人重组GDNF及其生物活性研究

从人胎脑组织中提取总ＲＮＡ,以ＲＴ－ＰＣＲ方法获取编码胶质细胞源神经营养因子（ＧＤＮＦ）成熟蛋白的ｃＤＮＡ。将人ＧＤＮＦｃＤＮＡ插入含Ｔ７启动子的质粒ｐＥＴ－２８ａ（＋）,构建表达质粒ｐＥＴ－ＧＤＮＦ,转化大场杆菌获得表达菌株ＢＬＧＤＮＦ,经诱导表达的ＧＤＮＦ形成包含体。凝胶自动扫描分析表明,表达量约占菌体总蛋白的３０％以上。用纯化的ＧＤＮＦ蛋白免疫新西兰兔制备了ＧＤＮＦ抗血清。纯化和复性的ＧＤＮ     

神经营养因子GDNF编码顺序的克隆及表达产物的纯化

根据基因库中的顺序,设计了胶质细胞源神经营养因子（ＧＤＮＦ）基因的ＰＣＲ引物,以此从人基因组ＤＮＡ中扩增并克隆了ＧＤＮＦ的编码序列,经ＤＮＡ测序确认后,该片段克隆到表达质粒ｐＥＴ－３ａ中,转化大肠杆菌ＢＬ２１（ＤＥ３）．培养的重组菌经ＩＰＴＧ诱导,在Ｔ７启动子调控下表达出ｈＧＤＮＦ蛋白．经电泳分析表明ＧＤＮＦ主要存在于细菌包涵体中．从培养菌中制备包涵体,经充分洗涤,溶解于含８ｍｏｌ／Ｌ尿素的变性缓冲液中．经ＳＰ－Ｓｅｐｈａｒｏｓｅ柱层析分离,梯度洗脱,以１５％ＳＤＳ－ＰＡＧＥ检查含ＧＤＮＦ的部分．将含单体ＧＤＮＦ部分进行复性,再次用ＳＰ－Ｓｅｐｈａｒｏｓｅ离子柱分离同源二体ＧＤＮＦ．最后经ＳＤＳ－ＰＡＧＥ制备电泳纯化,纯度大于９５％．经Ｎ端测序表明序列正确．经测定,每升培养菌可得约１０ｍｇ纯化的ＧＤＮＦ．     

胶质细胞源神经营养因子cDNA全序列的克隆及其生物学功能的研究

为了研究ＧＤＮＦ在神经系统中的生物学功能,通过ＲＴ－ＰＣＲ方法从大鼠睾丸总ＲＮＡ中扩增出ＧＤＮＦｃＤＮＡ全序列,序列分析表明与ＧｅｎＢａｎｋ中的顺序完全相同．将ＧＤＮＦｃＤＮＡ以非融合方式连接在真核表达载体ｐＥＧＦＰ－ＮⅠ的绿色荧光蛋白的上游,在ＣＭＶ启动子控制下表达．通过绿色荧光蛋白报告基因的表达表明ＧＤＮＦｃＤＮＡ能在真核细胞ＨｅＬａ中很好表达．采用裸ＤＮＡ转染方法研究ＧＤＮＦ对损伤的坐骨神经的修复作用,在雏鸡出生后３ｈ切断其右侧坐骨神经,将ｐＥＧＦＰ－ＧＤＮＦ与Ｌｉｐｏｆｅｃｔｉｎ的混合物注射到坐骨神经切断位点附近肌肉内,５ｄ后追补一次．２０ｄ后进行实验检测,观察到ＧＤＮＦｃＤＮＡ的转染阻止了切断神经侧的腰脊髓内［Ｌ４－Ｌ６］运动神经元的大量死亡,并显著促进了切断坐骨神经的再生．     

人PSP基因在昆虫细胞中的高效表达

应用昆虫杆状病毒表达系统（ＢＥＳ）在昆早细胞Ｔｎ－５Ｂ１－４中高效表达了人ｐｅｒｓｅｐｈｉｎ（ＰＳＰ）,ＳＤＳ－ＰＡＧＥ分析表达量占细胞可深性蛋白质的２０％左右,表达产物经Ｎｉ＾２＋－ＮＴＡ树脂亲和层析纯化后纯度达８５％以上。活性研究表明,昆早细胞表达的ＰＳＰ蛋白能显著促进脊髓神经元的存活。     

江浙蝮蛇神经生长因子在家蚕幼虫中的表达

江浙蝮蛇神经生长因子（ＮＧＦ）基因克隆于昆虫病毒转移栽体ｐＢａｃＰＡＫ８中,获得重组转移栽体ｐＢａｃ－ＰＡＫ－ＮＧ〈与线性化Ｂｍ－ＡａｃＰＡＫ６修饰病基因组ＤＮＡ共转染家蚕细胞,经过体内重组,筛选到重组病毒。用重组病毒洒家蚕幼虫,５天后收集血淋巴,经ＳＤＳ－ＰＡＧＥ检测及利用ＰＣ１２细胞进行ＮＧＦ生物活力测定证明,神经生长因子在家蚕幼虫中得到较高表达,且表达产物具有良好的生物学活性     

GDNF重组腺病毒增加MN9D细胞多巴胺合成与释放

有表达ＧＤＮＦ的重组腺病毒直接感染中脑多巴胺能神经元来源的ＭＮ９Ｄ细胞并经神经毒素ＭＰＰ＋处理．感染３６ｈ分别收获细胞及其培养基,用反相高效液相色谱方法测定多巴胺含量．结果显示,经ＧＤＮＦ重组腺病毒感染的ＭＮ９Ｄ细胞内及其培养基中的多巴胺水平分别增加５０．７％和５３．５％．给予神经毒素ＭＰＰ＋损伤后,细胞内多巴胺含量降低５３．５％,但同时给予ＧＤＮＦ腺病毒则可抑制这种降低,并使多巴胺水平增加１４１．３％．以上结果提示ＧＤＮＦ腺病毒可提高细胞内的多巴胺水平并促进其释放,同时还具有对抗神经毒素ＭＰＰ＋损伤作用,表明ＧＤＮＦ重组腺病毒用于帕金森氏病基因治疗具有良好的前景．     

GABA减缓缺氧引起的神经营养素mRNA表达量的变化

γ－氨基丁酸是中枢神经系统中的一种内源性抑制递质。近年已有实验证据揭示ＧＡＢＡ具有抵抗中枢神经元缺氧或缺血损伤的作用,但其机制尚不清楚。脑源性神经营养因子ＢＤＮＦ,神经生长因子ＮＧＦ和神经营养素－３是同一家族的成员,它们在海马脑区均有表达。为研究ＧＡＢＡ抗缺氧作用的机制,我们以培养的海马ＣＡ１神经元为材料,采用原位杂交的方法检测了缺氧后ＢＤＮＦｍＲＮＡ,ＮＧＦｍＲＮＡ和ＮＴ－３ｍＲＮＡ表达量的变化以及ＧＡＢＡ对这种变化的影响。结果显示,缺氧使ＮＧＦｍＲＮＡ和ＢＤＮＦｍＲＮＡ的表达量迅速上调且前者的变化极为剧烈,而ＮＴ－３ｍＲＮＡ的表达量显著下降;用２０μＭ的ＧＡＢＡ处理细胞后,上述缺氧引起的神经营养因子ｍＲＮＡ表达量的上升或下降幅度均被减缓,其中对ＮＧＦｍＲＮＡ表达量变化的影响最为显著。这些结果表明,ＧＡＢＡ具有稳定缺氧后ＢＤＮＦｍＲＮＡ,ＮＧＦｍＲＮＡ和ＮＴ－３ｍＲＮＡ表达水平的作用。推测这种作用与ＧＡＢＡ增加氯电导,维持钙稳态以及提高神经元的抗缺氧能力的效应有一定联系     

胶质细胞衍生的神经营养因子成熟肽基因的克隆、表达及纯化的研究

通过ＰＣＲ的方法克隆了胶质细胞衍生的神经营养因子（ｇｌｉａｃｅｌ－ｌｉｎｅｄｅｒｉｖｅｄｎｅｕ－ｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ,ＧＤＮＦ）成熟肽的基因,并将其连接到Ｅ．ｃｏｌｉ高效表达载体ｐＥＴ１６ｂ,在Ｅ．ｃｏｌｉ中获得高效表达．表达蛋白占菌体总蛋白２１％以上,以包涵体形式存在,经体外复性后用金属螯合亲和层析的方法得到具有较高纯度和活性ｒｈＧＤＮＦ．     

GDNF及BDNF对受损运动神经元的长期修复

为了研究胶质细胞源神经营养因子（ＧＤＮＦ） 及脑源神经营养因子（ＢＤＮＦ） 对切断轴突的新生运动神经元的长期维持存活及促进神经再生的作用, 我们选用出生时单侧切断坐骨神经的雏鸡模型, 用裸ＤＮＡ 转染方法, 在损伤神经附近的肌肉中转染ＧＤＮＦ ｃＤＮＡ 和ＢＤＮＦ ｃＤＮＡ 的真核表达载体,观察在体表达的神经营养因子对损伤的修复作用。结果显示,在体表达的ＧＤＮＦ 在８ 周内能使切断坐骨神经的腰脊髓运动神经元近９０ ％ 维持存活。切断的坐骨神经从断端向远体端再生,最长再生达９ ．５ｍ ｍ 。表达两个因子比单独表达ＧＤＮＦ 对运动神经元的存活无显著性差异。而两个因子协同作用对坐骨神经的再生更为有效,坐骨神经再生最长的可达１５ ．４ｍ ｍ 。     

GDNF重组腺病毒的构建及促进多巴胺能神经元存活的研究

利用体内同源重组原理,构建了能介导ＧＤＮＦ基因转移和表达的复制缺陷型重组腺病毒ＡｄＣＭＶｇｄｎｆ,其中ＧＤＮＦｃＤＮＡ插入腺病毒基因组的Ｅ１区并由ＣＭＶ启动子控制在人２９３细胞内通过同源重组包装生成重组腺病毒后,用形态学方法、病毒ＤＮＡ酶切分析、ＰＣＲ和ＲＴ－ＰＣＲ等方法进行鉴定正确．经测定病毒滴度达到１０１０ｐｆｕ／ｍｌ．用免疫沉淀方法从重组腺病毒感染的２９３细胞及其培养基上清中均检测到大量ＧＤＮＦ蛋白．用重组腺病毒直接感染或者用其条件培养基处理,分别使胚胎大鼠中脑原代多巴胺能神经元的数目增加８８．２％和９６．４％,明显增加多巴胺能神经元存活,对帕金森氏病基因治疗具有重要意义     

Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells

Amino acid sequence of neutral metalloprotease from Bac. brevis has been compared with that of Bac. amyloloquefaciens, Bac. cereus, Bac. subtilis, Bac. stearothermophilis, Bac. thermoproteolyticus (thermolysine). A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac. brevis enzyme. The sequence comparison allows to put Bac. brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac. cereus and Bac. stearothermophilus. Using automated Edman degradation the N-terminal sequence of Bac. brevis protease has been determined. It does not differ from the sequence predicted from the nucleotide sequence of the gene. It was shown that, when Bac. brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac. subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted. The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.     

Genetic analysis of the pAD1 hemolysin/bacteriocin determinant in Enterococcus faecalis: Tn917 insertional mutagenesis and cloning

Thirty-seven nonhemolytic/nonbacteriocinogenic mutations in Enterococcus (Streptococcus) faecalis plasmid pAD1 were generated by Tn917 insertion. All were found to belong to one of two complementation classes. Each class of mutants secreted either hemolysin/bacteriocin (Hly/Bac) component A or L into the culture medium. DNA encoding Hly/Bac was cloned in Escherichia coli in which both components of the hemolysin were expressed individually and collectively. The region encoding components A and L was further defined by deletion analysis and physically mapped. A total of approximately 8.4 kilobases of pAD1 DNA were observed to be required for hemolysin expression. Hly/Bac activity of the wild-type and the inactive L substance was observed to be heat stable. Active Hly/Bac resulting from incubating separately secreted components A and L was also found to be heat stable. The results indicate that component A activates component L and that activated component L possesses the Hly/Bac activity. Component A was also observed to be associated with host immunity to the Hly/Bac.     

GDNF缺失突变体的设计及其结构与功能关系(英文)

研究了GDNF结构与功能的关系 .基于鼠源GDNF的晶体结构 ,利用计算机SGIIndigo2(R4 4 0 0 )工作站和InsightⅡ (95.5)蛋白质分析软件模拟了人GDNF三维结构 ,设计了GDNF分子的两个缺失突变体ΔN1 2 8和ΔN78 90 .以野生型GDNFcDNA作为模板 ,用PCR法得到编码缺失突变体的DNA片段 .将大肠杆菌作为表达系统 ,使缺失突变体GDNF在大肠杆菌中表达 ,对表达产物纯化和复性后进行生物活性测定 .两株突变体在大肠肝菌中获得了高效表达 ,纯化后的GDNF突变体ΔN1 2 8可以与存在于KG 1a细胞表面的受体结合 ,但不能促进 8日龄鸡胚背根节突起的生长 .突变体ΔN78 90既不能与受体结合 ,同时也失去了促背根节突起生长的功能 .说明GDNF分子的N端氨基酸对分子的生物学活性很重要 ,但对分子与受体GDNFR α的结合并不是必需的 ,而分子中的螺旋区对分子与受体的结合以及生物学活性都必不可少 .     

Immunogenicity and safety of H1N1 influenza hemagglutinin protein expressed in a baculovirus system

Although most influenza vaccines are produced in eggs, new types of vaccines must be developed. In this study, the immunogenicity and safety of a baculovirus‐expressed hemagglutinin (HA) of H1N1 influenza virus (Korea/01/2009; designated “HA‐Bac‐K”) was compared with those of a commercially available baculovirus‐expressed HA (designated “HA‐Bac‐C”) and an Escherichia coli‐expressed HA (designated “HA‐E. Coli‐K”). HA‐Bac‐K succeeded in inducing hemagglutination inhibition and neutralization antibodies in mouse and ferret models. The different immunogenicities observed may be attributable to the different expression systems and purification protocols used. Our work suggests that HA expressed in a baculovirus system is an effective and safe candidate influenza vaccine.     

Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor-beta (TGF- beta), originally isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons, and more recently shown to be a potent neurotrophic factor for motorneurons. The biological activities and distribution of this molecule outside the central nervous system are presently unknown. We report here on the mRNA expression, biological activities and initial receptor binding characterization of GDNF and a shorter spliced variant termed GDNF beta in different organs and peripheral neurons of the developing rat. Both GDNF mRNA forms were found to be most highly expressed in developing skin, whisker pad, kidney, stomach and testis. Lower expression was also detected in developing skeletal muscle, ovary, lung, and adrenal gland. Developing spinal cord, superior cervical ganglion (SCG) and dorsal root ganglion (DRG) also expressed low levels of GDNF mRNA. Two days after nerve transection, GDNF mRNA levels increased dramatically in the sciatic nerve. Overall, GDNF mRNA expression was significantly higher in peripheral organs than in neuronal tissues. Expression of either GDNF mRNA isoform in insect cells resulted in the production of indistinguishable mature GDNF polypeptides. Purified recombinant GDNF promoted neurite outgrowth and survival of embryonic chick sympathetic neurons. GDNF produced robust bundle-like, fasciculated outgrowth from chick sympathetic ganglion explants. Although GDNF displayed only low activity on survival of newborn rat SCG neurons, this protein was found to increase the expression of vasoactive intestinal peptide and preprotachykinin-A mRNAs in cultured SCG neurons. GDNF also promoted survival of about half of the neurons in embryonic chick nodose ganglion and a small subpopulation of embryonic sensory neurons in chick dorsal root and rat trigeminal ganglia. Embryonic chick sympathetic neurons expressed receptors for GDNF with Kd 1-5 x 10(-9) M, as measured by saturation and displacement binding assays. Our findings indicate GDNF is a new neurotrophic factor for developing peripheral neurons and suggest possible non-neuronal roles for GDNF in the developing reproductive system.     

The genetic transfer, heredity and phenotypic expression of plasmid RP4::Mu cts genes in Bacillus cereus

The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.     

Immunohistochemical expression of two members of the GDNF family of growth factors and their receptors in the olfactory system

The glial cell line-derived (GDNF) family of trophic factors, GDNF, neurturin, persephin and artemin, are known to support the survival and regulate differentiation of many neuronal populations, including peripheral autonomic, enteric and sensory neurons. Members of this family of related ligands bind to specific GDNF family receptor (GFR) proteins, which complex and signal through the Ret receptor tyrosine kinase. We showed previously that GDNF protein was detectable in olfactory sensory neurons (OSNs) in the olfactory neuroepithelium (ON). In this immunohistochemical study, we localized GDNF, neurturin, GFRα1, GFRα2 and Ret in the adult rat ON and olfactory bulb. We found that GDNF and Ret were widely expressed by immature and mature OSNs, while neurturin was selectively expressed in a subpopulation of OSNs zonally restricted in the ON. The GFRs had differential expression, with mature OSNs and their axons preferentially expressing GFRα1, whereas progenitors and immature neurons more avidly expressed GFRα2. In the bulb, GDNF was highly expressed by the mitral and tufted cells, and by periglomerular cells, and its distribution generally resembled that of Ret, with the exception that Ret was far more predominant on fibers than cell bodies. Neurturin, in contrast, was present at lower levels and was more restricted in its expression to the axonal compartment. GFRα2 appeared to be the dominant accessory protein in the bulb. These data are supportive of two members of this neurotrophic family, GDNF and neurturin, playing different physiological roles in the olfactory neuronal system.     

膜蛋白LASS2的表达研究

利用多种原核表达系统、真核体外翻译系统和细菌/杆状病毒(Bac to Bac)的昆虫表达系统对一个具有重要生理功能的人的膜蛋白LASS2(Homo sapienslongevity assurance homologue 2 of yeastLAG1)进行表达研究。在原核表达系统中仅能够表达其羧基端胞外区片段却不能表达完整的LASS2蛋白,并制备了该片段的抗体。完整的LASS2蛋白能够在两种真核表达系统中进行表达,SDS-PAGE分析结果表明,表达产物分子量为约28kD的LASS蛋白, Western印迹分析也证实了这一结果, 并利用Ni-NTA树脂亲和层析将该蛋白纯化,纯度达到90%以上。