Characterization of a FeMo cofactor-deficient MoFe protein from a nifE-deleted strain （DJ35） of Azotobacter vinelandii

A MoFe protein （△nifE Av1） with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP （OP Av1）, △nifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis （PAGE） was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of △nifE Av1 both apparently decreased. When complemented with OP Fe protein, △nifE Av1 had no C2H2-reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism （CD） spectrum of △nifE Av1 at ～450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that △nifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-containing MoFe protein.     

The composition and distribution of metal clusters in the MoFe protein from a nifZ deletion strain （DJ 194） of Azotobacter vinelandii

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (△nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194).The results of Western blotting after anoxic native electrophoresis and SDS-PAGE showed that △nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Furthermore, △nifZ Avl was also similar to OP Av1 at the molybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (△ε) of circular dichroism (CD)at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the △nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and substrate (C2H2, H^ and N2)-reduction activity of △nifZ Av1 were 74% and 46%-50% of those of OP Av1, respectively. Furthermore, the △ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between △nifZ Avl and OP Av1 resulted from P-cluster rather than FeMoco, and from the half number of P-cluster in △nifZ Av1, but the composition or redoxstate of P-cluster in △nifZ Av1 were not changed. Thus it could propose that △nifZ Av1 is composed of two different αβsubunit pairs. One is a FeMoco-and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-containing pair. Since the deletion of nifZ gene leads to the deficiency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.     

Identification of a protein associated with circulative transmission of Barley yellow dwarf virus from cereal aphids, Schizaphis graminum and Sitobion avenae

Using 2-D electrophoresis and virus overlay assay,a 50-kDa protein(P50)exhibiting specific binding to purified virus particles of BYDV-GAV was found in the protein extracts from Schizaphis graminum and Sitobion avenae,two aphid species transmiting BYDV-GAV.P50 in the extracts of S.graminum was isolated by preparation electrophoresis and electro-eluted proteins from the gel slices for antiserum preparation.After feeding the antiserum through membrane,the transmission efficiencies of S.graminum and S.avenae for BYDV-GAV decreased significantly.It was suggested that P50 should be related with transmission process.Location of P50 was found at the plasma membrane surrounding the accessory salivary gland(ASG) in the head tissues of S.graminum by immunogold-labelling experiment.The ascertainment of the protein associated with virus transmission has a significance influence on further understanding the transmission mechanism and genetic engineering for resistant to vector transmission.     

Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 （HSC70） in Fenneropenaeus chinensis

The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5‘- and 3‘-untranslated region(5‘- and 3‘-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1--547 bp, but they did not exist in the region of 548--2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.     

Prokaryotic expression and characterization of a pea actin isoform （PEAcl） fused to GFP

Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells,it is difficult to purifyeach actin isoform in sufficient quantities for analysing itsphysicochemical properties. In the present study, apea(pisum Sativum L.)actin isoform (PEAc1)fused to His-tag at its amino terminus and GFP(green fluorescent protein)atits Carboxyl terminus were expressed in E. coli in inclusionbodies. The fusion protein (PEAc1-GFP)was highly purifiedwith the yield of above 2 mg/L culture by dissolving inclu-sions in 8 mol/L urea,renaturing by dialysis in a gradient of urea,and affinity binding to Ni-resin. The purified mono-meric PEAc1-GFP could efficiently bind on DNase I andinhibit the latter抯 enzyme activity. PEAc1-GFP could po-lymerise into green fluorescent filamentous structures(F-PEAc1-GFP),which could be labelled byTRITC-phalloidin,a specific agent for observing microfila-ments. The PEAc1-GFP polymerlzation curve was identicalwith that of chicken skeletal muscle actin. The critical con-centration for PEAc1-Gfp to polymerise into filaments is 0.24 μmol/L.The F-PEAc1-GFP could stimulate myosinMg-ATPase activity in a protein concentration dependantmanner (about 4 folds at 1 mg/mL F-PEAc1-GFP). The re-sults above show that the PEAc1 fused to GFP retained theassembly characteristic of actin, indicating that gene fusion,prokaryotic expression, denaturation and renaturation,andaffinity chromatography is a useful strategy for obtainingplant actin isoform proteins in a large amount.     

Genetic analysis and identification of a large leaf angles （lla） mutant in rice

Mutants are essential genetic muterials to elucidation of biological functions of genes involved.Characterization and isolation of genes in mutants is one of the research tasks in functional genomic era.T-DNA insertional mutagenesis has provided an efficient way to identify genes in plant species.in which the mutated genes could be rapidly isolated once the mutant was confirmed by T-DNA inscrtion.     

Synthesis and properties of a Pr（Ⅲ） complex with 2-acetylbenzimidazoledehyde-glycine Schiff-base ligand

Abstract The 2-acetyl-benzimidazoledehyde-glycine Schiff-base ligand and the corresponding Pr（Ⅲ） complex Pr2L3（NO3）3· 2CH3OH （L=C11H10N3O2） were synthesized in methanol and characterized by a series of methods, including chemical analysis, elemental analysis, TOF-MS, ^1H NMR, UV-, IR-, Raman spectra, thermal analysis, and the three-dimension fluorescence excitation and emission spectra. The Pr（Ⅲ） complex exhibits extraordinary water-solubility and the Pr（Ⅲ） hydroxide appears at pH≥13. The complex also possesses specific fluorescent properties. Thus, at the excitation wavelengths 200.0-280.0 and 260-350 nm the fluorescence bands were observed at 290.0 and 400.0 nm, respectively.     

Green fluorescent protein （GFP） as a vital marker for studying the interaction of Phytophthora sojae and soybean

Transgenic Phytophthora sojae strains that produce green fluorescent protein （GFP） were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae. The expression of GFP during different developmental stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the histopathologic events caused by the pathogen in soybean leaves, hypocotyls and roots were monitored. Meanwhile, the difference in resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results indicate that GFP can be stably expressed in zoosporangia, zoospores, cysts, hyphae and oospores of P. sojae. Using the GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host could be distinctly visualized. The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible cultivar Hefeng 35. These results show for the first time that this eukaryotic reporter can be used in P. sojae as a stable and vital marker, allowing the study of genetics of this hemibiotrophic pathogen.     

Enantioselective assay of S（＋）－ and R（－）－propafenone in human urine by using RP－HPLC with pre－column chiral derlvatization

The enantioselective assay for S(+)- and R(-)-propafenone (PPF) in human urine that developed in this work involves extraction of propafenone from human urine and using S(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-beta-D-glucopranosyl isothiocyanate, and quantitation by an RP-HPLC system with UV detection (lambda=220 nm). A baseline separation of propafenone enantiomers was achieved on a 5-microm reverse phase ODS column, with a mixture of acetic acid (25:12:0.02,v/v) as mobile phase. There was good linear relationship from 24.9 ng/ml to 1875.0 ng/ml for both of enantiomers. The regression equations of the standard curves based on C(S-PPF) (or C(R-PPF)) versus ratio of A(S-PPF)/A(S) (or A(R-PPF)/A(S)) were y=0.0032x-0.081, (r=0.999) for S-PPF and y=0.0033x+0.0039, (r=0.998) for R-PPF, respectively. The method's limit of detection was 12.5 ng/ml for both enantiomers, and the method's limit of quantitation was 28.2+/-0.52 ng/ml for S-PPF, 30.4+/-methanol:water:glacial 0.53 ng/ml for R-PPF (RSD<8%, n=5). The analytical method yielded average recovery of 98.9% and 100.4% for S-PPF and R-PPF, respectively. The relative standard deviation was no more than 6.11% and 6.22% for S-PPF and R-PPF, respectively. The method enabled study of metabolism of S(+)- and R(-)-propafenone in human urine. The results from 7 volunteers administered 150 mg racemic propafenone indicated that propafenone enantiomers undergo stereoselective metabolism and that in the human body, S(+)-propafenone is metabolized more extensively than R(-)-propafenone.     

幽门螺杆菌重组VacA蛋白的表达、纯化和鉴定

建立从幽门螺杆菌空泡毒素A(VacA)原核表达系统pET32a-vacA-E.coliBL21DE3中,表达、纯化和鉴定重组空泡毒素A(rVacA)蛋白的方法.采用不同浓度的IPTG(0.1、0.5和1.0 mmol.L-1)诱导原核表达系统表达rVacA,10%SDS-PAGE检测表达产量,Ni-NTA亲和层析法纯化rVacA,采用HPLC检测其纯度,Western-blotting进行免疫学鉴定.从已构建的VacA原核表达系统中成功表达和纯化了rVacA蛋白,产量约占细菌总蛋白的28.4%,纯度为82.3%.为后续进一步研究该蛋白的生物活性和相关检测试剂盒奠定基础.     

烛缸法分离培养及冷冻法保存幽门螺杆菌

目的研究幽门螺杆菌（H elicobacter py lori,H py lori）简易分离培养及保存方法,构建H py lori小型菌库。方法首先采用H py lori选择培养基,以烛缸法分离培养不同来源的标本,然后以革兰染色显微镜形态检查及生化试验鉴定为H py lori,最后把鉴定为H py lori的纯培养物,...     

一种大规模、快速的幽门螺杆菌脲酶分离纯化方法研究

幽门螺杆菌定植于人胃黏膜并产生大量的脲酶,报道一种大规模、快速的脲酶纯化方法.脲酶用去离子水振荡及冻融法抽提,采用Q Sepharose FF阴离子交换层析,Phenyl Sepharose HP疏水相互作用和SOURCE30S阳离子交换层析等方法纯化得到脲酶,通过酚红脲酶试剂检测酶活.总蛋白回收率为2.58%,纯化后脲酶纯度大于95%,SDS-PAGE电泳显示脲酶是由相对分子质量66 000的A亚基和30 000的B亚基组成.     

黑豆中抗真菌蛋白的纯化及活性鉴定

利用硫酸铵分级沉降、亲和色谱Affi-gel和离子交换色谱SP-Toyopearl从黑豆种子中分离出一种抗菌蛋白,SDS-聚丙烯酰胺凝胶电泳鉴定达到电泳纯.利用SDS-聚丙烯酰胺凝胶电泳标准蛋白迁移率与分子量对数的关系,计算出该抗菌蛋白的分子量为93.3 kDa,还原和非还原状态下均显示单一区带,说明该抗真菌蛋白为单倍体蛋白.抑菌试验结果表明,此纯化的蛋白对植物致病菌瓜果腐霉病菌、棉花枯萎病菌、苹果轮纹病菌、葡萄灰霉病菌和菜豆根腐病菌等5种真菌的生长具有不同的抑制作用.     

大豆（G．max）胰蛋白酶抑制剂SBTi－A_2新类型Ti~x的纯化及其性质研究

用丙酮脱脂、稀酸处理、硫酸铵盐析和ＤＥＡＥ－５２纤维素柱层析等方法从大豆（Ｇ．ｍａｘ）中分离纯化得一种新类型胰蛋白酶抑制剂Ｔｉｘ．ＳＤＳ－ＰＡＧＥ分析显示Ｔｉｘ与已知的大豆蛋白酶抑制剂Ｔｉａ的相对分子质量均为２１０００，且Ｎ－末端均为ＡｓＰ；亲和电泳分析显示Ｔｉｘ与Ｔｉａ均可与胰蛋白酶完全结合；ＢＡＥＥ法测定显示出Ｔｉｘ对胰蛋白酶活性的抑制略小于Ｔｉａ；两者经ＣＮＢｒ裂解后电泳分析得到相同带型；纯化样品的氨基酸组成分析显示Ｔｉｘ比Ｔｉａ多了两个碱性氨基酸，因而Ｔｉｘ带有更多的正电荷．研究结果初步证明Ｔｉｘ是Ｋｕｎｉｔｚ类型中一种新的胰蛋白酶抑制剂．     

金属硫蛋白(MT)的分离纯化与检测技术

综述了近年来在金属硫蛋白研究中应用的分离纯化和检测方法,对各种方法的适用性等特点作了评述。     

CagA阳性幽门螺杆菌感染与消化性溃疡、胃炎相关性512例临床分析

目的探讨CagA阳性Hp感染与消化性溃疡、胃炎等胃及十二指肠疾病的关系.方法采用ELISA法对Hp阳性(~(13)C-呼气试验为阳性)、胃黏膜快速尿素酶联免疫法阳性的512例胃十二指肠疾病患者及80例对照者(健康体检:~(13)C-呼气试验为阴性、胃黏膜快速尿素酶联免疫法阴性)行血清CagA抗体检测.512例胃十二指肠疾病患者中慢性浅表性胃炎(CSG)184例,慢性萎缩性胃炎(CAG)120例,十二指肠球溃疡(DU)100例,胃溃疡(GU)108例.结果 CagA抗体阳性检出率对照组为30%,慢性浅表性胃炎组阳性率为61.96%,慢性萎缩性胃炎组阳性率为63.33,十二指肠溃疡组阳性率为68.00%,胃溃疡组阳性率为74.07%.胃十二指肠疾病患者CagA阳性总检出率为66.02%,与对照组比较,差异具有统计学意义(P0.05).结论胃十二指肠疾病Hp阳性感染者中CagA抗体阳性总检出率明显高于对照组,提示CagA抗体阳性感染与胃十二指肠疾病密切相关;两组胃炎患者CagA抗体阳性检出率明显高于对照组,提示CagA抗体阳性感染与胃炎密切相关;胃十二指肠溃疡患者CagA抗体阳性检出率明显高于对照组,提示CagA抗体阳性感染与消化性溃疡密切相关.     

抗幽门螺杆菌便餐粉配方优化及其体外抑菌研究

为制备抗幽门螺杆菌便餐粉,将实验室制备的高免蛋液(由Ure B、BabA2和Fla A免疫产蛋鸡后制备而成,含Ure B-BabA2-FlaA-IgY)通过喷雾冷冻干燥技术获得高免蛋粉。在原有配方的基础上,通过单因素实验研究木糖醇添加量、麦芽糊精添加量和高免蛋粉添加量对便餐粉品质指标(休止角、堆积密度、结块率、流动性、挂壁)的影响。在单因素实验基础上,以口感评分为指标,通过正交试验设计获得便餐粉最佳组合。通过间接Elisa法分析便餐粉的效价稳定性,将便餐粉与幽门螺杆菌菌液共培养,测定OD_(600)值分析其抑菌效果。结果表明当麦芽糊精添加量为20%、木糖醇添加量为10%、高免蛋粉添加量为15%时,便餐粉的品质最佳,37℃条件下,便餐粉效价在4个月内无显著性变化,60 mg/m L的便餐粉可有效抑制幽门螺杆菌的生长。成功研制出抗幽门螺杆菌便餐粉。     

Pc 1 Duarte, a common polymorphism of a human brain protein, and its relationship to depressive disease and multiple sclerosis

Pc 1 Duarte, a common variant of a human brain-specific protein, is present in one-third of the control population and has a gene frequency of 0.17. There was a significantly increased frequency of mutant heterozygotes and homozygotes among individuals with depressive disease. This is consistent with a model in which Pc 1 Duarte is the major gene in depressive disease, acting in conjunction with an environmental threshold effect.