Eukaryotic Expression Of Truncated NSP2 And Its Role In PRRSV Replication

Porcine reproductive and respiratory syndrome(PRRS)is one of the most serious diseases in the swine industry.The pathogenic porcine reproductive and respiratory syndrome virus belongs to one member of the Nested Virus Arteriitis Virus Section,The genome is a single strand of positive-stranded RNA with at least 10 ORFs,that is,orf1 a,orf1b,orf2 a,orf2,orf3-7,and orf5 a gene.ORF1 encodes all non-structural proteins and 14 non-structural protein NSP1α,Nsp1β,Nsp2-6,Nsp7α,Nsp7β,Nsp8-12,Nsp2 s,Nsp2 is a multifunctional protein encoded by PRRSV,Including cysteinase region,hypervariable region,transmembrane region,Nsp2 has important biological significance in the replication and pathogenesis of PRRSV.This study carried out the PRRSV NSP2 analysis of the clinical disease in Guizhou Province between 2016 and 2017,analyzed the characteristics of the full-length NSP2 sequence of the clinical strain GZ-R in 2017,constructed a truncated NSP2 eukaryotic expression vector,and analyzed the expression of the truncated NSP2 in HEK293 cells,preliminary explored the role of truncated NSP2 in PRRSV replication,it aims to lay a foundation for understanding the PRRSV epidemic in our province and exploring the pathogenesis of PRRSV.PRRSV clinical case diagnosis.Clinical case samples collected in 2016-2017 were observed with clinical symptoms and dissection,histopathological observation and the RT-PCR detection of PRRSV NSP2 using PRRSV test kit of century yuan company.The pigs were mainly dyspnea,cyanosis,lymphocytic infiltration in lung and spleen,from the 24 suspected PRRSV disease samples,9 samples were positive for PRRSV NSP2 by RT-PCR,and the positive rate is 33.3%(8/24).Analysis of GZ-R NSP2 sequence of prevalent PRRSV strains in Guizhou.using molecular biology method,the full-length NSP2 of PRRSV positive sample GZ-R was sequence amplified,TA cloned and sequenced,the sequences were analyzedusing DANSar and NCBI online software.Results(1)The pMD18T-GZ-R-NSP2-L plasmid carrying the GZ-R NSP2full-length sequence and the pMD18T-GZ-R-NSP2-S plasmid carrying the GZ-R-NSP2 small fragment were obtained.The sequencing results showed that,the full length of GZ-R NSP2 is 2980 bp and encodes 993 amino acids,the small fragment of GZ-R NSP2 is 1131 bp and encodes 377 amino acids.The GZ-R NSP2 fragment has a large fragment N-terminal 1-945 bp and C-terminal 2795-2980 bp sequence,the 956-2794 bp sequence that lacks the large GZ-R NSP2 fragment,it is likely to be a new NSP2 subtype.(2)Analysis of GZ-R NSP2 whole-gene amino acid homology results showed that,the homology between the amino acid encoded by the NSP2 gene of the GZ-R strain and the 22 reference strains was 24.3% to 99.0%,the homology with the American strain was 54.1% and 99.0%.The homology of NSP2 gene with the classical American representative strain ATCC VR-2332 was 77.1%,and the homology with highly pathogenic strains HUN4 and JXA1 in China was98.4% and 98.5%,respectively,and NJ-1106.The amino acid sequence of the strain had the highest homology of 99.0%.Further compared with the amino acid sequence of ATCC VR-2332 Ch-1a HUN4HUN4JXA1 strain,we can find there are 291 amino acid deletions in the NSP2 gene of GZ-R,the deletion positions are consistent with the highly pathogenic PRRSV JXA1 and HUN4.Construction and expression analysis of truncated NSP2 eukaryotic expression vector.Truncated NSP2 eukaryotic expression plasmid was constructed using pcDNA3.1-5’Flag-NSP2-TL as a template by using conventional molecular methods.The truncated NSP2 eukaryotic expression plasmid was transfected into Marc145 cells by liposome 2000,analyzed the expression of truncated cells in the cells.Results: after the PCR,sequencing,restriction endonuclease digestion and IFA test,the eukaryotic expression vector of truncated Nsp2 was successfully constructed.(2)The results of IFA test show that,the expression of the recombinant proteins pFlag-Nsp2-147-323 aa and pFlag-Nsp2-47-323 aa in HEK 293 was detected whether labeled Flag antibody or specific NSP2 mAb.4.Study on the role of truncated Nsp in the replication of PRRS.Therecombinant plasmid pcDNA3.1-NSP2-147-323 aa and the recombinant plasmid pcDNA3.1-NSP2-47-323 aa was transfected into Marc145 cells by liposome,PRRSV was infected 24 h after transfection,the supernatant was collected 24 hours after infection to measure the TCID50.The result show that there was no significant difference in TCID50 between pcDNA3.1-NSP2-147-323 aaA group and empty vector group.Marc145 is the susceptible host cell of PRRSV,However,the low efficiency of transfection and expression of the cells may be the reason for the difference of the results.