AAV载体介导的蓬佩病模型小鼠体内基因治疗研究*

Objective: Pompe disease is a lysosomal glycogen storage disease caused by acid α-glucosidase (GAA) deficiency, which is characterized by glycogen accumulation in the heart, skeletal muscle, and central nervous system (CNS). AAV vector-mediated gene therapy is expected to be a breakthrough in the treatment of Pompe disease. In this study, AAV9 vector was used to mediate GAA gene transfer in Pompe disease model mice, and the changes of GAA protease activity, glycogen accumulation in tissues and pathological changes in mice after transgenic intervention were evaluated. Methods: Codon optimized GAA gene (coGAA) was carried by AAV9 vector, and the AAV vector was packaged by baculovirus production process. Adult Pompe model mice were given a single intravenous injection at the dose of 1.1×10¹³, 3.0×10¹³, 1.2×10¹⁴ vg/kg, and aged Pompe model mice were given a single intravenous injection at the dose of 3.0×10¹³ vg/kg. After reaching the end point of the experiment, the mice were euthanized, GAA protease activity was determined by fluorescence spectrophotometry, glycogen accumulation was observed by PAS staining, and pathological changes were detected by HE staining. Results: Five weeks after administration, GAA protein was widely expressed in all tissues of adult model mice, with higher expression levels in heart and liver, and lower expression levels in brain and spinal cord. After rAAV9-coGAA treatment, glycogen content in myocardium, skeletal muscle and brain decreased, and vacuolar degeneration in myocardium and skeletal muscle decreased significantly. After treatment, the tissue enzyme activity of the aged animals was significantly increased compared with that of the model mice. The vacuolar degeneration and inflammatory cell infiltration of the myocardium were decreased, but the pathological improvement of skeletal muscle was limited. Conclusion: A single intravenous injection of rAAV9-coGAA can enhance GAA enzyme activity, reduce glycogen accumulation and improve pathology in Pompe model mice. The therapeutic effect was dose-dependent, and the injection also had certain therapeutic effect on aged animals. This study laid a theoretical foundation for the clinical application of AAV9 mediated gene therapy via intravenous route in Pompe disease.     

细胞色素P450还原酶与CYP17的共表达及其功能分析*

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。     

植物口服疫苗的动物和临床实验*

利用转基因植物生产亚单位疫苗用于口服主动免疫具有安全、廉价和方便等优点。植物可以正确地表达细菌和病毒抗原基因,对动物及人类的临床实验研究表明：食用表达某种抗原的转基因植物可在实验动物或人群体内激起系统免疫和粘膜免疫,产生相应的特异性抗体,这些结果表明了植物口服疫苗的可行性。此外,在治疗自身免疫疾病以及癌症等方面,植物口服疫苗也具有值得关注的作用。     

生物法合成γ-氨基丁酸的研究策略*

γ-氨基丁酸(γ-aminobutyric acid,GABA)是一种极易溶于水的非蛋白质氨基酸,被广泛应用于食品和制药工业中,市场需求量极大。可通过化学合成法、植物富集法、微生物直接发酵法和生物转化法生产。近年来,因生物法合成GABA具有相对优势,受到研究者们的重视。对GABA的生产方法、生产GABA的微生物、微生物合成GABA的关键代谢途径和GAD酶的定向改造策略进行了论述。     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 *

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei)TK1501 β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86.63kDa和675.56U/mg。最适作用温度和pH分别为30℃和6.5。 Mg ²⁺和Ca ²⁺对β-葡糖苷酶酶活抑制作用最小,Cu ²⁺几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_max分别为1.44mmol/L和58.32mmol/(L·s),催化系数k_cat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501 β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

COVID-19疫苗研究现状*

2019年底于中国武汉暴发的新型冠状病毒肺炎疫情来势凶猛,迅速蔓延全球,并被世界卫生组织列为“国际关注的突发公共卫生事件”,给全人类的健康及经济发展造成难以估量的损害。新型冠状病毒对人群普遍易感且传染性强,在无特效药物及治疗手段的情况下,疫苗接种是防控COVID-19疫情最有效且最经济的途径。目前全球疫苗研发正在加速进行,各国之间通力合作,共同应对此次疫情。主要对目前正在研发的针对SARS-CoV-2的灭活疫苗、病毒载体疫苗、基因工程重组亚单位疫苗、核酸疫苗的研究进展进行综述。     

COVID-19疫苗研究现状*

2019年底于中国武汉暴发的新型冠状病毒肺炎疫情来势凶猛,迅速蔓延全球,并被世界卫生组织列为“国际关注的突发公共卫生事件”,给全人类的健康及经济发展造成难以估量的损害。新型冠状病毒对人群普遍易感且传染性强,在无特效药物及治疗手段的情况下,疫苗接种是防控COVID-19疫情最有效且最经济的途径。目前全球疫苗研发正在加速进行,各国之间通力合作,共同应对此次疫情。主要对目前正在研发的针对SARS-CoV-2的灭活疫苗、病毒载体疫苗、基因工程重组亚单位疫苗、核酸疫苗的研究进展进行综述。     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。     

大鼠SUMO特异性蛋白酶1催化区蛋白制备及功能鉴定*

目的: 制备大鼠SUMO特异性蛋白酶1(sentrin-specific protease,SENP1)催化结构域(SENP1C)蛋白,并鉴定其酶活性。方法: 分别以大鼠SENP1-pcDNA3.1和EGFP-pcDNA3.1重组体为模板,PCR扩增目的基因,克隆入pGEM-T载体;酶切鉴定后,再亚克隆入原核表达载体pET-28a;阳性重组体导入原核表达细胞BL-21,异丙基硫半乳糖苷(IPTG)诱导蛋白质表达;SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的表达。Ni-NTA吸附纯化蛋白质并透析处理,SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的纯度;1 μmol/L及5 μmol/L Tat-EGFP分别孵育HT22细胞不同时间,荧光显微镜下观察细胞转染情况。采用5 μmol/L Tat-SENP1C预孵育HT22细胞10 h,免疫印迹检测整体蛋白质的SUMO化水平;用5 μmol/L Tat-SENP1C预孵育HT22细胞或过表达Myc-Akt1和HA-SUMO1的HT22细胞10 h后,免疫沉淀和免疫印迹检测内源性和外源性Akt1与SUMO1的结合(SUMO化)。结果: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体成功构建,IPTG可以诱导蛋白质高表达;采用Ni-NTA纯化和透析可获得较高纯度的蛋白质;5 μmol/L Tat-EGFP孵育HT22 细胞10 h后,蛋白质穿膜效率较高;Tat-SENP1C重组蛋白可以显著降低HT22细胞中整体蛋白质的SUMO化以及内源性和外源性Akt1 SUMO化。结论: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体构建成功,且被IPTG诱导后可高效表达蛋白质;纯化的Tat-SENP1C蛋白具有较强的穿膜能力及酶活性。     

提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用 *

将B. circulans 251 β-CGTase应用于海藻糖制备,海藻糖转化率从50.4%提高至71.9%。为进一步提高底物的转化率,运用易错PCR-高通量筛选技术筛选对以麦芽糖为歧化反应受体的亲和性提高的B. circulans 251 β-CGTase突变体。利用低底物浓度的96孔板4,6-亚乙基-对硝基苯-α-D-麦芽七糖苷(EPS)显色法,最终筛选得到了一株对麦芽糖亲和性提高的突变体M234I。将野生型β-CGTase和突变体酶M234I进行蛋白质纯化,测定其酶学性质。结果表明,突变体的比活为345.25U/mg,野生型则为357.63U/mg;突变体M234I对麦芽糖的K_m为0.258 2mmol/L,仅为野生型(0.474 9mmol/L)的54.4%,对麦芽糖的亲和性显著提高;突变体的最适温度、最适pH较野生型未发生较大变化。以麦芽糊精(DE值16)为底物,将突变体M234I用于多酶复配体系生产海藻糖,酶反应结果表明海藻糖的转化率最高达74.9%,较野生型β-CGTase提高约3%。     

不同处理方法对灵芝β-葡聚糖降解效果的比较研究

本研究采用酸法、碱法、酶法和微波法对灵芝β-葡聚糖进行降解,通过降解率、产物分子量变化、产物聚合度分布等指标比较了不同方法的降解效果。结果表明,微波法降解率高达94%,处理后产物的分子量明显降低,寡糖产物聚合度分布广。酶法降解率约为40%,寡糖产物中含有DP2-5的成分。酸法及碱法降解率低于20%,寡糖产物少。研究表明,与其他3种方法相比,微波法降解率高、产物丰富、操作条件易于控制,是一种简单、高效的降解灵芝β-葡聚糖、制备灵芝β-葡寡糖的方法。     

Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization.

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity.     

Microscopic study on resorption of β-tricalcium phosphate materials by osteoclasts

Sintered compounds prepared with β-tricalcium phosphate (β-TCP) are commonly used as biocompatible materials for bone regenerative medicine. Although implanted β-TCP is gradually replaced with new bone after resorption by osteoclasts, exactly how osteoclasts resorb β-TCP is not well understood. To elucidate this mechanism, we analyzed the structure of β-TCP discs on which mouse mature osteoclasts were cultured using scanning electron microscopy. We found that β-TCP was resorbed by mature osteoclasts on one side of each disc, as evidenced by the formation of multiple spine-like crystals at the exposed areas. Because osteoclasts secrete acid to resorb bone minerals, we mimicked this acidification by dipping β-TCP slices into HCl solution (pH 2.0). However, no spine-like crystals appeared even though the size of each β-TCP particle was reduced. On dentin slices, osteoclasts formed clear actin rings, which are cytoskeletal structures characteristic of bone-resorbing osteoclasts. No clear actin rings were observed in osteoclasts cultured on β-TCP slices, although small actin dots were observed. Analysis by transmission electron microscopy showed that osteoclasts attached to β-TCP particles. These results suggest that osteoclasts resorb β-TCP particles independently of clear actin ring formation.     

Activation of Wnt11 by transforming growth factor-β drives mesenchymal gene expression through non-canonical Wnt protein signaling in renal epithelial cells

Transforming growth factor β1 (TGF-β) promotes renal interstitial fibrosis in vivo and the expression of mesenchymal genes in vitro; however, most of its direct targets in epithelial cells are still elusive. In a screen for genes directly activated by TGF-β, we found that components of the Wnt signaling pathway, especially Wnt11, were targets of activation by TGF-β and Smad3 in primary renal epithelial cells. In gain and loss of function experiments, Wnt11 mediated the actions of TGF-β through enhanced activation of mesenchymal marker genes, such as Zeb1, Snail1, Pai1, and αSMA, without affecting Smad3 phosphorylation. Inhibition of Wnt11 by receptor knockdown or treatment with Wnt inhibitors limited the effects of TGF-β on gene expression. We found no evidence that Wnt11 activated the canonical Wnt signaling pathway in renal epithelial cells; rather, the function of Wnt11 was mediated by the c-Jun N-terminal kinase (JNK) pathway. Consistent with the in vitro results, all the TGF-β, Wnt11, and JNK targets were activated in a unilateral ureteral obstruction (UUO) model of renal fibrosis in vivo. Our findings demonstrated cooperativity among the TGF-β, Wnt11, and JNK signaling pathways and suggest new targets for anti-fibrotic therapy in renal tissue.     

Unique disulfide bonds in epidermal growth factor (EGF) domains of β3 affect structure and function of αIIbβ3 and αvβ3 integrins in different manner

The β3 subunit of αIIbβ3 and αvβ3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbβ3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbβ3 and αvβ3. We created β3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbβ3 and αvβ3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbβ3 and αvβ3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbβ3 and αvβ3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbβ3. Activation of C523S/C544S αvβ3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S β3 in αIIbβ3 but not in αvβ3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in β3 differently affect the function of αIIbβ3 and αvβ3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbβ3 and αvβ3 and for Cys-523-Cys-544 only in αvβ3.