The Effects Of The Extracellular Region Domains Of CD163 And Sn Receptors In PRRSV-ADE Infection

Porcine reproductive and respiratory syndrome(PRRS)is one infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).The virus infected sows not only cause immune suppression,but also lead to persistent infection of pigs.Studies have shown that PRRSV-antibody complexes infection to macrophages upon FcγR-mediated is one reason of PRRSV persistent infection,but the mechanism is not very perfect.Our study indicated that the process of PRRSV-antibody complexes infection to macrophages may be associated with virus receptors.By studying the antibody-dependent enhancement(ADE)of CD163 molecule and Sn receptor in PRRSV infection,this study further complements the mechanisms of PRRSV persistent infection upon antibody-mediated and has important guiding significance for the development of new vaccines of PRRS.However,it is not very clear that the effects of CD163 molecule and Sn receptor on the molecular mechanisms of PRRSV infection to PAM cells and ADE of PRRSV infection,and it is also not determined that specific location of functional domains of CD163 molecule and Sn receptor play a role in PRRSV infection or PRRSV-antibody complex infection process.In this test 9 cysteine-rich(SRCR)domains of the extracellular region of CD163 molecule was divided into four segments,namely CD163-P1(SRCR1-2),CD163-P2(SRCR3-4),CD163-P3(SRCR5-6)and CD163-P4(SRCR7-9).17 domains of the extracellular region of Sn receptor was divided into nine segments,namely Sn1(1-150aa),Sn2(137-228aa),Sn3(236-406aa),Sn4(413-594aa),Sn5(599-791aa),Sn6(796-979aa),Sn7(981-1150aa),Sn8(1170-1248aa),Sn9(1441-1631aa).The constructed CD163 and Sn recombinant plasmids of different fragments were transformed into the expression strain BL21(DE3)to be expressed by prokaryote,and recombinant proteins were purified with different concentrations of urea.PAM cells were collected and plated out in triplicate 24-well plates,and then mixed different concentrations of different fragments of CD163 and Sn recombinant proteins with an equal volume of PRRSV or 4-fold diluted PRRSV-IgG complexes for 1h at 37℃.After infecting PAM cells,and setting the negative control group.We detected the PRRSV copies of infected cells with established PRRSV real-time quantitative PCR method,and used the Graph Pad Prism 5 software to handle with the datas that we got.In this study,PAM cells were collected from PRRSV antigen-antibody-negative pig,and cell total RNA was extracted.Two pairs of specific primers were designed according to Sn gene sequence(Gen Bank: JX070181.1)and the division of Sn extracellular domain to amplify two gene fragments of Sn6 and Sn8 by RT-PCR,of which two gene fragments length were respectively 735 bp and 567 bp.Then the two genes fragments were subcloned into the plasmid vector PET-32 a,and the recombinant plasmids PET-Sn6 and PET-Sn8 were successfully built.The constructed PET-Sn6 and PET-Sn8 recombinant plasmids and the conserved PET-CD163-P1,PET-CD163-P2,PET-CD163-P3,PET-CD163-P4,PET-Sn1,PET-Sn2,PET-Sn3,PET-Sn4,PET-Sn5,PET-Sn7 and PET-Sn9 recombinant plasmids were respectively transformed into the expression strain BL21(DE3).By optimizing the time and the concentration of IPTG inducing,we got a large number of expressed recombinant proteins CD163-P1,CD163-P2,CD163-P3 and CD163-P4,Sn1,Sn2,Sn3,Sn4,Sn5,Sn6,Sn7,Sn8 and Sn9.After washing in different concentrations of urea to purify recombinant proteins,then protein concentrations were measured by UV spectrophotometer.To determine the role of functional domains of CD163 and Sn receptors in PRRSV infection to PAM cells,we used the recombinant proteins of CD163-P1,CD163-P2,CD163-P3 and CD163-P4,Sn1,Sn2,Sn3,Sn4,Sn5,Sn6,Sn7,Sn8 Sn9 domains prepared from the experiment one,adjusting concentrations of each domain protein to 2mg/m L,1mg/m L,0.2mg/m L respectively in FBS-free 1640.And then the viral suspension of 200 TCID50 PRRSV Hn-1/06 strain(104.8 TCID50/m L)was mixed with equal volume different concentration recombinant proteins of CD163-P1,CD163-P2,CD163-P3 and CD163-P4,Sn1,Sn2,Sn3,Sn4,Sn5,Sn6,Sn7,Sn8,Sn9 domains for 1h at 37℃,after infection to PAM cells for another 1h,supplementing with 1640 media with 10% FBS for 48 h at 37℃,and setting the negative control group.PRRSV copies of infected cells were detected by established absolute fluorescence quantitative PCR method,and used the Graph Pad Prism 5 software to handle with the datas we got.The results showed that the viral copies of infected cells incubated with mixed viral suspension with different concentration recombinant proteins of CD163-P1,CD163-P2,CD163-P3 and CD163-P4,Sn1,Sn2,Sn3,Sn4,Sn5,Sn6,Sn7,Sn8,Sn9 domains were lower significantly than PRRSV infection to PAM cells alone group,indicating that different concentration recombinant proteins of CD163-P1,CD163-P2,CD163-P3 and CD163-P4,Sn1,Sn2,Sn3,Sn4,Sn5,Sn6,Sn7,Sn8,Sn9 domains produced a obvious competitive inhibition with the CD163 and Sn receptors existing in the PAM Cells,and the higher concentration of CD163-P1,CD163-P2,CD163-P3 and CD163-P4,Sn1,Sn2,Sn3,Sn4,Sn5,Sn6,Sn7,Sn8,Sn9 domain proteins,the stronger competitive inhibitory effect.For different dilution proteins of each segment of CD163,the inhibitory effect of CD163-P1 was most obvious in 2.0mg/m L concentration proteins of each fragment,the inhibitory effect of CD163-P3 was most obvious in 1.0mg/m L concentration proteins of each fragment,the inhibitory effect of CD163-P2 and CD163-P4 is more obvious in 0.2mg/m L concentration proteins of each fragment,but the whole rule was less obvious.For different dilution proteins of each segment of Sn,the competition inhibition of Sn6 and Sn8 fragment domains for different dilution proteins is more obvious compared with other fragments,suggesting Sn6 and Sn8 fragment domains may play a major role in the process of PRRSV infection to PAM cells,which provided some references for further studying the molecular mechanism of PRRSV infection to PAMs and PRRSV-ADE.On this basis,the purified IgG from an anti-PRRSV polyclonal antiserum was 2-fold diluted in FBS-free 1640 0.05 m L and mixed with an equal volume of the viral suspension of 200 TCID50 PRRSV Hn-1/06 strain,then mixed with the different concentration recombinant proteins of CD163-P1,CD163-P2,CD163-P3 and CD163-P4,Sn1,Sn2,Sn3,Sn4,Sn5,Sn6,Sn7,Sn8 Sn9 domains 0.1m L from the experiment one,after the mixtures were pretreated for 1 h at 37℃.Followed by infection to PAM cells for another 1h,supplementing with 1640 media with 10% FBS for 48 h at 37℃,and setting the negative control group.PRRSV copies of infected cells were detected by established absolute fluorescence quantitative PCR method,and used the Graph Pad Prism 5 software to handle with the datas we got.The results showed that the higher concentration proteins of each segment of CD163 developed a competitive binding effect with the CD163 receptor existing in the PAM Cells compare to PRRSV-IgG infection to PAM cells alone group,which inhibit PRRSV replication in the process of PRRSV-IgG infection to PAMs,and the higher the concentration was,the more obvious the inhibition effect was;However,for low levels of recombinant proteins of each segment of CD163,the viral copies of PRRSV were significantly increased.The results showed that it was not obvious for the rule of CD163-P1,CD163-P2,CD163-P3 and CD163-P4 each fragment in PRRSV-ADE infection.Compared with PRRSV-IgG infection to PAM cells control group,the higher concentration proteins for each segment of Sn had a significant inhibitory effect on PRRSV-IgG invasion into PAM,and the higher the concentration was,the more obvious the inhibitory effect was.For low levels of recombinant proteins of each segment of Sn,Sn6 and Sn8 domain proteins had a significant inhibitory effect on PRRSV-IgG invasion into PAM,but the PRRSV copies of other proteins were increased.The indicated indirectly that Sn6 and Sn8 domain fragments were involved in PRRSV-ADE infection,and the binding effect of Sn6 and Sn8 is more obvious.The results suggested that Sn6 and Sn8 fragments can affect PRRSV-IgG replication in the cell proliferation,indicating Sn6 and Sn8 fragments may be involved in the proliferation of immune complexes in cells,and play the major function.This study further explores the role of CD163 molecule and Sn receptor function domains in PRRSV-ADE infection,which provides a theoretical basis for further study the molecular mechanisms of PRRSV-antibody infection and has important guiding significance for research of PRRS new vaccines and screening of antiviral drugs.