LncRNA Expression Profiling And Preliminary Functional Exploration Of Brucella WbkC Protein Affecting RAW264.7 Cells

Brucellosis is a zoonotic infectious disease caused by Brucella,with abortion and infertility as the main clinical features,which seriously endangers animal husbandry production and public health safety.Brucella is a facultative intracellular parasite that has evolved a survival mechanism that escapes the"catch and kill"of the host in the target cell.Chronic infection can be formed by regulating the level of autophagy in target cells so that it can survive in host cells.Brucella lipopolysaccharide（LPS）is composed of lipid A,core oligosaccharide and O-side chain,and is an important virulence factor that induces immune responses in animals.The formyl transferase encoded by the wbkC gene plays an important role in the synthesis of the O-side chain of LPS.Long non-coding RNA（lncRNA）is involved in the regulation of various life activities and can regulated the translation of m RNA through modification This topic uses recombinant adenovirus to mediate the overexpression of Brucella melitensis（B.melitensis）wbkC protein in RAW264.7 cells,and uses transcriptome sequencing technology to study the lncRNA expression profile of B.melitensis wbkC protein affecting RAW264.7 cells.Through GO and KEGG functional enrichment analysis,it was preliminarily predicted that Brucella wbkC protein could affect the autophagy and immune functions of RAW264.7 cells.Main research contents of this subject are as follows:1.Prokaryotic expression of B.melitensis wbkC and preparation of polyclonal antibodiesThe B.melitensis wbkC gene was amplified by PCR,the recombinant plasmid p ET-28a-wbkC was constructed,and BL21 competent cells were transformed to express the His-wbkC fusion protein;the fusion protein was purified by Ni2+-NTA affinity chromatography,and New Zealand male rabbits were immunized four times.The polyclonal antibody of wbkC protein was prepared;the titer and specificity of the polyclonal antibody were determined by indirect ELISA and Western blot.Results:The recombinant fusion protein His-wbkC was expressed in the form of inclusion bodies at a high level after induction with 1.0 mmol/L IPTG at 37°C for 4 h;SDS-PAGE analysis showed a single clear wbkC protein;There is a specific band at 29 k Da;the titer of the antibody detected by indirect ELISA is between 1∶6 400 and 1∶12 800,and it has good specificity.2.Preparation and infection of recombinant adenovirus overexpressing B.melitensis wbkC geneThe B.melitensis wbkC gene was cloned into the adenovirus vector ADV4,the shuttle expression plasmid ADV4-wbkC was constructed,and the 293A cells were co-transfected with the backbone plasmid p GP-Ad-Pac,and the recombinant adenovirus particles Ad＿wbkC were collected,the titer of recombinant adenovirus was detected by micropan-cytopathic method,the recombinant adenoviruses Ad＿wbkC and Ad＿GFP were infected with RAW264.7 cells.The infection conditions were optimized,and q RT-PCR and Western blot were used to verify the overexpression of B.melitensis wbkC.Results:The titers of recombinant adenovirus Ad＿wbkC and Ad＿GFP were both1×10¹⁰ PFU/m L;Western blot showed that in the Ad＿wbkC test group,a specific band appeared at the molecular weight of 29 k Da;q RT-PCR showed that the Ad＿wbkC test group wbkC gene The expression level was about 1 510 times that of the control group Ad＿GFP,and the difference between the groups was extremely significant（p<0.01）.3.Analysis of B.melitensis wbkC protein affecting the expression profile of RAW264.7cells and mining of autophagy pathway lncRNARAW264.7 cells were infected with Ad＿GFP and Ad＿wbkC under optimized conditions,and total cell RNA was extracted by Trizol method.Using transcriptome sequencing technology,the lncRNA expression profile of B.melitensis wbkC protein affecting RAW264.7 cells was constructed.GO and KEGG functional enrichment analysis of lncRNA target genes was carried out,and the key endogenous lncRNA of B.melitensis wbkC protein affecting the autophagy pathway of RAW264.7 cells were preliminarily identified.Results:Compared with Ad＿GFP in the control group,32 lncRNA were up-regulated and 30 lncRNA were down-regulated in the Ad＿wbkC experimental group.The TOP5 pathways of GO functional enrichment analysis are:DNA binding（GO:0003677）,immune system process（GO:0002376）,nucleobase-containing compound biosynthesis（GO:0034654）,RNA biosynthesis（GO:0032774）,polymer biosynthesis process（GO:0009059）;The TOP5 pathways of KEGG signaling pathway enrichment analysis are:rheumatoid arthritis（ko05323）,systemic lupus erythematosus（ko05322）,tuberculosis（ko05152）,NOD-like receptor signaling pathway（ko04621）,phagosome（ko04145）;Screening of lncRNA related to the autophagy pathway,q RT-PCR verified that two differentially expressed lncRNA were consistent with the sequencing results.The key endogenous lncRNA of B.melitensis wbkC protein affecting autophagy in RAW264.7 cells were preliminarily identified as lncRNA 4933430A20Rik（ENSMUSG00000110116）and lncRNA B930036N10Rik（ENSMUSG00000091993）.Conclusion:This subject obtained B.melitensis wbkC protein through prokaryotic expression,and immunized rabbits to prepare wbkC protein polyclonal antibody,which provided specific antibodies for the subsequent intracellular overexpression test of B.melitensis wbkC protein;The recombinant adenovirus overexpressing wbkC was prepared to realize the overexpression of B.melitensis wbkC protein in RAW264.7 cells;The experiment constructed the lncRNA expression profile of B.melitensis wbkC protein affecting RAW264.7 cells.The analysis showed that 32 lncRNA were up-regulated and 30 lncRNA were down-regulated,the wbkC can affect activities such as autophagy and immune response in RAW264.7 cells.The identified the key endogenous lncRNA of B.melitensis wbkC protein affecting autophagy in RAW264.7cells:lncRNA 4933430A20Rik and lncRNA B930036N10Rik.It provides a new research idea for further exploring the molecular regulation mechanism of B.melitensis wbkC protein affecting autophagy in RAW264.7 cells.