人工哺育期腹泻婴猴奇异变形杆菌的分离与鉴定

目的对一株人工哺育期引发恒河猴婴猴腹泻的奇异变形杆菌进行了鉴定,为实验猕猴疾病检测、鉴别诊断提供参考依据。方法通过培养特性、菌落形态、染色、生化试验和血清学诊断鉴别等检查,对分离菌株进行初步鉴定,同时,对分离菌株进行致病性试验及药敏试验。结果通过表型生物学特性鉴定,并结合血清学诊断鉴别方法,确证该分离菌株为奇异变形杆菌,应用药敏试验筛选出了高度敏感的抗菌药,控制了该病的继续发生,致病性试验证明,该分离菌株对小白鼠有高致病性。结论分离到的奇异变形杆菌是导致本次婴猴腹泻死亡的病原菌,该菌为条件致病菌,对实验猕猴和研究人员均有潜在的危害,尽管该菌不是国家标准要求排除的病原菌,但该菌引发的传染病将对动物实验造成严重影响,故应引起高度重视。     

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.     

3α-羟类固醇脱氢酶基因的质粒载体构建和表达

目的 实现3α-羟类固醇脱氢酶基因在大肠埃希菌中的高可溶性表达.方法 从土壤中分离睾丸酮丛毛单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶(3α-HSD)基因,将它克隆到原核表达载体上进行诱导表达.提取细菌总蛋白进行SDS-PAGE分析并测定酶活性.结果 经核苷酸序列测定和酶切鉴定结果表明,成功地构建了重组质粒,IPTG诱导表达后,获得融合蛋白,SDS-PAGE初步测定目的蛋白的相对分子量约为29kDa,与预期理论值一致;酶活性测定结果表明菌体可溶性总蛋白HSD酶比活性为142.81 U/mg,是对照BL21的12.97倍.结论 该研究成功地构建了3α-羟类固醇脱氢酶基因高效原核表达系统,为利用基因工程手段大量制备3α-HSD的工作奠定了基础.     

大肠埃希菌Mn-SOD基因的克隆、表达及多克隆抗体制备

目的实现Mn-SOD基因在大肠埃希菌中的高可溶性表达,制备Mn-SOD的多克隆抗体。方法用PCR方法从一株野生型大肠埃希菌（E.coli）基因组中扩增Mn-SOD基因编码区．将它克隆到原核表达载体上进行大量表达和纯化,再用纯化的蛋白对新西兰大白兔进行背部多点注射,40d后取其血清,用Western-blot印迹实验测定抗体效果。结果SDS-PAGE分析表明SOD的表达量约为细菌总蛋白的50％;黄嘌呤氧化酶法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物酶比活为3921．77U／mg,是对照BL21的276．77倍;并制备了高效价的多克隆抗体。结论该研究成功地构建了大肠埃希菌Mn-SOD基因高效原核表达系统,所表达的Mn-SOD具有良好的免疫原性和免疫反应性。     

诺卡氏菌酰胺酶基因的分子克隆及在大肠杆菌中表达的初步研究

酰胺酶是一种重要的工业酶。利用生物信息学手段,在和已知酰胺酶基因序列分析比对的基础上,首次从Ncordiasp.YS-2002中成功地克隆得到酰胺酶基因ami,并对其基因序列及氨基酸序列的性质进行了分析。结果表明,所得酰胺酶基因ami片段大小共为1446bp,由启动子区、阅读框和回文结构终止区三部分构成。序列分析和进化树分析表明,Ncordiasp.YS-2002酰胺酶是一种比较特殊的酰胺酶,不含大多数酰胺酶共同具有的保守区序列。进一步将酰胺酶基因连接到pET-28a( )上,转入大肠杆菌BL21(DE3)中筛选获得重组菌株PEAB。酶活测定结果表明重组菌具有酰胺酶酶活,但较低,其原因可能是因为大量表达的产物主要以包涵体的形式存在。     

金龟子绿僵菌(Metarhizium anisopliae HN1)几丁质酶基因的克隆及高效表达

几丁质酶是昆虫病原真菌金龟子绿僵菌致病力的主要因子之一。本实验用RT-PCR方法,从本实验室分离筛选到的高毒力金龟子绿僵菌Metarhizium anisopliae HN1中,扩增得到几丁质酶基因全长,此基因全长为1275bp,登录号为DQ011865,经Blastn分析此基因序列与M. anisopliae E6的chi1基因（AF02749）同源率为96% 。以pET-22b(+)为基础载体,构建pET-chi重组表达载体,在大肠杆菌（Escherichia. coli ）BL 21中进行表达。经SDS-PAGE分析,获得了42kDa大小的重组目的蛋白,目的蛋白占表达总蛋白含量的63.3%。菌体经冷冻与超声波破碎后,按DNS法可测得几丁质酶的活性。     

E.coli木糖异构酶基因的克隆及表达条件的优化

采用PCR技术以大肠杆菌JM109基因组DNA为模板扩增得到木糖异构酶基因xylA,连接到载体pET-22b( ),得到重组质粒pET-22b( )-xylA。将此重组质粒转化到大肠杆菌菌株BL21(DE3)中,重组菌株经IPTG诱导后,通过半胱氨酸-咔唑法测得木糖异构酶活力。每mL发酵液中重组菌株显示出酶活力约为0.84 U。SDS-PAGE电泳结果显示出明显的5×104(相对分子质量)特异性蛋白质条带。     

Bacillus licheniformis6816碱性蛋白酶基因在E.coli中的克隆和表达

用PCR方法从地衣芽孢杆菌6816中扩增了碱性蛋白酶基因（apr),扩增的1.14kb的DNA片段插入到大肠杆菌载体pET-20b中,构建成重组分泌型表达载体pAPR1。pAPR1中碱性蛋白酶基因在大肠杆菌宿主JM109（DE3）中得到表达,SDS-PAGE分析显示融合表达产物的分子量为30kD,同核酸序列测定所推导的值相符,表达产物占细胞总蛋白的7.5%,重组菌的酶活比出发菌株提高了3.3倍,研究发现,重组的碱性蛋白酶在进入大肠杆菌周质空间时存在前肽自动脱落的现象。     

人成骨蛋白-1成熟肽基因在大肠杆菌中的克隆与表达

本实验报告了人成骨蛋白 - 1成熟肽基因在大肠杆菌中的克隆与高效表达。通过计算机软件分析 ,采用大肠杆菌偏爱密码子设计并分段合成了人成骨蛋白 - 1成熟肽编码基因片段。用 PCR技术扩增目的基因片段 ,先克隆到 p UC1 9载体上 ,测序正确后再将其克隆到 p BV2 2 0表达载体上 ,以DH5α为宿主菌 ,42℃ ,6 h诱导表达。经 SDS- PAGE鉴定分析 ,在约 1 6× 1 0 3处有一新的蛋白质条带 ,扫描分析表明 ,该条带占菌体总蛋白含量的 40 %左右。     

甜叶悬钩子(Rubus suavissimus S.Lee)中UDP-糖基转移酶基因的克隆、表达及序列分析

糖基化是植物次生代谢产物生物合成中重要的修饰反应。目前已报道的以二萜为底物的UDP-糖基转移酶(UGT)数量稀少。本研究基于甜叶悬钩子叶片的转录组数据,利用生物信息学分析手段,选定可能具有二萜类催化活性的UDP-糖基转移酶基因进行克隆。最终克隆得到18条UGT基因序列,在大肠杆菌中进行了异源表达,并对克隆的基因进行了初步的序列分析。本研究为进一步挖掘和验证甜叶悬钩子中UGT的功能奠定了基础。     

Cloning of the nitrile hydratase gene from Nocardia sp. in Escherichia coli and Pichia pastoris and its functional expression using site-directed mutagenesis

To obtain a recombinant Rhodococcus or Nocardia with not only higher enzymatic activity but also better operational stability and product-tolerance ability for bioconversion of acrylamide from acrylonitrile, an active and stable expression system of nitrile hydratase (NHase) was tried to construct as the technical platform of genetic manipulations. Two NHase genes, NHBA and NHBAX, from Nocardia YS-2002 were successfully cloned, based on bioinformatics design of PCR primers, and inserted into plasmid pUC18 and pET32a, respectively. Then, two recombinant Escherichia coli strains, JM105 (pUC18-NHBA) and BL21 (DE3) (pET32a-NHBAX) were constructed and their expressions of NHase were focused. The induction results showed that there was either no NHase activity in JM105 (pUC18-NHBA), or as low as 0.04 U (1 U=1 μmol acrylamide min⁻¹ mg⁻¹ dry cell) in BL21 (DE3) (pET32a-NHBAX). SDS-PAGE results showed that the -subunit of NHBA and NHBAX could not be efficiently expressed in both recombinant E. coli strains. The novel Pichia pastoris system was also applied to express NHase, but the expression level remained quite low (0.5–0.6 U) and the protein was unstable. For solving this problem, a possible genetic strategy, site-directed mutagenesis of the -subunit of the NHase was carried out. After the successful mutagenesis of the original rare start codon gtg into atg, a new recombinant strain, E. coli XL1-Blue (pUC18-NHBA^M), was screened and the NHase activity stably reached as high as 51 U under the same induction conditions.     

HIV-1型核衣壳蛋白P24截短体在大肠杆菌中的表达,纯化和鉴定

用聚合酶链式反应（ＰＣＲ）从ＨＩＶ １ｇａｇ基因中扩增出衣壳蛋白Ｐ２４截短体（ｔＰ２４）基因,插入质粒ｐＧＥＸ ４Ｔ３中构成重组表达质粒ｐＧＥＸ ｔｐ２４,将ｐＧＥＸ ｔｐ２４转化大肠杆菌ＢＬ２１后获得了高效表达,表达量占菌体总蛋白量的３４３８％。经Ｇｌｕｔａｔｈｉｏｎｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化的截短体Ｐ２４纯度为９２７７％。纯化的截短体Ｐ２４在间接ＥＬＩＳＡ和免疫印迹检测ＨＩＶ抗体阳性血清和正常人血清中,具有很高的抗原特异性和免疫反应性。     

猪源肠出血性大肠杆菌CD18株fedA基因的克隆及表达

目的:获得重庆猪源肠出血性大肠杆菌(EHEC)强毒株CD18株fedA基因表达产物。方法:根据GenBank中的fedA基因序列设计引物,从重庆猪源EHEC强毒株CD18株基因组中经PCR扩增目的片段,克隆到pUC19质粒,亚克隆到表达载体pET28b( )的SalⅠ-HindⅢ位点后转化大肠杆菌BL21(DE3),经IPTG诱导表达,Ni柱法纯化,SDS-PAGE及Western印迹检验表达产物。结果:CD18株fedA与文献报道的fedA的序列同源性为99.3%,推导的氨基酸序列的同源性为98.7%,表达产物在50～100mmol/L咪唑洗脱时出峰,SDS-PAGE显示其相对分子质量20000,Western印迹证明该蛋白条带能与分子标记蛋白His6抗体发生特异反应。结论:克隆到猪源EHECCD18株fedA基因,并在大肠杆菌中得到表达。     

A comparison of the dynamics of pantothenate synthetase from M. tuberculosis and E. coli: computational studies

Pantothenate synthetase (PS) catalyzes the final step of the pantothenate pathway, in which pantothenate is formed from pantoate and β-alanine in an ATP-dependent reaction. Mycobacterium tuberculosis PS (MTB PS) is functionally a dimer and a potential target for novel antitubercular drugs. Molecular dynamics simulations show that the functional dynamics of the enzyme are dominated by motions of a flexible gate loop in the N-terminal domain and of the C-terminal domain. The gate loop motions dominate in MTB PS while the C-terminal domain motion dominates in Escherichia coli PS. Simulations also show that the correlated motions of the domains are severely compromised in the monomeric forms. Mutations that reduce the mobility of the gate loop in MTB PS and increased it in E. coli PS were designed and validated through simulations.     

Cloning of the histidine transport genes from salmonella typhimurium and characterization of an analogous transport system in escherichia coli

The genes for the well-characterized high-affinity histidine transport system of S typhimurium have been cloned in λgt4. Genetic and physiological analyses of the analogous transport system of E coli were undertaken in order that available λ vectors, recombinant DNA techniques, and a genetic selection for transport function might be used to isolate the Salmonella genes. The presence of the transport genes on a 12.4 Kb cloned DNA fragment has been confirmed (1) genetically, by complementation studies; (2) physiologically, by the rates of histidine uptake by bacteria containing this DNA; and (3) by demonstrating that the cloned DNA codes for the previously identified transport proteins J and P. The isolated fragment carries the entire transport operon, the argT gene and the ubiX locus, but neither the purF gene nor the ack/pta loci.     

霍乱毒素B亚单位基因（CtxB）的克隆及其表达

从霍乱弧菌中抽提基因组ＤＮＡ,用ＰＣＥＲ方法获取霍乱毒素Ｂ亚单位基因（ＣｔｘＢ）。序列分析结果表明,ＣｔｘＢ基因编码１２４个氨基酸,其中编码６２位Ｔｈｒ的密码子与文献报道有差异。将ＣｔｘＢ基因插入质粒ｐＧＥＸ－４Ｔ－２,构建ｐＧＥＸ－ＣＴＸＢ表达质粒,转化大肠相菌ＢＬ２１（ＤＥ３０,筛选表达菌株ＣＴＸＢ／ＢＬ２１。工程株经ＩＰＴＧ诱导表达,可产生大量的表达蛋白,经ＳＤＳ－ＰＡＧＥ分析,融合蛋白分子     

甲磺酸培氟沙星抑制大肠杆菌RecQ解旋酶的体外DNA结合、解链和ATPase活性

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,参与DNA复制、修复、重组、转录及维持端粒稳定等细胞代谢过程,在维持染色体稳定性与完整性中起着重要作用．甲磺酸培氟沙星(pefloxacin mesylate,PFM)是一种新型氟喹诺酮类抗菌药物,对一些革兰氏阴性菌具有明显的杀菌效果,临床上已广泛使用．本研究利用荧光偏振、自由磷检测技术研究PFM对大肠杆菌RecQ解旋酶的DNA结合活性、解链活性、ATPase活性的影响．结果表明,低浓度PFM可促进大肠杆菌RecQ解旋酶与ssDNA、dsDNA结合,达到一定量后PFM则抑制酶与DNA底物的结合,这种影响与DNA底物有关;PFM对RecQ解旋酶的DNA解链活性和ATP酶活性都具有抑制作用,但其抑制的效果有极显著差异(P<0.01)：比较PFM对两种活性抑制的Ci值(对解链活性抑制的Ci值为(1.5±0.2) μmol/L,对ATP酶活性抑制的Ci值为(0.010±0.005) μmol/L)可知,PFM对大肠杆菌RecQ解旋酶ATPase活性的抑制强于其解链活性. 这些结果可为研究以DNA解旋酶为药物靶标的分子机理奠定相关理论基础．     

Morphology and Molecular Phylogeny of a New Haptorian Ciliate,Chaenea mirabilis sp. n., with Implications for the Evolution of the Dorsal Brush in Haptorians (Ciliophora,Litostomatea)

We discovered a new haptorian ciliate, Chaenea mirabilis sp. n., in brackish water collected near the town of Busan, Korea. Its morphology was studied using standard taxonomical methods and its phylogenetic relationships were assessed by phylogenetic analysis of the 18S rRNA gene. Chaenea mirabilis is distinguished from all congeners by the combination of the following traits: (i) a narrowly bursiform to flask‐shaped, 60–100 μm long body; (ii) 11–21 doughnut‐shaped or sometimes horseshoe‐shaped macronuclear nodules; (iii) two types of extrusomes: type I is rod‐shaped and 6–8 μm long, while type II is narrowly to broadly teardrop‐shaped and only 1.5–2 μm long; (iv) highly refractive special granules tightly arranged between the first and second brush row, forming a conspicuous bulge; and (v) 12–13 somatic kineties. In the 18S rRNA gene phylogeny, C. mirabilis clustered with full support with other congeners. However, there was no statistical support for classification of Chaenea into the families Fuscheriidae, Acropisthiidae, or Trachelophyllidae, but a sister relationship with the Lacrymariidae could not be excluded. Therefore, we establish a new family, Chaeneidae, within the order Lacrymariida. This affiliation is strongly corroborated by the distinctly subapical dorsal brush bearing cilium‐like bristles.     

Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定及在大肠杆菌中的表达

[目的]鉴定来源于吸水链霉菌的谷氨酰胺转胺酶基因;研究其在大肠杆菌系统的克隆与表达;分析该酶与其同源酶的活性中心氨基酸序列.[方法]从本实验室筛选的吸水链霉菌(Streptomyces hygroscopicus;CCTCC M203062)发酵液中,分离纯化得到谷氨酰胺转胺酶酶原(pro-MTGase),测得N-端前十个氨基酸序列并与其它链霉菌来源的相应基因序列比较设计引物,扩增得到pro-MTGase 基因,将该基因插入到表达载体pET-20b( )信号肽pelB下游,构建分泌型表达载体pET/pro-MTG,并转化不同的大肠杆菌宿主BL21(DE3)和Rosetta(DE3)pLysS.[结果]获得了pro-MTGase的完整基因序列,多重碱基序列比对表明其与S.platensis和S.caniferus的pro-MTGase基因同源性高达92%.利用Rosetta(DE3)pLysS通过降温至24℃诱导策略,获得部分胞外表达的酶原.SDS-PAGE显示,胞外表达重组蛋白的分子量约为44kDa,与吸水链霉菌表达的天然酶原相符.诱导4 h后发酵液中的重组酶原经胰蛋白酶活化为成熟酶后测得最高酶活为0.24U/mL.[结论]该研究是对吸水链霉菌的谷氨酰胺转胺酶基因的首次报道,也是国内首次利用大肠杆菌实现pro-MTGase的胞外可溶性表达.     

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.