1．3倍体HBV YIDD变异株真核表达载体构建及鉴定

构建HBV YIDD拉米夫定耐药株1．3倍全基因真核表达载体,为进一步探讨乙肝病毒变异株的生物学特性及筛选抗病毒药物奠定基础。参考GenBankHBV序列设计并合成一系列引物,以临床证实为拉米夫定耐药的病人HBV DNA为模板,通过PCR扩增得到HBV全基因组并克隆至pGEM—T Easy载体中,经测序证实聚合酶基因存在YIDD变异,然后以该病人的HBV全基因组为模板构建1．3倍全基因HBV—YIDD变异真核表达载体pcDNA3．1（＋）-1．3HBV。通过PCR扩增,酶切及测序证明pcDNA3．1（＋）-1．3HBV表达载体构建成功,该表达载体的构建为后期建立稳定表达HBV—YIDD变异的细胞模型提供材料。     

虾青素高产菌株的选育

采用激光（Laser）、紫外线（UV）、亚硝基胍（NTG）及其复合诱变法夫酵母菌,再用高浓度葡萄糖培养基选育经复合诱变的法夫酵母菌。结果表明：与原始出发菌相比,菌株经诱变后其虾青素产量有显著提高,其中复合诱变的是青素产量可达7.26μm/ml。诱变高产菌株遗传稳定性好。     

重组斑马鱼 p8蛋白的原核表达及纯化

目的：在大肠杆菌中重组表达斑马鱼p8蛋白并纯化。方法：PCR扩增斑马鱼p8蛋白基因编码区,连接到带有6×His标签的原核表达载体pET-28a中,构建重组表达质粒pET-28a-p8并转化大肠杆菌BL21（DE3）,用IPTG诱导表达;优化表达条件后用Ni^2＋柱纯化重组蛋白。结果：构建了pET-28a-p8重组质粒;目的蛋白在大肠杆菌中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的12．8×10^3。结论：获得了斑马鱼p8融合蛋白,为其生物学功能研究奠定了基础。     

Circ_0026579 alleviates LPS-induced WI-38 cells inflammation injury in infantile pneumonia

Circular RNA (circRNA) represents an important regulator in infantile pneumonia progression. To clarify the role of circ_0026579 in this disease, LPS was used to treat WI-38 cells to mimic inflammation injury. The levels of inflammatory factors were determined by ELISA assay. Cell proliferation and apoptosis were measured by MTT assay, EdU staining and flow cytometry. The protein levels of cyclinD1, cleaved-caspase-3 and insulin-like growth factor 2 (IGF2) were examined using Western blot analysis. Cell oxidative stress was assessed by detecting MDA level and SOD activity. The expression of circ_0026579, miR-24-3p and IGF2 were analyzed using quantitative real-time PCR, and the interaction between miR-24-3p and circ_0026579 or IGF2 was confirmed by dual-luciferase reporter assay and RIP assay. LPS induced inflammation in WI-38 cells. Circ_0026579 expression was promoted in LPS-induced WI-38 cells, and its knockdown alleviated LPS-induced WI-38 cells inflammation. MiR-24-3p was sponged by circ_0026579, and its expression was reduced by LPS. MiR-24-3p inhibitor reversed the regulation of circ_0026579 knockdown on LPS-induced WI-38 cells inflammation. IGF2 was targeted by miR-24-3p, and its expression could be enhanced by LPS. MiR-24-3p relieved the inflammation of WI-38 cells which could be abolished by IGF2 overexpression. Circ_0026579 positively regulated IGF2 expression through sponging miR-24-3p. Circ_0026579 knockdown alleviated LPS-induced WI-38 cells inflammation by miR-24-3p/IGF2 axis, suggesting that circ_0026579 might contribute to infantile pneumonia progression.     

运动与P53调节细胞信号转导通路研究进展

P53调节多个细胞信号转导通路,其功能与肿瘤抑制、细胞周期调控、能量代谢调节、促进线粒体生物发生、保持氧化应激平衡等有关,保持P53基因的稳态表达是预防肿瘤和延缓衰老的策略之一.体育锻炼能促进机体新陈代谢、延缓细胞衰老、减少细胞癌变几率,适宜的运动能够通过影响P53调节的多个细胞信号通路延续P53信号稳态.     

眼皮肤白化病患者酪氨酸酶基因突变的研究

目的：对临床诊断为眼皮肤白化病（OCA）患者的酪氨酸酶（TYR）基因进行突变筛查,了解我国大陆OCA患者TYR基因突变类型,探讨基因突变对人TYR蛋白结构和功能的影响。方法：应用PCR技术,扩增患者及其父母的TYR基因外显子、外显子-内含子交界区及启动子区;以DNA序列测定技术,进行突变筛查与鉴定;利用生物信息学方法,对突变引起蛋白结构和功能的改变进行预测与分析。结果：在15名患者的30个TYR等位基因内,查明11种突变;其中错义突变5种（W400L、R299H、E294K、R77Q和K142M）,无义突变3种（R116X、R278X和G295X）,插入突变2种（929insC和232insGGG）,剪切位点突变1种（IVS1-3 C〉G）;对4个突变W400L、R299H、929insC、232insGGG的生物信息学分析显示,突变的致病性与蛋白结构和功能的改变相关。结论：W400L占本研究所检出全部OCA1突变等位基因的30．0％（9／30）,可能为中国大陆人群中较常见的TYR基因突变类型;应用生物信息学分析方法对TYR基因突变的致病性做出一些合理可能的解释是可行的。     

肌抑素(Myostatin)基因突变体活性区的克隆、表达及活性的研究

肌抑素Myostatin是肌肉发育的重要抑制因子,肌抑素的突变,使其抑制功能的全部或几乎全部丧失,表现为肌肉细胞的增大和肌纤维束的增加。采用PCR技术,从肌抑素天然突变的双肌牛皮尔蒙特（Piedmontese）的基因组中扩增得到肌抑素突变体的活性区,并将其亚克隆到pMD18T载体上,利用基因重组技术,构建原核表达质粒pET30a(+)/action/Myostatin,在大肠杆菌中高效表达,采用亲和层析法纯化表达产物,并将其共孵育于离体培养的绵羊肌肉细胞,检测肌抑素突变体的生化活性,结果显示肌抑素的突变体具有促进肌肉细胞增生和增殖的功能。     

Raf激酶抑制剂及其耐药机制研究进展

Ras/Raf/MEK/ERK 通路是调节细胞生长与增殖的重要信号传导通路。在Ras/Raf/MEK/ERK 通路中某些成员的突变往往与恶性肿瘤的发生密切相关。B-Raf 激酶是该通路中Raf 家族最重要的亚型,其主要突变形式B-RafV600E 在黑色素瘤等多种肿瘤中高度表达。选择性B-RafV600E 抑制剂vemurafenib 和dabrafenib 的上市使得晚期黑色素瘤的治疗进入新纪元,但是耐药性和副作用依然限制了二者的使用。综述目前Raf 激酶抑制剂耐药性与副作用产生机制以及Raf 激酶抑制剂的最新研究进展。