Polyclonal antibody against an insect excitatory toxin BmKIT from Buthus Martensii karsch and detection of BmKIT expressed in transgenic cotton

An insect excitatory toxin gene from Buthus martensii Karseh （BmKIT） was cloned into the expression vector, pET-28a. BmKIT was expressed as inclusion bodies in Eseherichia coli BL21 （DE3） host cells. The authenticity of in vitro expressed protein was confirmed by Western blot. The inclusion body protein band in SDS-PAGE was excised and the protein, BmKIT, was extracted. Polyelonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed BmKIT and was used to quantify its presence in transgenic cotton.     

Nerve growth factor activity of several immunal related serine proteases

Rabbit was immuned by the previously purified protein with high nerve growth factor (NGF) bioactivity (NGF_like protease) from \%Agkistrodon halys Pallas\% and the antisera were collected. The polyclonal antibodies were tentatively purified and then used as ligands of an affinity column. The \%A.h.Pallas\% crude venom was fractionated by this affinity column and then by Mono Q on fast protein liquid chromatography (FPLC). As a result, fraction Ⅱ and fraction Ⅲ were purified respectively, whose N_terminal amino acid sequences show high homology with the serine proteases in snake venoms, as well as the previous NGF_like protease. However, they possessed different levels of NGF bioactivity. The NGF activity of the previous NGF_like protease is equivalent to that of NGF, while the activity of fraction Ⅱ seems relatively low in contrast to fraction Ⅲ which had no NGF activity.     

Characteristics of the binding features of Nelin with F-actin and screening Nelin interactive proteins

The interaction of nelin, a cardiac-specific expressed protein of human novel gene nelin, with F-actin was studied by both F-actin cosedimentation in vitro and colocalization assays. The results showed that nelin is a new F-actin binding protein and is colocolized with F-actin in cytoplasm of cells. Three new nelin binding proteins, filamin C subtype, titin N2B subtype and inter-alpha trypsin inhibitor heavy chain precursor (ITIH), were identified from human heart cDNA library using yeast two-hybrid screening. The binding activity of filamin C with nelin was confirmed by coimmunoprecipitation. Filamin C binds to nelin through its C-terminal region. It is indicated that nelin is a cytoskeleton associated protein and acts as a membrane-cytoskeleton associated protein involved in the formation of focal adhesions.     

Sequence analysis of the Microcystis aeruginosa FACHB-912 phytochrome gene supports positive selection in cyanobacteria

The phytochrome gene from Microcystis aeruginosa FACHB-912,isolated from algal blooms in Taihu Lake in China,was sequenced and found to code for a wavelength transition light receptor protein.The gene was composed of five distinct domains:a PAS fold domain,a GAF domain,a phytochrome domain,a His Kinase A(phosphoacceptor) domain,and a histidine kinase domain.Red-absorbing phytochrome,far-red-absorbing phytochrome,and photoconversion kinetics were monitored via the spectral characteristics of the protein.To detect positive selection,homologous sequences from cyanobacterium phytochrome genes were obtained from GenBank.Potential selective pressure was identified by phylogenetic analysis with maximum likelihood analyses.The frequency of the phytochrome gene being subjected to selective constraints suggests that positive selection is a potentially important mechanism that promotes the evolution of the cyanobacteria phytochrome gene.The sites identified in this study provide targets for further research on the structural-functional role of these residues,and on the correlation with the mechanism of algal blooms.     

Cloning and enzymology analysis of farnesyl pyrophosphate synthase gene from a superior strain of<Emphasis Type="Italic">Artemisia annua</Emphasis> L.

A cDNA(af1) encoding farnesyl pyrophosphate synthase AaFPS1(FPS,EC2.5.1.1/EC2.5.1.10) from a high yield Artemisia annua strain 025 has heen cloned from its cDNA library .Sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with molecular weight of 39 kD.Deduced aa sequence of the cDNA was similar to FPS from other plants,yeast and mammals,containing 5 conserved domains found in both prenyl trans-ferase and polyprenyl synthase,The expression of the cDNA in Escherichia coli showed measurable specific activity of FPS in vitro.The enzyme was purified by ion exchange chromatography and its kinetics was measured ,These results would further promote the molecular regulation of artemisnin biosynthesis.     

Interaction of hepatitis B virus with tumor suppressor gene p53: its significance and biological function

The mechanism of the interaction of hepatitis B virus (HBV) with tumor suppressor p53 and its role in the hepatocar-cinogenesis have been studied by PCR-directed sequencing, gel shift assays and in situ ultraviolet cross-linking assay. The biological function of the interaction of HBV with p53 gene was investigated by co-transfection of chloramphenicol acetyltransferase ( CAT) reporter gene. p53 and HBV DNA. and quantitative PCR. Among the 16 primary hepatocellular carcinoma (PHC) samples. 13 were HBV-DNA positive. 10 HBxAg positive and 9 p53 protein positive. The p53 gene point mutation was found in 5 samples, one of which had a G to T substitution located at codon 249. After analyzing the HBV genome by a computer program, a p53 response element binding sequence was found in HBV genome at upstream of enhancer I. from 1047 to 1059 nucleotides. This sequence could specifically bind to p53 protein, increase p53 protein accumulation in the PHC cells and stimulate the transactivating activity of p53 and HBV replication . The results also revealed that HBxAg could combine with p53 protein to form a complex in the cells and enhance CAT expression. Immunocytochemical staining showed that p53 protein complex was located in the cytoplasm and the process of p53 entry to nuclei was. in part, blocked. From our results, we conclude that the mutation of p53 gene at codon 249 is infrequent in HBV-associated PHC. the DNA-protein binding between HBV and p53. and the protein-protein binding between HBxAg and p53 might lead to the reduction or inactivation of p53 protein, which in turn resulting in HBV-associated hepatocarcinogenesis.     

2,3-Dihydroxybiphenyl dioxygenase gene was first discovered in Arthrobacter sp. strain PJ3

Bacterium strain PJ3,isolated from wastewater and identified as Arthrobacter sp. bacterium based on its 16S rDNA gene,could use carbazole as the sole carbon,nitrogen and energy source. The genomic library of strain PJ3 was constructed and a positive clone JM109(pUCW402) was screened out for the expression of dioxygenase by the ability to form yellow ring-fission product. A 2,3-dihydroxybiphenyl dioxygenase(23DHBD) gene of 933 bp was found in the 3360 bp exogenous fragment of pUCW402 by GenSCAN software and BLAST analysis. The phylogenetic analysis showed that 23DHBD from strain PJ3 formed a deep branch separate from a cluster containing most known 23DHBD in GenBank. Southern hybridization confirmed for the first time that the 23DHBD gene was from the genomic DNA of Arthrobacter sp. PJ3. In order to test the gene function,recombinant bacterium BL21(pETW-8) was constructed to express 23DHBD. The expression level in BL21(pETW-8) was highest compared with the recombinant bacteria JM109(pUCW402) and strain PJ3. We observed that 23DHBD was not absolute specific. The enzyme activity was higher with 2,3-dihydroxybiphenyl as a substrate than with catechol. The substrate specificity assay suggested that 23DHBD was essential for cleavage of bi-cyclic aromatic compounds during the course of aromatic compound biodegradation in Arthrobacter sp. strain PJ3.     

Cloning and expression of DnaJ homolog in carrot somatic embryo

As the co-chaperone of DnaK/Hsp70 protein, DnaJ/Hsp40 protein influences the synthesis and assembly of the protein complex by regulating ATPase activity of DnaK/Hsp70 protein. By employing the modified method of cDNA representational difference analysis, a homologous fragment of DnaJ was isolated from the deregulated carrot somatic embryos, and it was further used as the probe to screen the cDNA library of carrot somatic embryo deregulated for 12 h. As the result, DcJ1 gene, the homologous gene of DnaJ, was isolated from carrot. Sequence analysis showed that its coding region is 1257 bp, which codes 418 amino acids and comprises 3 highly-conserved characteristic domains. Southern blot analysis suggested that the DcJ1 gene seems to be a single copy in the genome, while Northern blot result indicated that DcJ1 expresses only in roots and its degree of expression changes obviously with the regulation-deregulation process. These results suggest that DcJ1 is correlated with the early development of carrot somatic embryo radicle.     

A novel peptide,selected from phage display library of random peptides,can efficiently target into human breast cancer cell

To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 （9 aa） phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate extra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the pepUde-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of peptide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by coculturing them with other human tumor cell lines and normal cells. The nucleoUde sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the pepUde to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC （the shifted sequence of CASPSGALRSC）, and after coculturing them with different cell lines, their targeting capacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of pepUdes was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.     

小鼠噬菌体抗体库的构建和N-肽结合单链抗体的筛选

成功构建一库容量为1.7×10⁸的小鼠噬菌体抗体库,并从中淘选出对N-肽有结合活性的单链抗体。首先从未免疫小鼠的脾脏中提取总RNA,分离出mRNA,经逆转录合成其总cDNA。随后通过PCR分别扩增出抗体重链和轻链可变区V_H、V_L的基因片断,再经重组PCR将两片断由一编码(Gly₄Ser)₃十五肽的接头连在一起,并克隆到噬菌质粒pCANTAB 5E中,电击转化大肠杆菌TG1。经辅助噬菌体M13KO7超感染回收全部重组噬菌体,此即噬菌体抗体库。N-肽是一种新发现的神经肽,其N端与阿片肽高度同源,可与阿片肽受体相互作用。对其研究可能有助于了解成瘾机制和有临床应用价值。将生物素化的N-肽与抗体库相互作用,用偶联有链霉亲和素的磁珠富集与N-肽结合的重组噬菌体,经四轮淘选,得到亲和力较高的单链抗体。     

一种导向性溶栓剂的基因构建及其在大肠杆菌中的表达

以针对人活化血小板表面糖蛋白GMP140的单克隆抗体SZ51的单链抗体作为导向分子,以单链尿激酶32kd变体(scu-PA-32k)作为效应分子,构建了一种新型的导向性溶栓剂。通过聚合酶链式反应(PCR),分别从SZ51的Fab片段基因中扩增出V_K和V_H区;从尿激酶原基因中扩增出scu-PA-32k基因。通过合适的linker及酶切位点,将V_K, V_H及scu-PA-32k基因相连接并装入表达载体pET-5a中的Nde I位点,在大肠杆菌中经IPTG诱导表达了重组蛋白。Western-Blotting检测到在8mol/L尿素存在下,该重组蛋白与抗尿激酶的多克隆抗体之间仍有弱的结合反应。表达产物经变复性处理和部分分离纯化后,在纤维蛋白平板上表现有较好的纤溶活性,且这种活性是通过激活纤维蛋白溶酶原为纤溶酶而实现的。重组蛋白纤溶活性的比活达到17500IU/mg,较好地保留了尿激酶的纤溶活性,重组蛋白的产率约每100g湿菌为1.5mg。     

GHRP-scu-PA-32K突变体的构建、表达及纯化产物的部分性质研究

在scu-PA-32K的cDNA分子基础上经定点突变,在N端紧接Leu¹之前引入编码GHRP四肽的寡核苷酸序列(GGTCATAGGCCT),构建了GHRP-scu-PA-32K的突变体cDNA。将它克隆到表达载体pCM-βneo中,与pCMβ-dhfr共转染CHO/DHFR-细胞。筛选到的稳定表达株在无血清培养基的表达量为580IU/(106细胞·24h)。经锌离子螯合亲和柱纯化的产物,SDS-PAGE显示为单一蛋白条带,分子量为33kD,质谱法测定分子量也为33kD。经血纤维蛋白平板法测定比活为150000IU/mg蛋白。对血纤维蛋白的亲和性,突变体为scu-PA-32K的4.5倍。     

产辅酶Q10的类球红细菌原生质体制备和再生研究

 辅酶Q₁₀(CoQ₁₀)是人类细胞自身产生的内源性辅酶因子,是一类天然抗氧化剂,目前已广泛应用于医药、食品、化妆品等诸多领域.基因组改组技术的核心内容为原生质体融合,为了提高基因组改组的效率,提高辅酶Q₁₀的产量,本研究对产辅酶Q₁₀的类球红细菌原生质体制备及再生条件进行了优化,通过生长曲线、正交试验等确定了原生质体制备及再生的适宜条件：溶菌酶浓度1mg/mL、作用温度37℃、作用时间1h以及再生培养基蔗糖浓度10%.在此条件下,类球红细菌原生质体形成率可达到96.1%,再生率可达到28.8%.这为进一步对该菌的基因组改组研究奠定了基础.     

利用基因组改组技术提高辅酶Q10产量

 辅酶Q₁₀(CoQ₁₀)是生物细胞呼吸链中的重要递氢体,已广泛应用于心脏病、肝炎、帕金森症等多种疾病的治疗中.为了提高微生物法生产CoQ₁₀的产量,本文利用基因组改组技术选育类球红细菌辅酶Q₁₀高产菌株.根据CoQ₁₀的合成途径及作用机理,确定了不同的抗性筛选标记物：罗红霉素、卡那霉素、对羟基苯甲酸、维生素K3和硫化钠.根据类球红细菌对标记物的耐受性确定了抗性筛选浓度.通过紫外线、紫外线/氯化锂、硫酸二乙酯、微波及钴60 5种诱变方式以及抗性培养基筛选获得了9株改良的突变株作为出发菌株.通过一轮基因组改组得到了几株高产菌株,其中PN13产CoQ₁₀的量可达到2.39mg/g,是原菌的2.52倍.     

豌豆cop1 cDNA的克隆、序列分析及其在大肠杆菌中的表达

以拟南芥cop1 cDNA为探针,从豌豆(Pisum sativum) cDNA文库中克隆到了豌豆cop1 cDNA。序列分析表明,它全长为2863bp,其中包括604bp 5′非编码区、243bp 3′非编码区和2016bp编码区,编码672个氨基酸。在大肠杆菌中实现了豌豆cop1基因的高效表达。对拟南芥、豌豆和番茄3种植物cop1的序列同源性比较表明,cop1可能是一种进化上很保守的蛋白质。     

浑善达克沙地C3和C4植物对干旱胁迫响应策略

 选取浑善达克沙地C₃植物羊草(Leymus chinensis)和C₄植物虎尾草(Chloris virgata)为研究对象,进行2种干旱胁迫处理,处理1将植物浇至饱和含水量后,使其自然干旱10d,之后复水至饱和含水量;处理2是在处理1的基础上,每天晚上让植物接受自然的大气凝结水.处理期间测定植物的叶片水势、比叶面积、气孔密度、氮含量及生物量等生理生态指标,探讨干旱胁迫及复水后不同光合功能型植物对干旱胁迫响应策略.研究表明,(1) 干旱胁迫显著降低了虎尾草的叶片水势,大气凝结水没有显著改变其叶片凌晨水势,干旱胁迫第10天时羊草叶片凌晨水势值显著降低,大气凝结水使羊草叶片的水势值保持在正常范围之内;(2) 不同干旱胁迫下,植物比叶面积和气孔密度与处理前相比呈下降趋势,但在复水后恢复到处理前水平;(3) 干旱胁迫显著提高了羊草叶片的氮含量,而虎尾草叶片氮水平变化不显著;(4) 干旱胁迫显著提高了羊草的根冠比,而虎尾草的根冠比影响不大,但干旱胁迫则显著提高了虎尾草的果重比.羊草在干旱胁迫下分配较多的生物量到根系,有利于水分的吸收,从而提高了其抗旱性,而虎尾草则分配较多的生物量到种子,使其不断扩大繁殖群体.     

琐琐葡萄多糖对PC12细胞损伤的神经保护作用

 体外培养PC12细胞,采用20μmol/L β淀粉样蛋白25-35(Aβ25-35)作用24h诱导细胞损伤,建立阿尔茨海默病(AD)细胞模型,研究琐琐葡萄多糖(VTP)对PC12细胞损伤的神经保护作用。设立对照组、模型组和VTP保护组(20,40,80μg/mL),CCK-8法检测各组细胞的存活率,乳酸脱氢酶(LDH)法检测细胞膜通透性及完整性,化学比色法测定细胞内超氧化物歧化酶(SOD)、丙二醛(MDA)含量,流式细胞术检测细胞凋亡率。结果显示,20,40,80μg/mL VTP可提高PC12细胞存活率,减少LDH渗漏,增加SOD活力,减少MDA含量,降低细胞凋亡率,与模型组比较有显著差异(P<0.01)。由此推论,VTP对Aβ25-35诱导的PC12细胞凋亡和氧化损伤具有明显的保护作用。     

利用十二肽库筛选心肌型脂肪酸结合蛋白特异性的亲和配体

采用噬菌体表面展示十二肽库对心肌型脂肪酸结合蛋白（H FABP）特异性亲和配体进行筛选, 经3轮筛选及序列比对后得到一个酶联免疫测定分析 (ELISA)检测阳性特征序列： W P N H H M L H K R W P. 对此序列进行肽合成, 并标记上荧光标记物芘, 经高效液相纯化、 冻干及质谱确定其分子量后制成荧光肽探针检测急性心肌梗塞病人血样, 结果均呈阳性. 结果表明, 所获得的肽探针具有较好的临床应用效果.心肌型脂肪酸结合蛋白; 噬菌体表面展示十二肽库; 亲和配体; 酶联免疫测定分析     

西帕依固龈液对IL-1β刺激人牙龈成纤维细胞产生PGE2的影响

 研究不同浓度西帕依固龈液对白细胞介素-1β(Interleukin-1β,IL-1β)刺激人牙龈成纤维细胞(Human Gingival Fibroblast,HGF)产生前列腺素E₂(Prostaglandin E₂,PGE₂)水平的影响。采用健康人牙龈组织原代培养的HGF细胞,以1ng/mL的IL-1β作为刺激因子,将HGF细胞按5×10⁴/mL的细胞密度接种到24孔培养板中,每孔200μL的细胞悬液。孵育24h贴壁后,弃原培养液,清洗2次后加药,分组为空白对照组(用含20mL/L的胎牛血清的DMEM培养液),IL-1β组(浓度为1ng/mL),IL-1β+西帕依固龈液组(西帕依固龈液终末浓度分别为12.5、25、50、100、200μg/mL),IL-1β+消炎痛(消炎痛浓度为100ng/mL)。用酶联免疫法测定PGE₂含量,观察不同浓度的西帕依固龈液(分别为12.5、25、50、100、200μg/mL)对HGF培养上清中PGE₂水平的影响。结果显示,5种不同浓度的西帕依固龈液均能显著抑制1ng/mL的IL-1β刺激后HGF产生PGE₂,随着浓度的增加,抑制效果也增加,但5种浓度的西帕依固龈液抑制效果均低于100ng/mL的消炎痛。由此得出,HGF具有合成和分泌PGE₂的功能,IL-1β能有效刺激HGF产生PGE₂;西帕依固龈液对IL-1β刺激HGF合成和分泌PGE₂具有显著抑制作用,其抑制作用在一定范围内呈浓度依赖性。