The Molecular Characterization Of Differentially Expressed OaLYPLA1 And OaPRDX6 From Ovis Aries Infected With Brucella

Brucellosis is a zoonosis which infects numerous species,widely distributing all over the world.In recent years,brucellosis has shown ascendant trend in domestic and abroad and has become a serious public health problem.At present,serological tests are used to detect antibodies in sera for diagnosing the brucellosis,however they are not able to discern the brucellosis vaccine inoculated sheep from the virulent Brucella infected ones.The differential diagnosis methods or molecular targets are focused on studying how to differ between the Brucella infection and the vaccine inoculation.In our lab,suppression subtractive hybridization(SSH)c DNA library of buffy coats from sheep(Ovis aries)infected with different virulent Brucella strains was constructed and the differentially transcribed genes were screened.Among these genes,a partial c DNA of LYPLA1 and PRDX6 were found.LYPLA1 also known as acyl-protein thioesterase 1(APT1)is a important protein with multifunctions.PRDX6 is a multifunctional protein and playing a major role in protection against oxidation,DNA damage,and lung phospholipid metabolism.More and more reports showed that PRDX6 is associated with diseases and as a novel systemic biomarker of some diseases.It is reported that both PRDX6 and LYPLA1 have the GXSXG motif which found in the active site of lipases,and the catalytic triad comprised of Ser,Asp,and His.Here the LYPLA1 and PRDX6 were studied and their biological activities and the differential expression were analysed,providing a theoretical and experimental basis for further studying their functions and the relationship with brucellosis.1.Molecular cloning,expression and m Ab preparation of LYPLA1 from Ovis ariesThe full-length c DNA of the LYPLA1 gene from Ovis aries(Oa LYPLA1)was cloned using rapid amplification of c DNA ends(RACE)technology and deposited in Gen Bank(accession number KJ000742).The full-length Oa LYPLA1 was 2457 bp with a 5′-untranslated region(UTR)of 24 bp,a 3′-UTR of 1740 bp with a poly(A)tail,and an open reading frame(ORF)of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da.The recombinant Oa LYPLA1 protein was expressed and purified,and its phospholipase activity was identified using egg yolk/agarose diffusion test.With the cell fusion technique,one hybridoma cell lines secreting monoclonal antibody(m Ab)against Oa LYPLA1 were obtained and named as 4A3,the isotype of the m Ab belonged to Ig G1.The specificity of the m Ab against the recombinant proteins and the native proteins extracted from tissues were analyzed using Western-blot.The results of the Western-blot analysis showed that the m Ab against Oa LYPLA1 were able to bind both of the recombinant and native proteins.2.Differential expression of LYPLA1 and PRDX6 from Ovis ariesThe tissue distribution analysis had been conducted and the resuts revealed that Oa LYPLA1 and PRDX6 from Ovis aries(Oa PRDX6)were constitutively expressed in tissues of sheep with the highest expression level of Oa LYPLA1 in the kidney and Oa PRDX6 in lung.Additionally,the m RNA levels of Oa LYPLA1 and Oa PRDX6 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were analyzed compared to untreated sheep.The m RNA levels of Oa LYPLA1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep and the m RNA transcription level of Oa PRDX6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis,while down-regulated in the group inoculated with a vaccine strain S2 of brucellosis.The results showed that the transcription levels of Oa LYPLA1 and Oa PRDX6 are both influenced by the infection of Brucella.It was implied that Oa LYPLA1 and Oa PRDX6 may have an important physiological role in the host response to Brucella.The function of Oa LYPLA1 and Oa PRDX6 in the host immune response to bacterial infection requires further study in the future.3.The establishment of LYPLA1 gene knockout RAW 264.7 cell modelThe CRISPR/Cas9 technology was used to construct a cell model knocked the LYPLA1 gene out.The PX459 recombinant plasmids with appropriately designed sg RNA and ORF sequence of Cas9 prorein was transfected into RAW 264.7 cells with electroporation.Finally,the cell without the LYPLA1 gene was screened and the genomic DNA and c DNA sequence of RAW 264.7 were analyzed via PCR and DNA sequencing to analyze the potential Off-target.The result showed that the CRISPR/Cas9 technology can be used for of establishing gene knockout RAW 264.7 cell model.It is shown that the LYPLA1 gene have influence in the macrophage killing ability.LYPLA1 gene was knocked out to increase macrophage killing ability towards intracellular Brucella S2 and suppress its killing ability towards intracellular Escherichia.4.The expression,biological activity and promoter analysis of Oa PRDX6The Oa PRDX6 with His6-tag was successfully expressed and purified.The DNA protection activity of Oa PRDX6 was identified using the method of Metal-catalyzed oxidation(MCO)assay.In order to investgate the regulatory mechanisms of Oa PRDX6.The transcriptional activity of promoter of Oa PRDX6 is investigated.A 3.4 kb 5′-flank region of the Oa PRDX6 gene with part sequences of m RNA was cloned and sequenced.The sequence was deposited in Gen Bank and assigned the accession number KM459018.Eight recombinant plasmids were constructed with truncated DNA fragment.The region from-108 nt to-36 nt of the promoter region of the Oa PRDX6 gene is identified contained basal transcriptional activity through the expression assay of the SEAP reporter gene.Here,the molecular characterization of Oa LYPLA1 and Oa PRDX6 was studied.The RAW 264.7 cell model without the LYPLA1 gene was established.This study provided a theoretical and experimental basis for further studying of the roles of LYPLA1 and PRDX6 during the infection of Brucella.