Experimental Research Of The Role Of CYP3A29 Gene In Pathogenesis Of Mycoplasmal Pneumonia Of Swine

Cytochromes P450 are a class of heme-thiolate enzymes that activate molecular oxygen used in the oxidation of a variety of substrates, which are responsible for synthesizing steroids, activating procarcinogens, and metabolizing pharmaceuticals. Recent research suggests that cytochromes P450s play an important role in anti-inflammation.CYP3A29 is one of CYP3 A subfamily, mycoplasmal pneumonia of swine is a chronic respiratory disease found at pig farms worldwide. Our preliminary study finds that CYP3A29 is closely related to the pathogenesis of mycoplasmal pneumonia of swine.Objective:To study the role of CYP3A29 gene in pathogenesis of mycoplasmal pneumonia of swine by establishing the porcine alveolar macrophages cell line which stably expressed CYP3A29 protein.Methods:The cDNA of CYP3A29 gene was amplified by RT-PCR and was then subcloned into plasmid pMD18-T vector to obtain a recombinant plasmid PMD18-T-CYP3A29, which was verified by DNA sequencing. CYP3A29 gene fragments and pcDNA3.1(+)/zeo vector fragments were purified after digestion with Xho I and Not I and were then ligated to each other using T4 DNA ligase to generate a pcDNA3.1(+)/zeo-CYP3A29 plasmid, which was verified by restriction enzyme digestion and DNA sequencing. Results sequencing and restriction enzyme digestion analysis showed that CYP3A29 gene was correctly cloned into pcDNA3.1(+)/zeo vector. The porcine alveolar macrophages cell lines 3D4/21 was transfected by recombinant plasmid pcDNA3.1(+)/zeo-CYP3A29 using lipofectamine 2000. After screened with Zeocin for four weeks, single clones were got out and methods such as RT-PCR and Western blot were used to detect the expression of CYP3A29 in PAM cells. Study the expression of IL-1β、 IL-6、IL-8、TNF-α、PPAR-γ among experimental group, control group and negative control group after mycoplasma hyopneuminiae stimulation at different time.Results:1 The complete CDS of CYP3A29 gene was cloned by RT-PCR.CDS of CYP3A29 was shared 99% identity with procine CYP3A29 in NCBI.2 Sequencing and restriction enzyme digestion analysis showed that CYP3A29 gene was correctly cloned into pcDNA3.1(+)/zeo vector.3 PAMs were screened with zeocin of different concentrations (50μg/ml、100μg/ml、 150μg/ml、200μg/m、300μg/ml),all of cells perish with 250μg/ml、300μg/ml zeocin on the third day and on the seventh day, all of cells perish with 200p,g/ml zeocin and most of cells were dying with 150μg/ml zeocin on the Eleventh Day4 PAMs were cultured in 24-well plates, which were transfected by recombinant plasmids with DNA consistencies of 125ng/well,250 ng/well,375 ng/well,500 ng/well, Best result is 500ng per well.5 The PAM cell lines with stable expression of CYP3A29 gene had been successfully obtained. CYP3A29 mRNA and protein can be also detected by RT-PCR and Wsetern-blot6 Mhp can markedly increased proinflammatory cytokine mRNA exresssion,the expression levels of IL-1β, IL-6, IL-8, TNF-a in experimental group which were significantly higher (P<0.01) occurred respectively at 4h,8h,4h,8h, while the control group and negative control group occurred respectively at 18h,12h,12h,12h (no significantly higher in negative control group). After the same time Mhp stimulation, the expression levels of IL-1β mRNAs in experimental group was significantly difference (P<0.01) than control group and negative control group at 4h,8h and 12h(P<0.05); meanwile the significant difference of IL-6 at 8h(P<0.05), and the significant difference of IL-8 at 4h(P<0.01),8h and 18h(P<0.05), the significant difference of TNF-a at 8h,12h,18h (P<0.01).7 The expression levels of PPAR-y were briefly rise at 18h,and The expression levels of PPAR-y in experimental group was less intensively than control group and negative control group.Conclusions:1 CYP3A29 gene was obtained and was correctly cloned into pcDNA3.1(+)/zeo vector.2 Cell transfection system and zeocin concentration were optimized.and the PAM cell lines with stable expression of CYP3A29 gene had been successfully obtained.3 PAMs with CYP3A29 gene showed high sensitivity to Mhp and there was a higher inflammatory reaction in experimental group which stably expressed CYP3A29 protein than control group and negative control group.so CYP3A29 gene may be relevant to the pathogenesis of mycoplasmal pneumonia of swine.4 Expression of PPAR-γ may be in reference to CYP3A29 gene.