小G蛋白BBS3b的原核表达纯化及其多克隆抗体制备北大核心

[目的]制备莱茵衣藻小G蛋白BBS3b的多克隆抗体。[方法]利用莱茵衣藻bbs3b基因的c DNA序列分别构建了带有GST和6×His标签的原核表达载体p GEX-2T-BBS3b和p ET-28a(+)-BBS3b并转化至大肠杆菌BL21(DE3)诱导表达,以12%SDS-PAGE鉴定。用纯化的GST-BBS3b融合蛋白免疫新西兰大白兔,采集第4次免疫后血液并分离血清,利用间接ELISA法进行效价测定,并将抗血清依次经过Protein A纯化和硝酸纤维素膜纯化,用Western Blotting检测抗体特异性。[结果]GST-BBS3b和6×His-BBS3b融合蛋白的分子量分别为46、20 k Da。用ELISA的方法测定抗血清效价为512 000,Western Blotting检测结果显示制备的多克隆抗体能够特异性识别莱茵衣藻BBS3b蛋白。[结论]制备的多克隆抗体效价为512 000,能够特异性识别莱茵衣藻中BBS3b蛋白,为利用莱茵衣藻作为模式生物进行BBS3b在纤毛信号传导中的作用机理研究奠定了基础。     

富硒螺旋藻中含硒藻蓝蛋白的纯化、结晶及初步晶体学研究

从富硒螺旋藻中提取含硒藻蓝蛋白,经凝胶色谱和离子交换色谱纯化,应用悬滴气相扩散法,采用(NH4)2SO4和PEG4000作沉淀剂,获得了该蛋白质晶体的两种晶型.晶型Ⅰ属于单斜晶系,晶胞参数a=10.80 nm,b=11.70 nm,c=18.40 nm,β=90.2°,晶体空间群属于P21.单位晶胞中每个晶体学不对称单位含12个(αβ)单体,晶体衍射的最高分辨率达0.28 nm.晶型Ⅱ为六方晶系,晶胞参数为a=b=15.5 nm,c=4.03 nm,晶体空间群属于P63.晶体衍射的最高分辨率达0.28 nm.单位晶胞中每个晶体学不对称单位含1个(αβ)单体.对分子在晶体中的可能堆积方式进行了讨论.     

儿童感染肺炎链球菌的 青霉素结合蛋白基因突变分析

目的分析儿童感染肺炎链球菌的青霉素结合蛋白基因突变与青霉素耐药水平之间的关系。方法自2012年1月至2014年12月期间分离的1 317株肺炎链球菌中随机抽取出青霉素MIC=2.0μg/mL、4.0μg/mL、≥8.0μg/mL各20株共60株作为实验菌株,采用PCR方法对实验菌株进行青霉素结合蛋白PBP1a、PBP1b、PBP2a、PBP2b、PBP2x、PBP3的基因扩增,扩增产物进一步纯化和测序,测序结果与青霉素敏感肺炎链球菌R6就国际上公认的PBPs保守序列进行比对分析。结果 60株肺炎链球菌的PBP2b、PBP1a、PBP2x、PBP2a基因的保守区或保守区附件均发现氨基酸突变,未发现PBP3与PBP1b突变。中介与耐药菌株基因突变位点存在重合,主要出现在单一的PBP1a序列的370STMK模体元件内Thr371Ser置换突变或伴有PBP2b序列的Thr451Ala/Ser和Ala624Gly置换突变,同时PBP2a序列的465SLN模体元件前置位发生Ser461Ala的置换突变。结论肺炎链球菌对青霉素中、高水平耐药菌株绝大部分合并有不同PBP序列中4~6个氨基酸的置换突变,但合并多个氨基酸置换突变并非就必然引起耐药水平的相应升高。中、高水平耐药与PBP1a、PBP2b、PBP2a的变异关系密切,其中PBP1a的STMK保守区域Thr371Ser置换是引起耐药的主要因素之一。     

巨桉和天竺桂幼树对不同浓度SO2的光合生理响应

二氧化硫是大气主要污染物之一,可对植物的关键生理过程光合作用产生重要影响。利用密闭环境控制室熏气处理,研究不同浓度(自然状态下浓度、0.5mg·L-1、1.5mg·L-1、3.0mg·L-1)SO2对盆栽巨桉和天竺桂幼树叶绿素含量、光响应曲线、光合特征参数、光合日变化及硫含量的影响。结果表明:(1)SO2胁迫显著减少了巨桉叶绿素a、b含量,且叶绿素a/b值显著降低,而天竺桂在SO2胁迫下叶绿素a、b含量显著增加,叶绿素a/b值无显著影响。(2)SO2胁迫显著抑制了两树种的净光合速率(Pn);在SO2胁迫下巨桉气孔导度(Gs)、胞间CO2浓度(Ci)和蒸腾速率(Tr)显著上升,而天竺桂的Gs和Tr显著被SO2抑制,Ci随SO2浓度的增加先升高后降低。(3)巨桉表观量子效率(AQY)、暗呼吸速率(Rd)、光补偿点(LCP)和光饱和点(LSP)及天竺桂Rd和LCP均随着SO2浓度的增加而先升高后降低,而天竺桂的AQY和LSP逐渐降低。(4)一天中,SO2胁迫显著提高了巨桉Pn、Gs和Tr,而对天竺桂Pn无显著影响,较低浓度SO2胁迫显著促进了天竺桂Gs和Tr,高浓度SO2胁迫则显著抑制其Gs和Tr;SO2胁迫显著抑制了两种植物的Ci。(5)SO2胁迫下,巨桉和天竺桂幼树叶片硫含量均显著增加。研究认为,巨桉对较低浓度的SO2胁迫有一定的适应能力,但对高浓度SO2胁迫的抗性不如天竺桂强,这可能与二者不同的叶片形态及生理特性有关。     

三裂蟛蜞菊中的倍半萜内酯成分及其化感作用

从外来种三裂蟛蜞菊(Wedelia trilobata)的全株中分离得到6个倍半萜内酯化合物,通过光谱分析,分别鉴定为6-异丁酰基-三叶拉色芹内酯(1)、1 β-acetoxy-4α,9α-dihydroxy-6 β-isobutyroxyprostatolide(2)、1 β,9α-diacetoxy-4α-hydroxy-6 β-isobutyroxyprostatolide(3a)、1 β,9α-diacetoxy-4α-hydroxy-6β-methacryl-oxyprostatolide(3b)、1 β,4α-dihydroxy-9α-tigloyloxy-6β-methylpropanoylprostatolide（4a)、9α-angeloyloxy-1 β,4α-dihydroxy-6 β-methylpro-panoylprostatolide(4b)。除1和3a外,其余4个化合物均是首次从该植物中得到。通过种子萌发和幼苗生长抑制试验发现化合物1、2、3a和3b的混合物以及4a和4b的混合物对萝卜、小白菜和西红柿的幼苗生长有较强的抑制作用。4a和4b的混合物对咸水虾有较高的毒性。     

中国人血清中丙型肝炎病毒聚合酶全长基因的克隆表达及鉴定

构建中国人丙型肝炎病毒(HCV)复制的RNA聚合酶原核表达载体pET30aNS5b,并在大肠杆菌中获得NS5B聚合酶蛋白的高效表达,为建立HCV NS5b聚合酶细胞外分子复制模型的方法创造条件.使用高保真Pfu DNA聚合酶进行反转录及套式PCR扩增,从我国HCV RNA阳性血清中扩增出HCV NS5b RNA多聚酶全基因序列,经BamHI 和SalI酶切,将其克隆至同样酶切的 pET-30a载体中;转化大肠杆菌BL21,IPTG诱导表达.用抗 HCV NS5b单克隆抗体做Western-Blot进行鉴定.结果表明构建了原核表达载体,pET30aNS5bpET30aNS5b明显表达出12-His-NS5b聚合酶蛋白.测序结果表明,与已发表的相关HCV NS5b RNA聚合酶序列比较,其核苷酸和氨基酸的同源性分别在69%～92.7%及88.8%～96.8%之间.在最佳表达条件下,可高效诱导表达融合蛋白(65kDa),最高表达量占菌体蛋白18.9%.Western-Blot结果显示表达蛋白为HCV NS5b酶.HCV聚合酶蛋白全长基因可以成功地克隆在pET-30a载体上并有效表达出目的蛋白,为研究建立HCV NS5b聚合酶细胞外分子复制模型奠定了基础.     

丙型肝炎病毒重要编码蛋白重组腺病毒载体的构建

旨在构建含HCV core、E1、E2、F、NS3、NS5a和NS5b基因的重组腺病毒载体。运用PCR技术扩增目的基因片段,通过酶切连接方法将上述7种基因片段克隆至穿梭质粒pAdTrack-TO4中,然后将重组质粒用PmeⅠ线性化后与骨架质粒pAdEasy-1在BJ5183细菌中同源重组。用PacⅠ酶把重组腺病毒载体线性化后转染HEK293细胞后产生重组腺病毒颗粒。利用穿梭质粒中带有绿色荧光蛋白GFP对其感染效率进行监测。结果显示,酶切鉴定和测序结果均证实含core、E1、E2、F、NS3、NS5a和NS5b基因的重组腺病毒构建成功,在感染的Huh7细胞中观察到绿色荧光蛋白GFP。成功制备了高滴度的腺病毒Ad-core、Ad-E1、Ad-E2、Ad-F、Ad-NS3、Ad-NS5a和Ad-NS5b,为后续研究奠定了基础。     

小G蛋白BBS3b的原核表达纯化及其多克隆抗体制备

[目的]制备莱茵衣藻小G蛋白BBS3b的多克隆抗体。[方法]利用莱茵衣藻bbs3b基因的c DNA序列分别构建了带有GST和6×His标签的原核表达载体p GEX-2T-BBS3b和p ET-28a(+)-BBS3b并转化至大肠杆菌BL21(DE3)诱导表达,以12%SDS-PAGE鉴定。用纯化的GST-BBS3b融合蛋白免疫新西兰大白兔,采集第4次免疫后血液并分离血清,利用间接ELISA法进行效价测定,并将抗血清依次经过Protein A纯化和硝酸纤维素膜纯化,用Western Blotting检测抗体特异性。[结果]GST-BBS3b和6×His-BBS3b融合蛋白的分子量分别为46、20 k Da。用ELISA的方法测定抗血清效价为512 000,Western Blotting检测结果显示制备的多克隆抗体能够特异性识别莱茵衣藻BBS3b蛋白。[结论]制备的多克隆抗体效价为512 000,能够特异性识别莱茵衣藻中BBS3b蛋白,为利用莱茵衣藻作为模式生物进行BBS3b在纤毛信号传导中的作用机理研究奠定了基础。     

金发草LEA 3 基因两个剪接体转化酿酒酵母的抗非生物胁迫功能分析

对金发草(Pogonatherum paniceum)第3组LEA蛋白(PpLEA3)基因两个剪接体进行分析,并利用酿酒酵母表达系统分析两个剪接体在不同非生物胁迫的响应差异.以PpLEA3基因两个剪接体(PpLEA3.a和PpLEA3.b)的重组载体pMD19-T-PpLEA3.a和pMD19-T-PpLEA3.b为模板,PCR法构建酵母表达载体pYES2-PpLEA3.a和pYES2-PpLEA3.b,并转化酿酒酵母细胞得到重组菌INV-PpLEA3.a和INV-PpLEA3.b.通过比较重组菌和对照菌(转空载体pYES2)在NaCl、NaHCO3、低温、干旱、UV胁迫下的恢复生长状况,结果表明两种重组菌胁迫后的生长情况明显好于对照菌,两个剪接体对非生物胁迫抵抗力的大小为:PpLEA3.a＞PpLEA3.b.两个剪接体在核酸序列上的差异导致了在蛋白亲水性和结构上的差异,最终导致了在抗逆能力方面的差异.     

长筒石蒜鳞茎中的生物碱类成分(英文)

从长筒石蒜鳞茎的乙醇提取物中分离得到7个生物碱类化合物,经理化方法和波谱分析,分别鉴定为galanthamine(1),lycoramlne(2),6β-hydroxycrinamine(3a),6α-hydroxycrinamine(3b),(-)-amarbellisine(4),tazettine(5),macowine(6).其中化合物3a～6为首次从该植物中分离得到.     

Specific stimulation of α2-6 sialyltransferase activity by a novel cytosolic factor from rat colon

A factor present in the 100 000 g supernatant from the homogenate of rat colon stimulated the activity of purified GaIβ1-4GlcNAc α2,6 sialyltransferase [α2-6ST(N)] from rat liver and α2-6ST(N) from either liver microsomes or Golgi membrane. The stimulation of α2-6ST(N) activity by the colon factor using protein acceptors was about four-fold and highly reproducible when the reaction product of the α2-6ST(N) was assayed by either precipitation or affinity chromatography. In contrast, the colon factor did not stimulate the GaIβ1-4GlcNAc α2,3 sialyltransferase [α2-3ST (N)], from rat jejunum microsomes or purified Galβ1-3GalNAc α2,3 sialyltransferase [α2-3ST (O)] from porcine liver, or purified β1,4 galactosyltransferase (GT) from bovine milk. In addition to rat colon, the 100 000 g supernatant from the homogenates of rat brain and kidney also stimulated the α2-6ST(N) activity. The stimulation of α2-6ST(N) by the colon factor resulted in a decrease in the K_m (by about two-fold) and an increase in V_max (about 2- to 3-fold) for desialylated α₁ acid glycoprotein and CMP-[¹⁴C]N-acetylneuraminic acid. The stimulation of α2-6ST(N) activity by the colon factor was temperature dependent, protease sensitive and was inhibited by CTP, but did not need the presence of either metal ions or detergent. The cytosolic factor was partially purified by ion-exchange chromatography with the retention of the activator activity in the peaks containing low molecular weight proteins, but the activity was lost on attempts to further purification. A specific marked stimulation of the α2-6ST(N) activity by cytosolic factors in certain tissues might suggest a physiological role for these factors in the regulation of α2-6ST(N) activity.     

Purification and properties of α-amino--caprolactam racemase from Achromobacter obae

We have purified a unique enzyme, α-amino--caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with M_r 50 000 and a sedimentation coefficient (s_20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is 9.0. D- and L-α-amino--caprolactams are the only substrates. The K_m values for the D- and L-isomers are, 8 and 6 mM, respectively.     

Penicillium sp. 23 alpha-galactosidase: purification and substrate specificity

α-Galactosidase, a glycoprotein with carbohydrate and protein in ratio 1:6, has been isolated from liquid culture of micromycete Penicillium sp. 23 and purified to homogeneous state by ammonium sulphate precipitation followed by ion exchange and gel-filtration chromatography on TSK-gels. The Penicillium sp. 23 α-galactosidase specificity against a series of natural and synthetic substrates has been studied. The enzyme was found to exhibit strict specificity towards the glycon and hydrolyze exclusively α- -galactosides such as p-nitrophenyl-α- -galactopyranoside (p-NPhGal), melibiose, raffinose and stachyose. The configuration at C1 and C4 atoms of substrate as well as substitution at C2 and C6 of substrate made an important contribution to the interaction with the enzyme. The tested α-galactosidase exerted the highest affinity (K_m) with respect to the synthetic substrate p-NPhGal and maximal rate of hydrolysis (V_max), about 10 times higher, comparing with natural substrates (melibiose, raffinose and stachiose). The Penicillium sp. 23 α-galactosidase possesses wide specificity towards α-galactosidase hydrolysis link type, splitting off at varying rates the terminal galactose from disaccharides, attached by α-1,2-, α-1,3- and α-1,6-links. The enzyme is ineffective towards disaccharides with α-1,4-link. The enzyme showed potential to splitting off α-1,3-bound terminal galactose residues from antigens of the human blood group B(III) erythrocytes.     

Synthesis of Aspartyl Tripeptide Esters in Relation to Structural Features of Sweet Peptides

The distribution of multiple forms of β-amylase in some varieties or species of soybean seeds was examined by the gel isoelectric focusing method. Seven components (1′, 1, 2, 3, 4, 5 and 6) were found. Their respective isoelectric points were 5.07, 5.15, 5.25, 5.40, 5.55, 5.70 and 5.93±0.04. The varieties or species of soybean seeds were separated into two types by their zymograph: the low pI type and high pI type. Component 6 was purified from commercial defatted soybean meal containing all seven components by ion-exchange column chromatography and by gel filtration, and compared with previously purified components 2 and 4. Components 2, 4 and 6 had the same molecular weight and immunological properties but some differences were found in their amino acid compositions and enzymatic properties. The C-terminal amino acid of components 2 and 6 was glycine but that of component 4 was alanine. It was concluded from these results that differences between components 2, 4 and 6 were caused by charged amino acid substitution.     

Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63

Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min^–1 mg^–1) and convert dibenzofuran to 2,2,3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.     

Physicochemical and Light Scattering Studies on Ribosome Particles

The light scattering technique has been used to measure the molecular weight of Escherichia coli ribosomes. The 30S, 50S, and 70S components have been isolated and purified. The refractive index increment dn/dc was found to have the same value, (0.20 ±0.01) cm³/g, for the three species. The molecular weights are (1.0 ±0.1)·10⁶, (1.7 ±0.1)·10⁶, and (2.9 ±0.3)·10⁶ daltons respectively. Some information about the dimensions in solution (radius of gyration) and the interaction constant (second virial coefficient) have been obtained, and their significance is discussed.     

Phytase from Aspergillus terreus

1) Aspergillus terreus No. 9A-1 was cultivated by a shaking method and the optimal cultural conditions for the phytase production were concluded as follows: Composition of medium; rice bran 30 g, ammonium sulfate 3 g, distilled water 1.0 liter; initial pH 5.5; shaking condition; 50 ml of medium/500 ml vol. flask; 120 oscil./min, 90 hr.

2) Phytase from Asp. terreus was purified by ammonium sulfate precipitation, acetone precipitation and chromatography on SE-Sephadex C-50 and Sephadex G-200 columns. The enzyme was purified about 520-folds with the yield of 20% from the broth. The purified enzyme was homogeneous by column chromatography, ultracentrifugation and electrophoresis.

3) This purified preparation of phytase showed following properties, a) Optimal pH for the reaction was 4.5; b) optimal temperature for the reaction was about 70°C; c) the enzyme was stable in the range of pH from 1.2 to 9.0     

Purification of 5β-reductase from hepatic cytosol fraction of chicken

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5β-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5β-reduced metabolites derived from [4-¹⁴C]testosterone. During the purification procedures, 17β-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5β-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3α- and 3β-hydroxysteroid dehydrogenases were able to be removed from 5β-reductase fraction. The final enzyme preparation was apparently homogenous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5β-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. K_m of 5β-reductase for NADPH was estimated as 4.22 × 10⁻⁶M and for testosterone, 4.60 × 10⁻⁶M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5β-reductase were examined.     

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K_m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V_max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.     

Identification and characterization of N-acetylglucosamine-6-O-sulfate-specific β1,4-galactosyltransferase in human colorectal mucosa

6-Sulfo-sialyl Lewis X structure is attributable to recognition between lymphocytes and high endothelial venules. However, the biosynthetic pathway still remains unclear. We found that a β-galactosyltransferase (βGalT) in human colorectal mucosa preferentially acts on GlcNAc-6-O-sulfate (6S-GN). 6S-GN:β4GalT was partially purified by UDP-hexanolamine-Sepharose and asialo-agalacto-ovomucin-Sepharose chromatographies. The optimum pH of this enzyme was found to be 6.5–7.5 and the Michaelis constants for 6S-GN and UDP-Gal were 0.43 mM and 16 μM, respectively. The enzymatic activity was dependent on divalent cations and the substrate specificity was not affected by α-lactalbumin. This is the first demonstration of the occurrence of 6S-GN:β4GalT.