猪PBD-1稳定转染细胞系的建立

目的:构建稳定表达猪PBD-1的转基因细胞系,为稳定生产猪PBD-1并将其应用于新型兽药的开发提供生产细胞源。方法:真核表达载体pIRES2-EGFP-PBD-1经酶切和测序鉴定后,用脂质体介导法转染marc-145细胞,通过不同浓度的G418加压筛选,和进一步采用单个克隆法筛选,建立稳定转染的marc-145细胞系,用RT-PCR及抑菌试验检测PBD-1的表达。结果:建立了稳定转染的marc-145细胞系,成功地表达目的基因,其培养上清液及细胞冻融液具有抑菌作用。结论:利用真核表达载体pIRES2-EGFP-PBD-1稳定转染marc-145细胞系,为进一步研究猪PBD-1的生物学特性及其在兽用生物制品上的应用奠定了基础。     

猪繁殖与呼吸综合征病毒在Marc-145细胞微载体上培养条件的研究

通过研究搅拌程序,细胞接种密度,微载体浓度与细胞生长的关系,探索Marc-145细胞在微载体上的生长条件,并测定猪繁殖与呼吸综合征病毒在微载体培养的细胞上的TCID50。结果表明:细胞接种密度为4.28×105 cells/mL时,病毒滴度可达到107.5TCID50,这说明利用微载体繁殖PRRSV是可行的,为猪繁殖与呼吸综合征疫苗的大规模生产奠定实验基础。     

猪细小病毒感染Marc-145细胞后对其分泌白细胞介素18转录时相的影响

为了解猪细小病毒(PPV)感染Marc-145细胞后引起细胞炎性反应特别是白细胞介素18(IL-18)的反应,探讨宿主-病毒之间的作用关系,运用荧光定量PCR技术,测定和分析了PPV感染Marc-145细胞后引起的病毒DNA含量的变化和IL-18的分泌水平.结果表明,IL-18的表达量在1h无明显变化、在3,24h增加...     

bta-microRNA-145对胰岛素样生长因子1受体-磷脂酰肌醇3激酶-蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路相关基因表达的影响及其潜在靶标的揭示

本文旨在研究bta-microRNA-145(bta-miR-145)对胰岛素样生长因子1受体-磷脂酰肌醇3激酶-蛋白激酶B/哺乳动物雷帕霉素靶蛋白(IGF1 R-PI3 K-Akt/mTOR)信号通路相关基因表达的影响,为从microRNA的角度研究奶牛的泌乳调控提供依据.以原代奶牛乳腺上皮细胞为研究对象,分别转染bta-miR-145过表达模拟物(mimic)、bta-miR-145抑制表达模拟物(inhibitor),实时定量PCR检测与IGF1 R-PI3 K-Akt/mTOR信号通路相关的15个基因表达的变化.结果表明:bta-miR-145过表达和抑制表达没有引起预测靶标胰岛素样生长因子1受体(IGF1R)、胰岛素受体底物1(IRS1)、β-酪蛋白(CSN2)基因的mRNA表达量发生显著变化(P＞0.05);btamiR-145过表达可引起真核生物翻译起始因子4E(EIF4E)基因的mRNA表达量的显著下调(P＜0.05),抑制表达引起EIF4E基因的mRNA表达量显著上调(P＜0.05),并且通过生物信息学分析发现牛EIF4E的3'非翻译区(3'UTR)上存在bta-miR-145的可能结合位点.以上结果提示,bta-miR-145对IGF1 R-PI3K-Akt/mTOR信号通路具有一定的调节作用,EIF4E为bta-miR-145的潜在靶标.     

中药体外对Marc-145细胞的影响及抗PRRSV突变株的作用

将板蓝根、甘草和鱼腥草3种单味中药各含量为100mg/mL的中药粗提物作连续倍比稀释后,采用CPE和MTT法将其在Marc-145细胞上进行最大安全质量浓度和对细胞增殖的影响试验,同时采用体外细胞培养的方法在Marc-145细胞上进行抗猪繁殖与呼吸综合征病毒突变株(PRRSV PX株)作用试验。结果显示,3种中药粗提物最大安全作用质量浓度板蓝根和甘草均为3.125g/L、鱼腥草为1.563g/L;板蓝根、甘草、鱼腥草分别在3.125～0.049、3.125～0.195、1.563～0.391g/L时测得细胞D值大于细胞对照组,对细胞分裂增殖均有促进效果,最佳作用质量浓度均为1.563g/L;3种中药粗提物有不同程度的抗PRRSV PX株作用,板蓝根和甘草粗提物阻断、抑制PRRSV PX株感染及直接灭活PRRSV PX株效果最好的质量浓度;阻断为3.125、3.125g/L、抑制为1.563、0.391g/L:阻断、直接灭活为0.782、0.782g/L,鱼腥草粗提物在0.782g/L时阻断和抑制PRRSV PX株效果为好,但其对PRRSV PX株基本没有直接灭活作用。     

对华北地区500kV输变电工程水土保持的认识与建议

为了研究输变电工程建设过程中扰动土地面积分布特点,得出水土流失防治重点,为输变电工程水土流失防治工作提供技术支撑,以华北地区29项500 kV输变电工程为研究对象,统计分析变电站防治分区和输电线路防治分区不同地貌类型条件下各防治分区扰动土地面积空间分布特点和成因,并确定水土流失防治重点;通过分析植被恢复、铺设土工布和水泥灌浆桩基础等水土保持实用技术在不同地貌类型条件下防治水土流失和节约水土保持投资方面的实施效果,提出华北地区输变电工程水土流失防护重点部位与措施建议。     

MicroRNA‐145 regulates immune cytokines via targeting FSCN1 in Staphylococcus aureus‐induced mastitis in dairy cows

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus‐induced mastitis. In the present study, we overexpressed and suppressed miR‐145 to investigate the function of miR‐145 in Mac‐T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac‐T cytokines and in cell proliferation. We found that overexpression of miR‐145 in Mac‐T cells significantly reduced the secretion of IL‐12 and TNF‐α, but increased the secretion of IFN‐γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real‐time PCR (qRT‐PCR), Western blotting and luciferase multiplex verification techniques, we found that miR‐145 targeted and regulated FSCN1. Knock‐down of FSCN1 significantly increased the secretion of IL‐12, while the secretion of TNF‐α was significantly downregulated in Mac‐T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta‐miR‐145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.     

鱼腥草多糖对MA104和MARC145细胞活力影响的比较研究

研究鱼腥草多糖(houttuynia cordata polysaccharide,HCP)对MA104和MARC145细胞活力的影响,筛选梯度浓度的鱼腥草多糖对MA104和MARC145细胞促生长最佳浓度剂量范围和毒性剂量.通过水提醇沉法提取HCP,得糖率为8.18％;分别采用MTT法和细胞平板计数法绘制MA104和...     

荧光假单孢菌的生防遗传改良

综述了荧光假单孢菌生物防治方面的分子生物学研究进展，提出了通过遗传工程手段进一步改良的可行途径。     

Effect of Nsp9 Gene on Replication of PRRSV in Marc-145 Cells

In order to evaluate whether Nsp9 could enhance the replication of porcine reproductive and respiratory syndrome virus (PRRSV), pIRES2-EGFP-Nsp9 plasmid containing whole Nsp9 genome were transfected into Marc-145 cells,Real-time PCR and Western blotting were used to evaluate the expression of N protein after PRRSV was inoculated. The results showed that the level of N protein in the Nsp9 transfected cells was significantly higher than control group (P<0.05),which was 1.5 times higher. Meanwhile,the expression at protein level had the same trend as the mRNA level. When the dose of plasmid was increasing,the expression of N protein both at the mRNA and protein levels were increased. In conclusion,Nsp9 gene could enhance the replication of PRRSV in Marc-145 cells.