Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig

The Research of the Function of Near N-terminal Structral Domain of Sialic Acid and the Effect of Lps on IFN-α Generated from PAM Infected with PRRSV

Author ZhangZhiYuan
Tutor XiaPingAn;CuiBaoAn
School Henan Agricultural University
Course Preventive Veterinary Medicine
Keywords porcine reproductive and respiratory syndrome virus Sialic acid adhesin PAM cell IFN-α LPS
CLC S858.28
Type Master's thesis
Year 2011
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The porcine reproductive and respiratory syndrome(PRRS) is a viral infectious disease ,which is caused by the infection of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV predominantly invades into monocytes/ macrophages.The results show that it is closely related to the virus receptor on the porcine macrophages,and sialic acid adhesion receptor plays a critical role in the process of PRRSV infection PAM cell.The extracellular domain of sialic acid adhesion receptor participated the adsorption of PRRSV and PAM. This study is mainly investigated the effect of the first structural domain of sialic acid adhesion receptor N-terminal on the process of PRRSV infection on PAM. And it lays the foundation for the sialic acid adhesion receptor endocytosis on PRRSV.In the natural conditions,Gram-negative bacterial disease ,such as Haemophilus, always happens secondary after porcine infected by PRRSV. The role that endotoxin played in antiviral mechanism has not been yet cleared,which is produced from Gram-negative bacterial in vivo. In this experiment,we discussed the effects of LPS to IFN-αand viral replication in health PAM and in PAM infected with PRRSV ,and it prepares the ground for depth discussion of the role that endotoxin plays in antiviral immune response.Obtaining PAM cells by lung filling and extracting total RNA of PAM cells.Designing a pair of specificity primers of the first structural domain of sialic acid adhesion receptor N-terminal.Amplifying the first domain gene fragment of amino terminal by RT-PCR and the size is 450bp. The gene fragment was subcloned into pET-32a vector,and we constructed a recombinant plasmid Sn150.The plasmid was expressed successfully in IPTG induction. And we obtained a recombinant protein Sn150 ,which molecular weight is about 36.6KD, consistent with the expected protein molecular weight.Through further optimized conditions of expression and recombinant protein was extensively expressed. Renaturing and purifying recombinant protein by urea. The purified recombinant protein was inoculated for mice to obtain the multiclonalantibodies against Sn150 recombinant fusion protein. The multiclonalantibodies were tested by indirect ELISA, and the results shows that the titer of multiclonalantibodies titer was 1:12800. Western blotting experiment was implemented with specific multiclonalantibodies. The result shows that specific multiclonalantibodies could specific binding with its recombinant protein, and it is suggested that the recombinant protein has good immunogenicity.According to the PRRSV andβ-actin gene sequences included in GenBank, we designed specific primers and established the method of relative quantitative to detect PRRSV.Andβ-actin is set as a reference gene to establish relative quantitative method. Through the test of real-time PCR method ,we obtained a specific dissolution peak of the primers, it revealed that the primers’ specificity are well.The PAM cells were cultured in the 24-hole cell culture plate. After 6 hours, the mouse anti-Sn150 serum was added to the PAM cells. While the PAM cells were closed with the serums after an hour,we inoculated PRRSV ,respectively cultivating 6h, 12h, 15h, 18h, 24h, and collected cells and supernatants. The dynamic transcription of the virus in the PAM cells was detected by real time-PCR. At the same time, healthy PAM cells were recognized as negative control. The result showed that the PRRSV mRNA transcription level of test group closed by mouse anti-Sn150 specific antibodies at each time have been higher than that of the control group corresponding to each time,obviously and the specific antibodies of anti-Sn150 enhanced the replication of PRRSV in PAMs.Therefore,it noted that the progress that PRRSV into PAM may exist more complex regulatory mechanism.The PAM cells were cultured in the 24-hole cell culture plate with virus solution including 200 TCID50. After adsorbed one hour, cells were treated with 100ng/ml LPS,and then set up two controls,which were inoculated virus and health cells, incubated 6h, 12h, 15h, 18h, 24h respectively.After that, collecting cells and supernatants,detecting the replication copies of PRRSV mRNA with the established real-time PCR method.The results show that compared to PRRSV infecting PAM untreated with LPS,the transcription level of PRRSV was delayed for the peak in PAM treated with LPS after infected with PRRSV,and the transcription of PRRSV mRNA slightly up-regulated.They illustrated that LPS could increase the proliferation of PRRSV in PAMs.According to the IFN-αgene sequence included in GenBank, we designed specific primers and established the method of relative quantitative to detect IFN-α.Andβ-actin is set as a reference gene.Through the method ,we obtained a specific dissolution peak of the primers, it revealed that the primers’ specificity are well.The PAM cells were cultured in the 24-hole cell culture plate with virus solution including 200 TCID50. After adsorbed one hour, cells were treated with 100ng/ml LPS,and then set up two controls,which were inoculated virus group and health cells group, incubated 6h,12h,15h,18h,24h respectively.After that,collecting cells and supernatants. The dynamic transcription of IFN-αin the PAM cells were detected by real time-PCR.The results displayed that compared to the health PAM,the transcription level of IFN-αin health PAMs induced by LPS has no significant change at different times.And the comparison between PAM infected with PRRSV stimulated with LPS and unstimulated with LPS,the transcription level of IFN-αreached to the peak later,and the level of IFN-αmRNA dropped significantly.And then,it showed that LPS inhibited production of IFN-αin PAMs.As a result of PAM infected with PRRSV producing much more IFN-αthan health PAMs,PRRSV could induce the cells generating IFN-α. Revealing that LPS could induce the inhibiting effect of organism on antiviral immune response.

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