Study On Prokaryotic And Yeast Expression Of PoIFN-?2/?/? From Congjiang Xiang-pig And Its Anti-PRRSV Activity

Porcine reproductive and respiratory syndrome virus?PRRSV?is a serious contact infectious disease affecting the global pig industry.When PRRSV infects cells,it induces both innate and adaptive immune responses,with the main features of gestational sow reproductive dysfunction,weaned pig pneumonia,severe respiratory symptoms,and increased mortality in growing pigs.Interferon?IFN?is produced and released by pathogen-stimulated host cells and exhibits direct antiviral activity by inhibiting viral replication and mediating the corresponding cellular immune function.In this study,molecular biology,genetic engineering,cell biology and other experimental techniques were used to obtain the gene sequences of IFN-?2,IFN-?and IFN-??CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-??coding regions from Congjiang Xiang-pig in Guizhou.The nucleotide sequence and coding amino acid sequence were analyzed by bioinformatics,and the prokaryotic expression vector carrying CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?gene and Saccharomyces cerevisiae?S.cerevisiae?expression vector were constructed.Prokaryotic and yeast expression of CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?were performed.The effect of recombinant protein CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?expressed by S.cerevisiae on PRRSV replication was analyzed at the level of mRNA and protein.1.Amplification of CJ-poIFN-?2/?/?gene and its bioinformatics analysisIn this study,three pairs of primer-specific amplification CJ-poIFN-?,CJ-poIFN-?and CJ-poIFN-?coding region were designed.After the full length of the coding region of the CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?gene was amplified by RT-PCR,it was ligated to the Cloning plasmid pUCm-T to obtain a recombinant plasmid CJ-poIFN-?2/?/?-pUCm-T.The sequencing results showed that the nucleotide sequences of the CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?gene were 546,561 and 501 bp.Bioinformatics analysis of the obtained gene sequences revealed that:1)CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?were homologously analyzed with other IFN nucleotide sequences published in GenBank,and the results showed that CJ-poIFN-?2 and the reference species IFN-?2 nucleotide homology are 69-98.7%,and the amino acid homology is 46.1-97.8%.Among them,CJ-poIFN-?2 has the highest homology with pig IFN-?2?GenBank NO:NM₀01130219?.It has the lowest homology with mouse?GenBank NO:X01969?.CJ-poIFN-?shares 65.2-100%homology with the reference species IFN-?,and amino acid homology is 8.1-100%,among which CJ-poIFN-?Meishan pig?GenBank NO:AY687281?and Bama Pig?GenBank NO:KJ147517?has the highest amino acid homology and the lowest amino acid homology with mouse?GenBank NO:K00020?.The homology of CJ-poIFN-?with the reference species IFN-?nucleotide is 42.4-99.8%,and the amino acid homology is 8.4-93.4%,among which CJ-poIFN-?and Jianbaixiang pig?GenBank NO:JF906510?,the IFN-?nucleotides of Chenghua pig?GenBank NO:AF493993?,Tibetan Pig?GenBank NO:AY293733?and Meishan pig?GenBank NO:DQ666352?have the highest homology,and it has low homology with mouse?GenBank NO:X01969?.2).The secondary structure of CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?gene coding protein is mainly alpha-helix and random coil.It does not contain transmembrane domain and has two signal peptide digestion sites.CJ-poIFN-?2 and CJ-poIFN-?were unstable proteins,and CJ-poIFN-?was stable protein.3)CJ-poIFN-?2 consists of 181 amino acids with 22 Thr-phosphoric sites and 1 O-glycosylation site,without N-glycosylation sites.CJ-poIFN-?consists of 187 amino acids,47 Thr phosphorylation sites and 3 N-glycosylation sites,without O-glycosylation sites.CJ-poIFN-?consists of 167 amino acids,including 54 Thr phosphorylation sites,2 N-glycosylation sites and 3 O-glycosylation sites.This study laid the foundation for enriching the knowledge of Congjiang Xiang-pig and studying the bioactivity of CJ-poIFN.2.Prokaryotic expression of CJ-poIFN-?2/?/?and preparation of polyclonal antibodyIn this study,the coding region of CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?gene was obtained by RT-PCR and cloned into prokaryotic expression plasmid pET28a.The recombinant plasmid CJ-poIFN-?2/?/?-pET28a was obtained and transformed into Escherichia coli BL21?DE3?for induced expression.SDS-PAGE and Western Blotting methods were used to detect the supernatant of the bacterial culture carrying the recombinant plasmid CJ-poIFN-?2/?/?-pET28a,the induction of bacterial liquid precipitation and the expression of uninduced bacterial liquid.Anti-CJ-poIFN-?serum was prepared by immunizing mice with the recombinant protein purified by nickel column.The titer and reactivity of anti-CJ-poIFN-?serum were analyzed by ELISA and Western Blotting.The results showed that:1)No specific band was found in the recombinant protein CJ-poIFN-?2 when SDS-PAGE was performed.2)The induced bacterial cell-precipitated sample carrying the recombinant plasmid CJ-poIFN-?-pET28a had a specific band at about 24 kD,which was consistent with the expected results,indicating that the recombinant protein CJ-poIFN-?mainly exists in the form of inclusion bodies.3)SDS-PAGE results of IPTG-induced bacterial solution carrying recombinant plasmid CJ-poIFN-?-pET28a showed that the specific bands of 19.4kD could be seen in the expected band position,indicating that the recombinant protein existed mainly in the form of inclusion bodies.19.4 kD could be seen in Western blotting.The results of purification by nickel column showed that the elution effect of 400 mmol/L imidazole was better.4)The purified recombinant protein CJ-poIFN-?was subcutaneously immunized with 50?g/mice to prepare polyclonal antibodies against CJ-poIFN-?.ELISA results showed that the titer of high immunized serum against CJ-poIFN-?protein was 1:25,600.Western Blotting results showed that the specific band of about 19.4 kD,indicating that the prepared polyclonal antibodies from CJ-poIFN-?mice had good reactivity.This study laid a foundation for further research on the biological activity of CJ-poIFN and speeding up the effective utilization of quality resources from Congjiang Xiang-pig.3.Expression of CJ-poIFN-?2/?/?in S.cerevisiaeIn this study,specific primers carrying His tag were designed for the obtained sequence of CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?gene.The expression plasmid CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?gene of S.cerevisiae was constructed and transformed into INVSc1,and the positive strain CJ-poIFN-?2/?/?-pYES2-INVSc1 was obtained by screening.The positive strains were induced and cultured in the SC-U inducing medium,and the bacteria were collected at12 h,24 h,48 h,72 h,and 96 h respectively for OD₆₀₀00 determination,the expression of recombinant protein was detected by SDS-PAGE and Western Blotting method and the specific poIFN-?2/?/?ELISA kit.SDS-PAGE results showed that the expression of CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?in S.cerevisiae was low.CJ-poIFN-?2/?/?-pYES2-INVSc1-96 h was selected for ELISA and Western blotting verification.ELISA results showed that the concentrations of CJ-poIFN-?,CJ-poIFN-?and CJ-poIFN-?expressed by S.cerevisiae were 45.08,15.95 and 25.63pg/mL respectively after 96 h of induction culture.Western Blotting results showed that CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?expressed by S.cerevisiae had a specific band at 43-55kD.This study laid a foundation for further study on the effect of recombinant protein expressed by S.cerevisiae on PRRSV replication.4.Study on the anti-PRRSV activity of CJ-poIFN-?2/?/?expressed by S.cerevisiaeIn this study,the supernatant of CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?expressed by S.cerevisiae was pretreated for 24 h and PRRSV was inoculated into Marc-145 cells.The activity of CJ-poIFN-?2/?/?-anti-PRRSV expressed by S.cerevisiae was calculated by Reed-Muench method.CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?supernatants purified by S.cerevisiae were pretreated with Marc-145 cells for 24 h,then inoculated with PRRSV for 24 h to analyze the effect of CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?expressed by S.cerevisiae on the transcription and translation of PRRSV N gene.The effect of CJ-poIFN expressed by S.cerevisiae on the transcription levels of IFN-stimulated genes OAS and Mx1 was analyzed by applying S.cerevisiae-expressing CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?supernatant to PK15 cells for24 h.The results showed that 1)CJ-poIFN-?2,CJ-poIFN-?and CJ-poIFN-?expressed by S.cerevisiae can effectively inhibit the proliferation of PRRSV by diluting 2^2.4,2².35.35 and 2².21.21 times,respectively.2)CJ-poIFN-?2 and CJ-poIFN-?expressed by S.cerevisiae can effectively inhibit the mRNA and translation level of PRRSV N gene.3)CJ-poIFN-?expressed by S.cerevisiae can effectively up-regulate the mRNA level of Mx1 gene,and CJ-poIFN-?2 expressed by S.cerevisiae can effectively up-regulate the mRNA levels of OAS and Mx1 genes.