The Effect Of FcγR Ⅰ On ADE Of PRRSV Infection And The Influence Of LPS On Infection Of PRRSV

The porcine reproductive and respiratory syndrome(PRRS) is a viral infectiousdisease,which is caused by the infection of porcine reproductive and respiratorysyndrome virus (PRRSV). In vivo, PRRSV predominantly invades into monocytes/macrophages. FcγRⅠ（CD64）were expressed on monocytes/macrophages and hadhigh affinity with IgG. This study mainly investigated the effect of the extracellulardomain of FcγRⅠ during the process of PRRSV infection on PAM. And it lays thefoundation for the immune fuction of FcγRⅠ. In the natural conditions,Gram-negative bacterial diseases,such as haemophilus, always happen secondarilyafter porcine being infected by PRRSV, and Gram-negative bacteria can produce agreat many of endotoxin in vivo. Through study of the mRNA transcriptional leveland variety of the PRRSV copy-number in two cases which put the induction of LPSfirst before PAM being infected by PRRSV,or secondly after that, so as to lay afoundation for the further research on the pathogenic mechanism of mixed infectionof PRRSV and bacteria.Obtaining PAM cells from healthy lung filling and extracting total RNA of PAMcells. Designing FY a pair of specificity primers of the FcγRⅠextacellar domainwhich were eliminated form the signaling peptides. Amplifying the gene fragment byRT-PCR and the size is about450bp. The gene fragment was subcloned into pET-32avector,and a recombinant plasmid FY was constructed.The plasmid was expressedsuccessfully in IPTG induction. And we obtained a recombinant protein FY,whichmolecular weight is about47.2KD, consistent with the expected protein molecularweight.Through further optimized conditions of expression,recombinant protein wasextensively expressed. Renaturing and purifying recombinant protein by urea. Thepurified recombinant protein was inoculated for mice to obtain themulticlonalantibodies against FY recombinant fusion protein. Themulticlonalantibodies were tested by indirect ELISA, and the result showed that thetiter of multiclonalantibodies was1:12800. Western blotting experiment wasimplemented with specific multiclonalantibodies. The result indicated that specificmulticlonalantibodies could specificly bind with their recombinant protein, and it issuggested that the recombinant protein has good immunogenicity.In this study, purified rabbit IgG (3.0mg/mL) was using to immunize pigs insubcutaneously, every2.2mL, using the agar diffusion test for detection of serumanti-rabbit IgG antibody titer until antibody titers reached1:32nasal inoculation ofPRRSV, after inoculation4d,6d,9d,13d,17d,20d,23d,26d,39,47d,51days, bloodsamples were collected antiserum, fluorescence quantitative RT-PCR method detected PRRSV virus nucleic acids, and detection of anti-PRRSV antibody titer by ELISA theresults show that immunity to infection4-39days after the infection of pigs can bedetected in serum to PRRSV nucleic acid inoculation in the control group only in the4-26d detects a virus nucleic acid, immune to infection viremia than those infectedcontrol group, the duration of eight days, and immune infection group4d,6d,9d,13d,17d,23d,26d,39days of PRRSV mRNA level compared with inoculation in thecontrol group slightly increased. Immune serum ELISA antibody detection in9d,13d,17d,23d are PRRSV antibody positive, and positive antibodies in the body for15days; inoculation control serum ELISA antibody detection in6d,9d,13d,17d arepositive, positive antibody in the body for12days; which then poison control groupslightly higher than in the13d and17d when the immune serum antibody titer, theabove results indicate that pigs PRRSV non-specific IgG can promote virus infection.In this study, the first final concentration of1.30mg/mL, PRRSV-negative healthypig IgG handle PAM cells at37°C12h, then100TCID50of PRRSV virus solution0.2mL inoculated PAM cells, and set up access to poison control group, byfluorescence quantitative RT-PCR method were determined after infection,12h,24h,36h,48h of PRRSV copy number changes, the results of PRRSV-negative healthy pigIgG treated PRRSV copy number compared with inoculation in the control groupsignificantly increased in all time periods, including12h the highest copy number is11.7times the control group,48h is3.5times the control group, indicating thatPRRSV non-specific pig IgG of FcγR Ⅰ, to promote the replication of PRRSV inPAM cells.In this study, the recombinant protein of the extracellular region of the mouseanti-pig of FcγR I antibody1h after inoculation of PRRSV by adding PAM Cells werecultured12h,24h,36h,48h, cells were collected and the supernatant, mouse-negativeserum to join the PAM cells1h inoculated with PRRSV as a control, real-time PCRmethod to detect the virus copys in PAM cells, the results show that each time mouseanti-pig of FcγR I extracellular domain of recombinant protein antibody treatment ofPRRSV copy number of the experimental group and the negative serum treatedcompared to each time period were increased, and in which24h up to6.3times higher.This study, the recombinant protein of the extracellular region of the mouse anti-pigof FcγR I antibody PAM cell function after1h culture12h,24h,36h,48h, cells werecollected and the supernatant, while doing a rat negative serum treatment group andthe healthy cells in the control group, PAM cells using real-time PCR method fordetection of IFN-α, TNF-alpha, IL-10mRNA level. The results showed that specificantibody test group and negative serum treatment compared to the control group at12h,24h,36h,48h, IFN-alpha mRNA increased by an average, of which up to3.9times higher12h; specific antibody in the test group and negative serum compared tothe control group compared to the level of TNF-α mRNA in each time period wereincreased, which12h,24h significantly increased; deal with specific antibodies in thetest group and the negative serum control group, IL-10mRNA transcription level ofeach time period were significantly reduced. The above experimental description ofPAM cells, activation of FcγR Ⅰ, can promote of IFN-α and of TNF-α mRNAtranscription, while the inhibition of IL-10mRNA in transcription, thus speculate that PRRSV infection early of FcγR Ⅰ, referral guide of the PAM cell phagocytic role inthe promotion of virus multiplication, the late anti-viral factors play to inhibit viralproliferation. This experiment laid the foundation for the role of in-depth study ofFcγR Ⅰin PAM cell antiviral immune.Then the PRRSV was added into cultured PAM cells24-well cell culture plates,200μL/hole cultured12h,adding RPMI1640nutrient solution with a final concentration of100ng/mL LPS (group V+L); adding a final concentration of100ng/mL of LPS of RPMI1640nutrition liquid junction into cultured PAM cells in24well cell culture plates, cultured12h, each well added the virus solution200μL (group L+V), hosted a viral infection control group (group V), timing at the time of adding virus solution, at the same time set the LPS-induced control group (group LPS). Cultured12h,24h,36h,48h the cells and the supernatant were collected, and at the same time set the healthy cells control group. Using real-time PCR to detect PRRSV copy number in PAM cells and IFN-α, IL-10mRNA transcription level. The results showed that the copynumber in group V+L was significantly lower compared with the group V at each time; PRRSV copy number in group L+V was significantly lower than the group V ateach time; IFN-αmRNA levels in group LPS at48h was significantly improved compared to an healthy cells controls, the remaining time period or less; The difference ofIL-10mRNA levels in group LPS with the healthy cells control group was not significant at all times; So I consluted LPS significantly inhibited PRRSV in PAM cells in proliferation, LPS slight to promote IFN-α mRNA transcription in the health of PAM cells, had no effect on the IL-10mRNA transcription in health PAM. IFN-alpha mRNAlevels in group V were lower compared to healthy cells control group at all time, especially36h and48h significantly reduced. The IL-10mRNA levels in group V were significantly higher compared to healthy cells control group in each time period. The IFN-alpha mRNA levels in group V+L are significantly increased compared to the group V at24h,36h,48h, up to11times at48h, and the overall was upward trend, IL-10mRNA level in group V+L are significantly reduced compared to group V at24h,36h,48h, and the overall was downward trend; The IFN-α mRNA level in group L+Vof the were significantly increased compared to group V at each time, and the overallwas downward trend, The IL-10mRNA levels in group L+V were significantly reduced compared to group V in each time period. The above studies have shown that LPS-induced IFN-alpha mRNA transcription effect was strengthen when PRRSV exist, and significantly inhibited IL-10mRNA transcription, thereby strengthening the antiviral immune effect in PAM cells. This research laid the foundation for more in-depth investigate pathogenic mechanisms of the PRRSV and bacterial infection.