Gene cloning and characterization of a novel recombinant antifungal chitinase from papaya (Carica papaya)

A chitinase cDNA clone (CpCHI, 1002 bp) was isolated from papaya fruit, which encoded a 275 amino acid protein containing a 28 amino acid signal peptide in the N-terminal end. The predicted molecular mass of the mature protein was 26.2 kDa, and its pI value was 6.32. On the basis of its amino acid sequence homology with other plant chitinases, it was classified as a class IV chitinase. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed an optimal reaction temperature at 30 degrees C and a broad optimal pH ranging from 5.0 to 9.0. The recombinant enzyme was quite stable, retaining >64% activity for 3 weeks at 30 degrees C. The spore germination of Alternaria brassicicola could be completely inhibited by a 76 nM level of recombinant CpCHI. Recombinant CpCHI also showed antibacterial activity in which 50% of E. coli was inhibited by a 2.5 microM concentration of the enzyme.     

Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio)

A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.     

Protein from red cabbage (Brassica oleracea) seeds with antifungal, antibacterial, and anticancer activities

A 30 kDa antifungal protein was purified from red cabbage ( Brassica oleracea ) seeds. It exhibited a molecular mass and N-terminal amino acid sequence disinct from those of previously isolated Brassica antifungal proteins. The protocol used entailed ion exchange chromatography on Q-Sepharose and SP-Sepharose followed by fast protein liquid chromatography on Mono S. The protein hindered mycelial growth in Mycosphaerella arachidicola (with an IC50=5 μM), Setospaeria turcica, and Bipolaris maydis. It also inhibited the yeast Candida albicans with an IC50=96 μM. It exerted its antifungal action by permeabilizing the fungal membrane as evidenced by staining with Sytox green. The antifungal activity was stable from pH 3 to 11 and from 0 to 65 °C. It manifested antibacterial activity against Pseudomonas aeruginosa (IC50=53 μM). Furthermore, after 48 h of culture, it suppressed proliferation of nasopharyngeal cancer and hepatoma cells with IC50=50 and 90 μM, respectively.     

重组海参溶菌酶C端基因(SjLys-C)的可溶性表达和抑菌活性分析

为进一步研究海参(Stichopus japonicus)溶菌酶基因(Sjys)(Genbank登录号:EF036468)中不司片段表达产物的生物特性,本研究通过对其cDNA片段的分析,发现C端基因区域所对应的蛋白质序列中含有非酶活性.根据已知的海参溶菌酶的cDNA序列,设计山含有Nco Ⅰ和EcoR Ⅰ酶切位点的特异性引物,从新鲜的海参肠中提取总RNA,以其为模板利用RT-PCR扩增出长度为259 bp的溶菌酶C端(SjLys-C)基因.将该目的基因连接到pET-32a(+)载体上,构建重组质粒pET-32a(+)-SjLys-C,再转化至大肠杆菌(Escherichia coli)Rosetta(DE3)pLysS,成功地构建了重组蛋白SjLys-C的基因工程菌.利用该工程菌诱导发酵,结果显示它能高效表达出26 kD左右的重组蚩白SjLys-C.经过Western blot分析,该重组蛋白在26 kD左右能够与Penta-His抗体发生特异性免疫反应.对纯化的重组蛋白SjLys-C进行了抑菌特性的分析,结果发现它对溶壁微球菌(Micrococcus lysodeikticus)和副溶血弧菌(Vibrio parahae molytic us)有较高的抑菌活性.此外,将该重组蛋白经100℃、40 min处理后,其抑菌能力提高了5％～21％.研究结果表明,重组蛋白SjLys-C基因工程菌能够制备出具有可溶性的、并具有抑菌活性的重组蛋白SjLys-C,在农业和医药等行业中有潜在应用和开发价值.     

水稻几丁质酶的原核表达、复性及体外抑菌活性的研究

利用RT-PCR反应,从纹枯菌诱导的水稻叶片cDNA中克隆了1个水稻几丁质酶基因RC7的全长编码序列（ORF）。通过亚克隆方法,RC7与原核表达载体pGEX-4T-1上的GST融合,构建成GST-RC7重组蛋白的原核表达载体pGST-RC7,然后,将其转化到大肠杆菌细胞中。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导,获得了以包涵体形式高效表达的GST-RC7。经过洗涤、溶解（变性）、复性及纯化等处理,包涵体中GST-RC7得到溶解、复性和纯化。纯化的复性GST-RC7蛋白具有几丁质酶活性。对6种重要作物病原真菌进行体外抑菌活性分析的结果表明, RC7可显著抑制水稻纹枯病菌、小麦纹枯菌、小麦赤霉菌、棉花黄萎菌、烟草赤星菌的菌丝生长,说明RC7可作为上述植物病害抗性基因育种的重要基因。     

Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli

An amarantin 11S globulin cDNA encoding one of the most important storage proteins of amaranth seeds, with a high content of essential amino acids, was expressed in Escherichia coli. A good level of expression of recombinant amarantin with a molecular weight of 59 kDa was obtained. The recombinant protein was extracted by ammonium sulfate precipitation and purified to homogeneity using ion-exchange chromatography and reversed phase high-performance liquid chromatography. The expressed protein exhibited electrophoretic, immunochemical, and surface hydrophobicity properties similar to those of native amarantin from amaranth seed. Also, the recombinant protein was refolded in vitro using two different methods.     

亚非马蜂溶血肽基因与增强型绿色荧光蛋白基因融合在昆虫细胞中的表达

构建亚非马蜂(Polisteshebraeus)溶血肽基因重组转移载体pBacHT-GFPTPhMT,转化含穿梭载体Bacmid的受体菌Escherichiacoli(DH10Bac)中,得重组穿梭载体Bacmid-GFPTPhM,再用Lipofectin介导其DNA转染粉纹夜蛾(Trichoplusiani)细胞系Tn。SDS-PAGE分析表明,感染Bacmid-GFPTPhM的Tn-5B1-4细胞的表达产物在约为34kD处出现特异性条带,表达量约占细胞总蛋白的3%。Westernblot显示表达产物具有良好的免疫活性。感染Bacmid-GFPTPhM的细胞表达时相动态分析进一步表明,与增强型绿色荧光基因融合的亚非马蜂溶血肽基因的重组病毒已在昆虫细胞中进行了成功地表达,感染后72h表达量可达最高水平。     

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.     

Cloning and characterization of a plant defensin VaD1 from azuki bean

A recombinant mungbean defensin VrD1 was previously shown to exhibit antifungal and bruchid-resistant activity. To study the function and regulation of VrD1, genomic DNAs of plant defensins were isolated from Vigna radiata VC6089A and azuki bean Vigna angularis Kao Hsiung No. 6. The azuki bean defensin genomic DNA VaD1 was sequenced and converted to VaD1 cDNA. VaD1 defensin was purified from Vigna angularis Kao Hsiung No. 6 to apparent homogeneity. The complete amino acid sequence of the purified VaD1 was determined and was found to be exactly the same as the sequence deduced from VaD1 cDNA. VaD1 is a basic protein containing 46 amino acids with four conserved disulfide bonds and shares high sequence homology (78.3%) with VrD1. VaD1 inhibited the growth of Fusarium oxysporum, Fusarium oxysporum f. sp. pisi, Staphylococcus epidermidis, and Salmonella typhimurium. VaD1 also inhibited in vitro protein synthesis and bruchid larval development, but was less active than the recombinant VrD1.     

Molecular cloning, characterization, and expression of a cDNA coding Copper/Zinc superoxide dismutase from black porgy

A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reaction (PCR) based technique from cDNA synthesized from black porgy, Acanthopagrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed slightly higher identity (72.8-78.1%) with shark and swordfish Cu/Zn-SOD than with Cu/Zn-SOD from mammalian (68.1-70.7%) and plant (55.5-56.5%) sources. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondria targeting sequence, which suggests that the black porgy cDNA clone encodes a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDNA clone can express active Cu/Zn-SOD enzyme in E. coli.     

绵羊肺炎支原体(Mycoplasma ovipneumoniae)丙酮酸脱氢酶E1-α亚单位基因(pdha)的克隆、表达及其免疫学活性测定

丙酮酸脱氢酶α-亚单位(PDHA)在病原体丙酮酸脱氢酶的催化过程中发挥着重要作用。为表达绵羊肺炎支原体(Mycoplasma ovipneumoniae)PDHA蛋白并测定其免疫学活性,应用PCR方法扩增出绵羊肺炎支原体pdha基因并对其序列进行分析,将pdha基因中色氨酸密码子TGA优化为TGG后进行全基因合成,插入到pET32-a(+)载体上,构建了pET32-a(+)-pdha重组质粒,将重组质粒转化到大肠杆菌(Escherichia coli)BL21中诱导表达PDHA蛋白,并通过免疫印迹及小鼠(Mus musculus)免疫试验对其免疫学活性进行测定。结果pdha基因全长1125bp,编码375aa,(G+C)%为34.76%,第304~306位、379~381位、586~588位、592~594位、625~627位、811~813位、889~891位及964~966位TGA在支原体中编码色氨酸而不是作为终止密码子;基因序列比对及进化树分析显示,绵羊肺炎支原体pdha基因与10种支原体的pdha基因序列同源性为32.6%~85.3%,氨基酸序列同源性为39.3%~90.6%,基因序列和氨基酸序列均与猪肺炎支原体(M.hyopneumoniae)有同源性,分别为85.3%和90.6%;绵羊肺炎支原体pdha基因在33℃、IPTG0.25mmol/L诱导6h的表达条件下,表达量最高;重组的PDHA蛋白可与绵羊肺炎支原体高免血清具有免疫印迹条带,在免疫小鼠后血清抗体效价与对照组相比,均显著升高(P<0.05)。本实验首次成功克隆表达了绵羊肺炎支原体pdha基因,并证明其重组PDHA蛋白具有较好的免疫学活性。为绵羊支原体肺炎基因工程疫苗及诊断研究提供候选靶标。     

甘蓝型油菜中黑芥子酶基因在大肠杆菌中重组质粒的构建和表达

实验通过RT-PCR程序从提取的油菜总RNA中扩增出硫代葡萄糖苷水解酶（又称黑芥子酶,EC3.2.1.147）基因,酶解后插入大肠杆菌表达载体（Escherichia coli）pGEX-4T-1,获得克隆菌株。基因测序在 GenBank中的登陆号为EF583560,翻译的氨基酸序列与已报道的黑芥子酶（GenBank中的登陆号为Q00326）中的一段有一个氨基酸不同,同源性达到99%。经过IPTG诱导表达,在91KDa左右处有表达量很高的一条带。     

cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.     

白菜NAC转录因子BrcCUC3基因克隆及其对叶缘和主枝发育的影响

植物NAC (矮牵牛NAM基因、拟南芥ATAF1/2和CUC2基因)转录因子CUP-SHAPED COTYLEDON(CUC)亚家族成员在植物茎尖分生组织形态建成、器官边界分离、叶发育方面发挥着重要作用.采用基因同源克隆的方法获得了白菜(Brassica rapa ssp.chinensis)基因BrcCUC3,并转化拟南芥(Arabidopsis thaliana)做了初步的功能鉴定.研究结果表明,白菜Brc CUC3的编码区长1 008 bp,编码335个氨基酸,基因结构分析显示BrcCUC3包含3个外显子、2个内含子,内含子剪接位点符合GT-AG规则.氨基酸序列分析表明,BrcCUC3蛋白具有典型的植物NAC domain结构域.BrcCUC3编码区氨基酸序列与其它植物的CUC3蛋白有很高的一致性,尤其与甘蓝(Brasica oleracea)、萝卜(Raphanus sativus)和拟南芥CUC3蛋白高度一致,一致性分别达到98％,97％和83％.在不同物种CUC3的系统进化树上,BrcCUC3归属于双子叶植物分支的十字花科亚组,由不同植物20条CUC3编码区氨基酸序列所建立的系统进化树与真实的植物进化基本一致.荧光定量PCR分析结果表明,BrcCUC3在白菜叶深裂株系叶片中的表达量比叶全缘叶片中的高.利用根癌农杆菌(A grobacterium tumefaciens)浸花法转化拟南芥,获得了转BrcCUC3基因的拟南芥植株.过表达BrcCUC3的转基因拟南芥呈现叶缘出现裂刻、主枝增加的新表型.初步说明该基因参与叶形和主枝的发育调控,为揭示白菜叶形发育分子调控机制和通过基因工程创制植物叶形新种质提供分子依据.     

Functional proteomic analysis of rice bran esterases/lipases and characterization of a novel recombinant esterase

An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.     

Expression and characterization of sweet potato invertase in Pichia pastoris

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.     

Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.     

重组鸡β-防御素5 - β-防御素10双分子蛋白的构建及其理化特性分析

鸡抗菌肽属禽β-防御素（AvBD）类,是鸡先天性免疫的重要组成部分。研究将AvBD10基因定向插入到AvBD5-pGEX SalⅠ和NotⅠ双酶切位点上,构建了AvBD5-pGEX- AvBD10双基因共表达重组载体。将重组质粒转化大肠杆菌 (Escherichia coli ) BL21,于37 ℃不同时间进行诱导表达,SDS-PAGE检测外源基因的表达。结果表明,重组AvBD5-AvBD10双分子融合蛋白的分子量约为36 kD,重组双分子蛋白占菌体总蛋白的35%,重组菌表达产物以包涵体形式存在。重组双分子蛋白经纯化后,分别以对数生长中期的大肠杆菌[BL21(DE3-) 株]与致病性链球菌［Streptococcus（CAB株）］为检测菌,利用薄层平皿琼脂糖孔穴扩散法测定了重组双分子蛋白的抗菌活性,结果表明,重组双分子蛋白对这两种细菌都具有抗菌活性。并且对温度和pH有很高的稳定性,在-70～100 ℃或pH 3～12处理30 min仍具有抗菌活性。     

Molecular cloning, recombinant expression, and immunological characterization of a novel allergen from tartary buckwheat

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.