Preliminary Identification Of Key Endogenous LncRNA Of Bile Secretion Of HepG2 Cells Affected By Swine Hepatitis E Virus ORF3 Protein

Swine hepatitis E（SHE）is a zoonotic infectious disease caused by swine hepatitis E virus（SHEV）.As the key virulence protein of SHEV,open reading frame 3（ORF3）plays an important role in its pathogenesis.Jaundice,as the most important clinical symptom of SHE,is caused by bile secretion dysfunction,and its internal mechanism is still unclear.Long non-coding RNA（lnc RNA）,as a new post transcriptional regulatory molecule,its abnormal expression will affect the occurrence and development of many diseases.To explore whether SHEV ORF3 protein regulates the secretion of bile by affecting the key endogenous lnc RNA of cells,which leads to the occurrence of jaundice symptoms.In this study,we used adenovirus to mediated the overexpression of SHEV ORF3 protein in Hep G2 cells.Full transcriptome sequencing technology was used to study the effect of SHEV ORF3 protein on the lncrna expression profile of Hep G2 cells.Through the functional enrichment analysis of GO and KEGG,the key endogenous lnc RNA of Bile Secretion of Hep G2 Cells Affected by SHEV ORF3 Protein was preliminarily identified.The main research contents are as follows:1.Prokaryotic expression of SHEV ORF3 protein and preparation of polyclonal antibody.The SHEV ORF3 gene was amplified by PCR,the prokaryotic expression recombinant plasmid p ET-28a-ORF3 was constructed,and the His-ORF3 fusion protein was expressed in E.coli BL21（DE3）competent cells.Fusion protein was purified by Ni²⁺-NTA superfluid column affinity chromatography,and antiserum was prepared by immunizing New Zealand male rabbits.The titer of serum antibody was determined by indirect ELISA,and the specificity of polyclonal antibody was tested.The results showed that the length of ORF3gene amplified by PCR was 345 bp,and the prokaryotic expression recombinant plasmid p ET-28a-ORF3 was successfully constructed.When E.coli BL21（DE3）competent cells containing recombinant plasmid were induced at 37℃for 6 hours,the expression of fusion protein was the largest,and different IPTG induction concentrations had little effect on the expression of fusion protein.Western blotting confirmed that the protein was the target protein with His-tag tag,and the purity of the target protein was more than 80%.The titer of polyclonal antiserum is between 1:12 800-1:25 600,which has good specificity.2.Packaging preparation and target cell infection of recombinant adenovirus overexpressing SHEV ORF3 gene.The recombinant adenovirus shuttle plasmid ADV4-ORF3 was constructed by linking SHEV ORF3 gene with ADV4 vector.It was mixed with p GP-Ad-Pac（skeleton plasmid）and co transfected into 293A cells.The recombinant adenovirus was collected,amplified,purified and detected.Hep G2 cells were infected with recombinant adenovirus to explore the multiplicity of infection（MOI）and infection time.The expression levels of SHEV ORF3gene and protein were detected by observing enhanced green fluorescent protein（EGFP）,q RT-PCR and Western blotting.The results showed that the recombinant adenovirus shuttle plasmid ADV4-ORF3 was successfully constructed,and the recombinant adenovirus titer was about 5×10⁸PFU/m L.When recombinant adenovirus infected Hep G2 cells with MOI of 15:1for 36 hours,the overexpression effect of ORF3 protein was the best and the cell morphology was normal.Green fluorescence signals were observed from a large area of cells under the inverted fluorescence microscope.q RT-PCR showed that the expression of ORF3 gene in the experimental group（Ad＿ORF3）was significantly（p<0.01）higher than that in the control group（Ad＿GFP）.Western blotting showed that there were specific bands at the size of about13.7 k Da in the swimming lane of the experimental group,indicating that SHEV ORF3protein was successfully overexpressed in Hep G2 cells.3.Analysis of lnc RNA expression profile of Hep G2 cells affected by SHEV ORF3protein and preliminary excavation of lnc RNA related to bile secretion.Hep G2 cells were infected with adenovirus under the best conditions,and the experimental group（Ad＿ORF3）and the control group（Ad＿GFP）were set up.The total RNA was extracted by Trizol method,and the lnc RNA expression profile of SHEV ORF3 protein affecting Hep G2 cells was established by using whole transcriptome sequencing technology.The lnc RNA target genes were analyzed by GO and KEGG function enrichment,the key endogenous lnc RNA of Bile Secretion of Hep G2 Cells Affected by SHEV ORF3 protein was preliminarily identified.The results showed that the expression density map of each sample of high-throughput sequencing accorded with the normal distribution,and the expression trend of biological repeated samples was consistent,which met the requirements of subsequent data analysis.Compared with the control group,319 significantly differentially expressed lnc RNAs were identified in the experimental group,including 124 known＿lnc RNA and 195novel＿lnc RNA,of which 158 were up-regulated and 161 were down regulated.Differentially expressed lnc RNAs are mainly involved in 1 868 biological processes,415 cellular component and 533 molecular functions.They are mainly enriched in signal pathways such as carbohydrate digestion and absorption（ko04973）,complement and coagulation cascades（ko04610）and pentose and glucuronate interconversions（ko00040）.Two known＿lnc RNA and four novel＿lnc RNA related to bile secretion（ko04976）signal pathway were screened and verified by q RT-PCR.The differential expression results of three novels＿lnc RNA were consistent with the sequencing results.It was preliminarily identified that the key endogenous lnc RNAs of SHEV ORF3 protein affecting the bile secretion of Hep G2 cells were lnc RNA UBC（MSTRG.6881.4）,lnc RNA UBC（MSTRG.6881.9）and lnc RNA UBC（MSTRG.6881.12）.In conclusion,this study successfully realized the prokaryotic expression of SHEV ORF3 protein and the preparation of polyclonal antibody,which provided specific test antibody for the subsequent intracellular overexpression test of SHEV ORF3 protein.High titer overexpression adenovirus was prepared by packaging,which mediated the overexpression of SHEV ORF3 protein in Hep G2 cells.It was preliminarily identified that the key endogenous lncrnas of SHEV ORF3 protein affecting bile secretion of Hep G2 cells were lnc RNA UBC（MSTRG.6881.4）,lnc RNA UBC（MSTRG.6881.9）and lnc RNA UBC（MSTRG.6881.12）.This study laid a theoretical foundation for further explaining the molecular regulation mechanism of SHEV ORF3 protein affecting bile secretion of Hep G2cells,and also provided a certain reference basis for later revealing the pathogenic mechanism of SHEV.