VD_3羟基化酶的定向研究进展

维生素D3羟基化酶(Vdh)作为细胞色素酶P450s(CYP)蛋白家族成员,催化VD3形成有生物活性的1α,25(OH)2VD3。但是,由于VD3并不是Vdh的天然底物,Vdh羟基化活性较低。采用定向构建Vdh重组质粒的方法对Vdh催化活性位点给予优化,从而提高其羟基化能力。就目前对Vdh的结构特性和定向研究进行综述。     

荧光假单胞菌香兰素脱氢酶基因功能的验证

验证了荧光假单胞菌(Pseudomonas fluorescensATCC13525)香兰素脱氢酶基因(vanillin dehydrogenasegene,vdh)的功能。基因vdh表达产物(Vdh)的活性测定结果显示Vdh具有很高的活性,而且不经IPTG诱导的Vdh也具有同样高的活性。经过4 h的体外酶促反应,重组蛋白Vdh能把95%以上的香兰素转化为香兰素酸,从而验证了vdh基因的表达产物具有香兰素脱氢酶的功能。同时发现NAD 是从香兰素到香兰素酸体外转化必不可少的因素。     

4-羟基苯乙酸-3-羟化酶A重组表达菌株的构建及其对羟基酪醇的生物转化研究

目的:构建表达4-羟基苯乙酸-3-羟化酶A(4-hydroxyphenylacetate-3-hydroxylase A,HHA)的重组菌株,进而以酪醇为底物,利用重组菌株转化生产羟基酪醇。方法:选择大肠杆菌BL21(DE3)为模板来扩增HHA基因,经酶切后连接到表达载体p ET-28a中,获得重组表达载体p ET28a-HHA,将重组载体转化到感受态细胞BL21(DE3),在重组菌液中加入适量酪醇,利用胞内的重组酶对酪醇加羟基合成羟基酪醇,细胞离心后,分别采用薄层层析法和气质联用法检测上清液中羟基酪醇的转化结果。结果:IPTG诱导表达后,经SDS-PAGE分析获得分子质量分别为58.8k Da和18.5k Da的两条蛋白质条带。薄层层析法和气质联用法均检测到催化产物羟基酪醇的生成。结论:成功构建了表达HHA的重组菌株,该菌株可有效将酪醇转化为羟基酪醇。     

骨形成蛋白10成熟肽在大肠杆菌中的表达纯化及活性分析

目的:以生物制备骨形成蛋白10(BMP10)为目标,研究BMP10成熟肽在大肠杆菌中的表达及活性。方法:以人源BMP10成熟肽基因为模板,PCR获得N端带有组氨酸标签(6×His)的融合基因6×His-m BMP10,构建p ET28a/m BMP10表达载体;热击转染大肠杆菌BL21(DE3)菌株,卡那霉素抗性筛选获得重组表达菌株BL21/p ET28a-6×His-m BMP10,IPTG诱导表达后利用SDS-PAGE电泳、Western印迹对蛋白进行分析;超声波破碎菌体,收集包涵体,镍柱亲和层析纯化获得电泳纯目的蛋白;透析复性后,非还原SDS-PAGE检验目标蛋白的二聚体形成;通过体外细胞实验检测蛋白活性。结果:纯化得到纯度90%以上的m BMP10,复性后二聚体得率约为40%;活性实验测得P19细胞的Smad6蛋白表达上调3倍左右。结论:通过大肠杆菌表达体系获得具有生物活性的BMP10,为后续作用机理研究奠定了实验基础。     

绿色木霉TvALP-linker-TvRBL融合基因的构建与原核表达

在Tv ALP基因和Tv RBL基因间加入一段柔性链接头(linker)基因序列,构建融合基因。生物信息学分析表明该融合基因含有一段信号肽序列。利用RT-PCR技术从绿色木霉菌丝体总RNA中扩增出Tv RBL基因、Tv ALP基因和去除信号肽的ΔTvALP基因,分别克隆到原核表达载体p ET30a,构建重组质粒p ET30a-Tv ALP-linker-Tv RBL和p ET30a-ΔTv ALP-linker-Tv RBL,转化大肠杆菌BL21(DE3)p Lys S,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达。SDS-PAGE电泳分析结果表明,去除信号肽的表达质粒p ET30a-ΔTv ALP-linker-Tv RBL在大肠杆菌BL21(DE3)p Lys S中获得了表达,在60 k D处有一条蛋白质特异条带,与预测的目的产物蛋白条带大小一致。     

葡萄糖3-脱氢酶工程菌的构建、表达及其合成3-酮对硝基苯井冈霉胺

采用基因挖掘技术,从NCBI数据库发现来源于Glaciecola polaris的葡萄糖3-脱氢酶基因,该基因经密码子优化后合成,构建p ET28b-GpG3DH重组表达载体,转入大肠杆菌E. coli BL21中进行重组表达,结果显示:GpG3DH实现了部分可溶表达,其分子量为5. 5×104。随后,优化了GpG3DH的表达条件,结果发现,重组菌在TB培养基中37℃培养至OD600为0. 8时,加入IPTG至终浓度为0. 5 mmol/L,16℃诱导表达10 h,GpG3DH酶活为2. 83×10-2U/m L。进一步将重组菌用于3-酮对硝基苯井冈霉胺的细胞转化法合成,细胞用10 mmol/L的EDTA处理,在p H为7. 5、25℃的条件下转化32 h,3-酮对硝基苯井冈霉胺的产率(Y3-KpNPV)为0. 54。     

肺炎支原体铁吸收调节蛋白基因的克隆表达与纯化

[目的]克隆肺炎支原体铁吸收调节蛋白(Fur)基因并纯化Fur蛋白,为研究其生物学功能奠定基础。[方法]利用Clustal Omega分析肺炎支原体Fur蛋白及其同源序列并用MEGA6.0构建进化树,通过PCR扩增Fur基因并对其进行双酶切,然后连接到p ET28a,得到重组载体p ET28a-Fur,将其转化大肠杆菌BL21(DE3),利用IPTG诱导Fur蛋白表达,并通过亲和层析纯化Fur蛋白。[结果]多序列比对和进化树分析表明Mp含有一个Fur蛋白。克隆得到大小为477 bp的Fur基因,编码159个氨基酸。得到重组表达质粒p ET28a-Fur,该质粒能在大肠杆菌中能高效表达,并纯化得到重组Fur蛋白。[结论]成功克隆获得Mp Fur基因,在大肠杆菌中高效表达并获得高纯度Fur蛋白。     

沙眼衣原体Ahpc蛋白的原核表达、纯化及活性分析

本研究根据Ct Ahpc基因序列设计特异性引物,以Ct DNA为模版PCR扩增Ahpc基因到大小为585 bp,将其连接到p ET28a获得重组质粒p ET28a-Ahpc,将该质粒转化到大肠杆菌BL21(DE3)获得重组菌BL/p ET28a-Ahpc,利用IPTG诱导重组Ahpc蛋白表达,目的蛋白分子量大小约为26 k D。利用Ni-NAT亲和层析法纯化得到的重组Ahpc蛋白。氧化铁二甲酚橙法检测发现Ahpc蛋白能够分解双氧水和叔丁基过氧化氢,具有分解过氧化合物的活性。测定重组大肠杆菌在百草枯所致氧化胁迫下的生长曲线,发现表达Ahpc蛋白的重组菌的生长情况比对照好。本研究成功表达和纯化得到沙眼衣原体Ahpc蛋白并测定其活性,为解析沙眼衣原体的抗氧化机制提供实验证据。     

重组芋螺毒素His-Xa-MrVIB分泌表达研究

[目的]利用简单快速的基因工程法来生产富含二硫键的芋螺毒素Mr VIB,寻找有效合成具有天然活性芋螺毒素的新途径。[方法]人工设计合成芋螺毒素Mr VIB基因引物来构建表达载体p ET22b(+)/His-Xa-Mr VIB,将其转化大肠杆菌BL21(DE3)plys S进行诱导表达。再利用Ni-NTA琼脂糖柱进行亲和层析纯化重组蛋白,Tricine-SDS-PAGE电泳分析重组蛋白表达形式。[结果]重组芋螺毒素His-Xa-Mr VIB(r His-Xa-Mr VIB)在大肠杆菌中获得有效分泌表达,经一步亲和层析获得纯度大于90%的重组芋螺毒素。[结论]基因工程方法能够有效分泌表达芋螺毒素Mr VIB,解决化学合成芋螺毒素产量低、成本高、难以纯化等问题。     

PPK和GMAS共表达重组菌株的构建及其在L-茶氨酸合成中的应用

构建了共表达ATP再生和L-茶氨酸合成酶的重组大肠杆菌菌株,并将其应用于L-茶氨酸的合成中。合成多聚磷酸盐激酶(PPK)和γ-谷氨酰甲胺合成酶(GMAS)基因序列,分别利用p ETDuet-1和p ET-21a(+)载体,构建共表达重组质粒p ETDuet-ppk+gmas和p ET21a-ppk+gmas。将上述两种重组质粒转入大肠杆菌BL21(DE3)中,获得重组菌株TPG和APG。IPTG诱导表达后,SDS-PAGE结果表明,PPK和GMAS在两种重组菌中均可溶性表达。当用于催化L-茶氨酸合成时,来自APG的GMAS-PPK要优于TPG。利用APG所产的酶进行L-茶氨酸合成,在37℃、p H 7.0条件下,使用催化量ATP可实现L-茶氨酸的摩尔产率为86.0%。该结果一方面扩展了酶法ATP再生系统的应用,另一方面为生物催化合成L-茶氨酸提供了一种有效方法。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.