猪囊尾蚴dUTPase基因的克隆、表达及其产物的酶学活性分析

【目的】克隆、表达猪囊尾蚴dUTPase基因并分析其酶学活性。【方法】通过RT-PCR的方法克隆猪囊尾蚴dUTPase基因,将其与pET载体连接并在原核系统中高效表达;表达产物经Ni柱纯化后进行酶学活性测定。【结果】序列分析表明猪囊尾蚴dUTPase基因与六钩蚴的dUTPase基因核苷酸同源性为100%,而且其氨基酸序列中同样存在5个保守基序。SDS-PAGE显示在21kD附近出现与目的蛋白大小相符的特异条带,表达产物经纯化后dUTPase的含量为2.863mg·mL-1。酶学活性试验证实重组dUTPase能特异性降解dUTP,同时EDTA可以抑制dUTPase的活性;而Mg2+可以增强猪囊尾蚴dUTPase的活性。【结论】成功克隆表达了猪囊尾蚴dUTPase基因并鉴定了它的酶学活性,为进一步研究dUTPase在猪囊尾蚴中的生物学功能奠定了基础,同时也为抗猪囊尾蚴病的药物设计和开发提供了研究基础。     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production,oxidative stress,and caspase-3 activation

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.     

应用缺口末端标记法(TUNEL)分析犬瘟热病毒(CDV)诱导的细胞凋亡

利用犬瘟热病毒弱毒标准株ＯＰ－ＣＤＶ感染敏感细胞系非洲绿猴肾细胞,应用缺口末端标记法分析感染细胞的凋亡。结果在感染后４８ｈ的Ｖｅｒｏ细胞中检出了凋亡阳性细胞,而对照细胞内未检测到凋亡细胞,感染后２４ｈ的细胞尚未出现细胞病变,这一时期的细胞内也未检测到凋亡细胞,表明ＣＤＶ能诱导细胞凋亡,且凋亡细胞的检出时间与细胞病变的出现时间呈正向相关。     

玉米CMS-S小孢子败育过程中的细胞程序性死亡

以玉米（Zea mays L.）S型细胞质雄性不育系(CMS-S)的一对近等基因系S-Mo17^Rf3Rf3 和S-Mo17^rf3rf3为材料，采用TdT介导的dUTP DNA末端标记（TUNEL）、细胞色素C免疫原位杂交和DNA寡聚核小体片段电泳等方法，分别在细胞学水平和DNA水平上研究了玉米CMS-S小孢子败育的细胞程序性死亡（PCD）过程。结果表明，在花粉母细胞减数分裂后的四分体解离时期，不育花药的绒粘层细胞较可育花药提前裂解；在不育系S-Mo17^rf3rf3花药和花粉S-rf3中均明显出现PCD过程的DNA片段化以及线粒体细胞色素C外渗的现象，证明了玉米CMS-S的花粉败育与花药绒粘层细胞的提前凋亡和小孢子细胞的程序性死亡有关。     

急性病毒性坏死病毒dUTPase基因的克隆、表达及其产物的酶学活性分析

根据已测定完成的栉孔扇贝急性病毒性坏死病毒(acute viral necrosis virus,AVNV)基因组序列,设计特异性引物,以提取的发病扇贝组织总DNA为模板,PCR扩增得到编码AVNV dUTPase的开放阅读框ORF074,将产物克隆至原核表达载体pET32a(+)中,构建表达质粒pET32a-dut。然后,将其转化至E.coli BL21(DE3)进行诱导表达。SDS-PAGE检测显示,诱导表达蛋白分子量约为46 ku,与预期表达蛋白大小一致。经Western-blotting及质谱分析鉴定,所表达蛋白即为重组dUTPase。表达产物经Co²⁺柱纯化后进行酶学活性测定。结果显示,重组dUTPase能特异性催化dUTP,EDTA可以抑制dUTPase的活性,而Mg²⁺可以增强其活性。     

Extracellular and intracellular expression pattern of nuclear isoform of dUTP pyrophosphatase in mammalian cells after chemical carcinogen exposure

AIM: To study the extracellular and intracellular expression pattern of dUTP pyrophosphatase (nuclear isoform, DUT-N) in mammalian cells after exposure to different chemical carcinogens.METHODS: Human amniotic epithelial cells (FL cells), hepatoma cells (HepG2 cells) and cervical cancer cells (HeLa cells) were treated with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG, N-nitroso alkylating agent) and benzo［a］pyrene-7, 8-dihydroxy-9, 10-epoxide (BPDE, the metabolic activated form of Benzo［a］pyrene). SDS-PAGE and immunoblotting were used to examine the extracellular and intracellular protein levels of DUT-N.RESULTS: Immunoblotting showed that DUT-N appeared in the culture medium of FL cells, but not HeLa cells, after MNNG and BPDE exposure. MNNG, not BPDE induced the appearance of this protein in the culture medium of HepG2 cells. Although MNNG and BPDE treatment both induced the appearance of DUT-N, the intracellular protein level was down-regulated in the former and up-regulated in the latter.CONCLUSION: The appearance of DUT-N in the culture medium after chemical carcinogen treatment is not a phenomenon induced by specific carcinogen, but there are some common features between different carcinogens or DNA-damaging agents for given cell lines, such as FL cell. Meanwhile, the cellular mechanism of DUT-N appearance remains different from one carcinogen to another.