activated by flagellin fljb. recently, an ever increasing number of studies have described the adjuvant property of flagellin in the context of a broad range of recombinant vaccines. we tested the ability of rgp5-fljb to act as an adjuvant by administering the fusion protein rgp5-fljb to mice, and found that immunization with the fusion protein induced a significantly-enhanced gp5-specific and prrsv-specific igg response compared with immunization with rgp alone, indicating that the polyclonal antibodies against rgp were able to react with prrsv and fljb is an efficacious adjuvant for the induction of antigen-specific igg production. imiquimod, a synthetic tlr- agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccines. in this study, the gp5-specific igg titer following rgp5 + r co-administration was significantly upregulated after the third immunization and persisted for almost 5 weeks. the robust igg1/igg2a ratio post-third immunization in the rgp5-fljb group, similar to that of the rgp5 + r848/alum co-administration groups, indicated that a strong gp5-specific th response was induced by the fusion protein rgp5-fljb. the intranasal administration of other antigens in combination with flagellin has been shown to significantly increase the antigen-specific iga titer, not only in the mucosal compartment but also in the serum. additionally, previous studies found that immunization via the intranasal but not the subcutaneous route elicited sporozoite neutralizing antibodies capable of inhibiting > 90 % of sporozoite invasion. these findings suggest that a comparison of different administration routes of flagellin is needed in future studies.  we designed a truncated rgp gene and prepared his-rgp gst-rgp and rgp5-fljb proteins with a prokaryotic expression system. the immunoreactivites of his-rgp and fusion protein rgp5-fljb were confirmed by western blotting. after the removal of endotoxin in our rgp and rgp5-fljb preparations, we verified the activation of an innate immune response via a tlr-5-specific bioactivity assay and investigated the adjuvant activity of fljb via administration of rgp5-fljb protein to mice. our findings confirm that fljb could serve as an excellent immunoadjuvant for the production of gp5-specific and prrsv-specific igg antibodies and the induction of a robust humoral immune response. 
purified fusion protein rgp5-fljb is capable of activating the innate immune response, as demonstrated by the results of our tlr-5-specific bioactivity assay, and fljb has adjuvant activity, as shown by the results from our administration of rgp5-fljb in a mouse model. histidine -tagged rgp and fusion protein rgp5-fljb were induced with isopropyl-β-d-thiogalactoside, verified by sds-page and western blotting, and purified via ni-nta affinity columns. the results show that immunization with the fusion protein rgp5-fljb induced a significantly enhanced gp5-specific and prrsv-specific igg response that persisted for almost 5 weeks. co-administration of the rgp with r or alum also yielded a higher igg response than administration of rgp alone. our findings confirm that fljb could serve as an excellent adjuvant for the production of gp5-specific and prrsv-specific igg antibodies as part of an induction of a robust humoral immune response. porcine reproductive and respiratory syndrome is characterized by severe reproductive failure and severe pneumonia in neonatal pigs and is caused by prrs virus. flagellin, a toll-like receptor agonist, is an effective inducer of innate immune responses. a truncated rgp gene lacking the signal peptide and transmembrane sequences was amplified and inserted into prokaryotic expression vectors, pcoldi or pgex-6p- salmonella typhimurium flagellin fljb was amplified and inserted into the plasmid pcold-rgp generating recombinant plasmid pcold-rgp5-fljb. the igg1/igg2a ratio in the rgp5-fljb immunization group was significantly higher than that in the rgp alone group and was equivalent to the response in the rgp5 + alum immunization group, suggesting a strong th immune response was induced by the fusion protein. the tlr-5-specific bioactivity of fusion protein rgp5-fljb was determined by detecting the expression levels of the cytokine il- in hek293-mtlr cells by sandwich elisa. prrsvglycoprotein 5flagellinfusion proteinvaccineissue-copyright-statement© the author 2015
 porcine reproductive and respiratory syndrome is characterized by severe reproductive failure and severe pneumonia in neonatal pigs and is caused by prrs virus. glycoprotein from prrsv is a key inducer of neutralizing antibodies. flagellin, a toll-like receptor agonist, is an effective inducer of innate immune responses. this study presents a novel prrsv vaccine candidate based on the adjuvant effect of salmonella typhimurium fljb fused with prrsv gp  a truncated rgp gene lacking the signal peptide and transmembrane sequences was amplified and inserted into prokaryotic expression vectors, pcoldi or pgex-6p- salmonella typhimurium flagellin fljb was amplified and inserted into the plasmid pcold-rgp generating recombinant plasmid pcold-rgp5-fljb. histidine -tagged rgp and fusion protein rgp5-fljb were induced with isopropyl-β-d-thiogalactoside, verified by sds-page and western blotting, and purified via ni-nta affinity columns. the tlr-5-specific bioactivity of fusion protein rgp5-fljb was determined by detecting the expression levels of the cytokine il- in hek293-mtlr cells by sandwich elisa. the purified endotoxin-free proteins were administered intraperitoneally in a c3h/hej mouse model. the results show that immunization with the fusion protein rgp5-fljb induced a significantly enhanced gp5-specific and prrsv-specific igg response that persisted for almost 5 weeks. co-administration of the rgp with r or alum also yielded a higher igg response than administration of rgp alone. the igg1/igg2a ratio in the rgp5-fljb immunization group was significantly higher than that in the rgp alone group and was equivalent to the response in the rgp5 + alum immunization group, suggesting a strong th immune response was induced by the fusion protein.  purified fusion protein rgp5-fljb is capable of activating the innate immune response, as demonstrated by the results of our tlr-5-specific bioactivity assay, and fljb has adjuvant activity, as shown by the results from our administration of rgp5-fljb in a mouse model. our findings confirm that fljb could serve as an excellent adjuvant for the production of gp5-specific and prrsv-specific igg antibodies as part of an induction of a robust humoral immune response. keywords prrsvglycoprotein 5flagellinfusion proteinvaccineissue-copyright-statement© the author 2015 