| The mucosal immune system is an important part of the entire immune network in human and animal body, and it is structurally and functionally divided into sites for antigen uptake and processing at inductive sites. The mucosal immune system play an important role in resisting to infection, First of all, mucosal s-IgA can form a barrier to pathogens at the mucosal surface by preventing the initial attachment of the pathogen and its infiltration of the surface layers or by binding to and neutralizing toxins that the pathogen produces, which is the first line of defending infection; Second, the mucosal immune system has a common mucosal immune mechanism which make systemic mucosal immunity status consistent. Against, the mucosal immune system must also be capable of inducing effective cell-mediated and antibody-mediated immune responses towards selected antigens. The information indicates that the mucosal immune is an only management strategies and a one of new technologies in the field of epidemics prevention.Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, causing economically significant impacts on the swine industry worldwide. However none of the current commercial vaccines is able to completely prevent respiratory infection, trans-placental transmission, pig-to-pig transmission the virus nor maintaining immune protection in sows. We share the information related to PRRSV and review the available options for PRRS control strategies based on pathogenic characteristics, the immune properties and biological characteristics of PRRSV. In this study, the Nisin controlled expression (NICE) system of Lactococcus lactis was selected as vector of recombinant protein expression to express ORF6gene, the aim was to develop the gene engineering L.1acti mucosal vaccines of PRRSV, and evaluate immune activity and adjuvant effect. The results of research are summarized as following.1. In this study, the ORF6structural proteins gene of PRRSV)was amplified by RT-PCR. The ORF6gene was subcloned into the vector PMD19-T and sequenced, the gene consistion of515bp and the nucleotide sequence is not mutate. One pairs of primers was designed according to the nucleotide sequence and the vector PNZ8149characteristics. The DNA of the ORF6gene was amplified by PCR using PMD19-T-M as template, the PCR product with Sac I and Nco I restriction enzyme sites was inserted into vector PNZ8149, and transformed into L.1acti NZ3900by electroporation, result in recombinant strain PNZ8149/NZ3900-M/PRRS. Then, the recombinant protein was detected by Western blot and IFA experiments after the bacteria was respectively induced2h,4h,6h,8h,10h,12h by10ng/mL Nisin. The result indicated that the molecular weight of the expressed recombinant protein was about19kDa, protein bands is relatively clear at6h, the protein maintain the antigenicity of PRRSV as expected, moreover the protein was secreted and located on the surface of the bacteria. The result showed that we successfully constructed the food-grade recombinant L.1acti PNZ8149/NZ3900-M/PRRS in which the lactose operon, Furthermore, the recombinant protein was located on the surface of the bacteria and had reactogenicity with antibody against PRRSV. The study is expected to lay a theoretical foundation on developing of the gene engineering L. lacti s mucosal vaccines.2. To evaluate the changes of immune response of recombinant L.1acti PNZ8149/NZ3900-M/PRRS strain that expresses ORF6gene of PRRSV, BALB/c mice were immunized with the bacterial supernatant. Six to eight week old BAI B/C mice were randomly divided into four groups (group I, Ⅱ,Ⅲand Ⅳ, respectively), with each group consists of30animals. The control group I were immunized intranasally (i.n.) with50ul PBS, the control group Ⅱ were immunized i.n.with50ul L.lactis cell suspension (1011CFU) carrying the control vector, the experimental groupⅢ were vaccinated i.n. administration of50ul recombinant L.1actis cell suspension (1011CFU), the experimental groupIV were vaccinated orally (o.r) administration of50ul recombinant L.1acti s cell suspension (10"CFU). The mice were were vaccinated on days1,2,3,11,12, and13and on days21, 22, and23.)-The mice were respectively sacrificed on day23,30,37,44, and51, respectively. Blood samples, lung lavage fluid and intestinal washes were obtained on different days after immunization, IgG in serum and s-IgA in lung lavage fluid and intestinal washes were analyzed by using a similar enzyme-linked immunosorbent assay (ELISA). For the purpose of evaluating of changes about cytokines IL-2、IL-4、IL-5、 IL-10and IFN-y, the spleen collected on day37were analyzed.The results clearly shows that mice immunized with recombinant strain PNZ8149/NZ3900-M/PRRS could induce remarkable special s-IgA in intestinal washes and lung lavage fluid, at the whole experimental session, compared with the control group Ⅰ and Ⅱ, the level of special s-IgA showed significantly increase ((P<0.01), the production of the s-IgA antibody in lung lavage fluid from the i.n immunized mice was higher significantly than that from the o.r immunized mice ((P<0.01), the production of the s-IgA antibody in intestinal washes from the i.n immunized mice was higher than the o.r immunized mice ((P<0.05). The s-IgA antibody from the immunized mice get it’s maximum number and maintain a stable trend at the whole experimental session, it is possible that the measurement time of s-IgA is late or the detection cycle is short. Cytokines IL-5responses play an important role in inducing s-IgA antibody responses, therefore, we examined the activity of IL-5in splenocytes on day37, there was significant production of IL-5in mice administered with recombinant strain by the way of oral and intranasal, but the production of IL-5in intranasal immunized mice was no significant compared to that from the oral immunized mice ((P>0.05).To assess cell responses induced by recombinant L.1actis expressing ORF6gene of PRRSV, we investigated the activity of IL-2and IFN-y in splenocytes on day37. IL-2and IFN-y contents of the experimental groupIIIandIV were significant higher than those of the control group I and II (P<0.01), furthermore the i.n immunized mice was higher significantly than that from the o.r immunized mice (P<0.01), IL-2and IFN-y contents in splenocytes from mice immunized intranasally was respectively349.848pg/ml and173.276pg/ml. as well as, IL-2and IFN-y contents in splenocytes from mice immunized orally was respectively335.455pg/ml and162.913pg/ml. Notably, it is worthy to note that cytokines of IL-4contents was not different to the all groups. IgG anti-PRRSV was detected in serum, in contrast, the level of special IgG was higher significantly following experimental groupⅢ and Ⅳ compared to the control group Ⅰ and Ⅱ((P<0.01) at all time points the i.n immunized mice get it’s maximum number on day30, which lasted at least4weeks post-immunization; as well as the o.r immunized mice get it’s maximum number on day44, following decreased rapidly. Regretful, the titer of IgG was not investigated, for the reason that whether is able to resistant to the virus.All the results indicated that mice immunized by the recombinant L.1actis expressing ORF6gene of PRRSV can significantly induce mucosal immune responses, humoral immunity responses and enhanced Thl immunity response against the PRRSV, in contrast, Intranasal immunization is superior to oral immunization.3. Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvant for immune responses, particularly in mice. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant, but data on mucosal immune responses induced by CpG ODN plus the recombinant L.1actis are scarce. To investigate the immune responses induced by intranasal immunization, with some doses of CpG ODN plus the recombinant L.lactis expressing ORF6gene of PRRSV antigen. Six to eight week old BAI B/C mice were randomly divided into three groups (group Ⅰ, Ⅱ, andⅢ, respectively), with each group consists of30animals. The control group Ⅰ were immunized i.n. administration of50ul recombinant L.1actis cell suspension (1011CFU), the experimental groupⅡ were immunized i.n.10ul CpG ODN plus50ul administration of recombinant L.1actis cell suspension, the control groupⅢ were vaccinated i.n.10ul CpG ODN. Immunization dose, immunological methods and indicators detected in common with the second part of the test. The results clearly show that CpG ODN promoted the induction of IgG antibody in serum and s-IgA antibody in intestinal washes and lung lavage fluid. And the production of the s-IgA antibody in lung lavage fluid of immunized mice was higher than that in intestinal washes; the activity of cytokines IL-2and IFN-γ in splenocytes on day37were investigated, in compared with the control group Ⅰ, the activity of cytokines IL-2and IFN-γ in CpG ODN plus recombinant L.1actis were increased, and higher significantly than that of the control groupⅢ(p<0.01). Results from this study indicate that stimulatory CpG ODN may be effective as a mucosal adjuvant with the recombinant L.1actis expressing ORF6gene of PRRSV antigen. The study is expected to lay a theoretical foundation on the adjuvant about recombinant strain vaccine. |
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