Preliminary Exploration Of Interaction Mechanism For P-Body Assembly Protein LSm1,DCP2 And PRRSV

PRRSV(Porcine reproductive and respiratory syndrome virus,PRRSV)is highly variant and infectious single stranded positive strand RNA virus,which has not been effectively controlled and has brought great losses to the world pig industry.P-Body(processing bodies,P-Body)is m RNA processing bodies,which is a m RNA-protein complex that is involved in the regulation of gene transcription in the cytoplasm.It is a kind of subcellular structure without envelope.And it mainly involves in the important life activities such as translation inhibition,RNA mediated gene silencing,m RNA degradation and so on.The assembly of P-body can affect the replication of the virus in the cell,and the replication of the virus affects the assembly of P-body in turn.LSm1 and DCP2 are located in P-body,which are landmark protein of P-body and can affect the replication of many viruses.In order to explore the interaction PRRSV replication and host P-body,we used the experimental technique of molecular biology,genetic engineering,cell biology,and so on,to obtain PRRSV nucleocapsid protein N,P-Body landmark protein DCP2 and LSm1 gene sequence,analyze amino acid sequence of the nucleotide sequence and coding,construct the prokaryotic expression plasmid carrying N,LSm1,DCP2.We induced expression recombinant plasmid and purified recombinant protein.And immunized rabbits to gain hyperimmune serum.Finally,We preliminarily explored the effects of PRRSV replication to host DCP2 and LSm1 on gene transcription level.1.Amplification of PRRSV N gene and gain of its encoding protein hyperimmune serum.PCR amplified the full length of the N gene coding region,which was connected to the prokaryotic expression plasmid p Cold I,and obtained the recombinant plasmid p Cold I-N.Sequencing showed that the nucleotide sequence length of N gene was 372 bp.The results of sequence analysis showed that PRRSV strain E11105 and the North American type strains VR-2332,Lelystad European type strains virus(LV),China outbreaks highly pathogenic PRRSV strains JXA1 in 2006,China strains CH-1a nucleotide sequence homology were 93.3%,34.7%,99.2%,95.4%,amino acid sequence homology was 94.4% 15.3%,94.4%,99.2%,showed that E11105 strain belonged to North American type strains,which close relationship with high pathogenicity PRRSV in China.Prediction results showed that N protein didn't have signal peptide,which was non secreted protein,and had 5 B cell epitope.The molecular weight of recombinant N protein was about 16.7 k D,which mainly was the form of insoluble protein,but also soluble protein.The recombinant N protein was purified to immunize rabbits,and the hyperimmune serum of N protein was successfully obtained.There was higher titer of rabbit serum with soluble recombinant N protein than no soluble recombinant N protein,namely 1:12800 and1:3200.The study results laid the foundation for studying the functional properties of PRRSV N protein and the pathogenesis of PRRSV.2.Amplification of P-Body Assembly protein DCP2 gene and gain of its hyperimmune serum.After DCP2 was amplified by PCR,we connected it to the cloned plasmid and sequenced.The results showed that we obtained the two transcription variants full-length of DCP2 gene encoding region,respectively X1 and X2 variants,their length are 1263 bp and 1158 bp.The encoding amino acids of DCP2 X1 transcript variants gene were more than 36 than X2 from 314 th amino acids to 349 th amino acids position.We selected the longer X1 transcript variants to analyze.The results showed that DCP2 transcript variants X1 nucleotide sequence and it encoding amino acid sequence was high similarity and conserved in mammals,which was more than 94%.The DCP2 transcription variants X1 protein of Marc145 cell had no transmembrane structure and signal peptide,and it was non secretory protein,which had good dominant epitope.The DCP2 transcript variants X1 gene was cloned into the prokaryotic expression vector p Cold I,and induced expression the recombinant protein.The result showed that it was no soluble protein,and the recombinant DCP2 transcript variants X1 protein was purified to immunize rabbits.We successfully obtained the hyperimmune serum of recombinant DCP2 transcript variants X1 protein,and Its titer was 1:6400.The results laid a solid foundation for the further study the function of DCP2 and its role and mechanism in viral replication.3.Amplification of P-Body Assembly protein LSm1 gene and construction of prokaryotic expression plasmid.After two rounds of nested PCR amplification,we successfully obtained the LSm1 gene encoding region full-length from Marc145 cells,and cloned it into p Cold I prokaryotic expression vector.The sequencing result showed that we successfully obtained 402 bp LSm1 gene sequence from Marc145,which could encode 133 amino acids.The nucleotide sequence of LSm1 gene from Marc145 cells was high similarity with the human,following the marine mammals,the terrestrial wild animal and livestock,from 92.8% to 99.8%,and the encoding amino acid is not the law,but the amino acid sequence similarity is high,from 97.8% to 99.3%.It is predicted that LSm1 protein had no transmembrane structure and signal peptide,and it was non secretory protein,which had 4 good B cell dominant epitopes.Induce expression result showed that the Recombinant Prokaryotic expression plasmid was no expression recombinant LSm1 protein.By the study,we inferred that LSm1 protein transcription and expression levels may be low in cells,because c DNA of cells must pass by nested PCR to appear LSm1 band.LSm1 recombinant expression protein was not expression in the system of the study.We need to consider to change expression system,use truncated expression,fusion expression,and other methods to get the high concentration recombinant prokaryotic expression LSm1 protein.This test provides a reference for the amplification of the LSm1 gene.and laid a solid foundation for gain artificial recombinant expression protein and specific antibody of LSm1 and the functional mechanism of LSm1 at the cellular level and its role in viral replication.4.Effects of PRRSV replication to the LSm1 and DCP2 genes transcription level in host.We inoculated the cells with PRRSV,collected cells respectively in 0h,4h,8h 12 h,24h,36 h,48h and extracted total RNA reversed transcription to c DNA.and detected PRRSV N,Marc145 LSm1 and DCP2 gene transcription level by q PCR.The results showed that with the replication of PRRSV in Marc145 cells,the transcription level of LSm1 in host cells increased significantly in each period,while the DCP2 transcription level of 0-8 was significantly decreased,and 8-48 h was significantly increased.These results indicate that PRRSV replication has a certain effect to the transcription level of P-Body marker protein LSm1 and DCP2 in host cells.The results provide a solid foundation for further study on the relationship between PRRSV replication and LSm1 and DCP2.