L—山梨酮脱氢酶基因在大肠杆菌中的克隆和表达

参照已知的Ｌ－山梨酮脱氢酶基因序列,合成了两个引物序列,以ＡｃｅｔｏｂａｃｔｅｒｌｉｑｕｅｆｉｃｉｅｎｓＩＦ０１２２５８的染色体ＤＮＡ为模板进行ＰＣＲ反应,克隆得到了Ｌ－山梨酮脱氢酶基因,经酶切验证与预期结果相同,序列测定结果也与已知序列一致,采用ＰＣＲ方法在此基因的两端加上了Ｅ．ｃｏＲＩ和ＨｉｎｄＩＩ两个酶切位点,经Ｅ．ｃｏＲＩ和ＨｉｎｄＩＩ酶切后去掉了两端的多余序列后,将此片段连接到ｐＫＫＨｈ     

犬瘟热病毒H基因原核表达载体的构建

利用本实验以前构建的含有犬瘟热病毒H基因的pMD18-T质粒,根据pMD18-T-H序列设计带有XbalI和HindⅢ酶切位点的引物,对H基因进行PCR扩增,得到约1800bp左右的片段。该片段被克隆到pMD18-Tsimple载体上,用XbalI和HindⅢ进行单、双酶切鉴定．筛选阳性克隆。将阳性克隆再用XbalI和HindⅢ进行双酶切,纯化回收CDVH基因片段。将原核表达载体pPROEXTMHTa、pet-30b用同样的方法酶切,回收载体片段。将CDVH基因片段分别用T4连接酶连接到回收的pPROEXTMHTa、pet-30b载体上,构建原核表达载体质粒pPROEXTMHTa-H和pet-30b-H。再用XbalI和HindⅢ进行单、双酶切鉴定阳性载体。本实验为下一步H蛋白表达、纯化并作为CDV诊断用抗原及CD的免疫预防奠定了基础。     

菜甜菜碱醛脱氢酶基因的克隆和序列分析

以耐盐的菠菜mRNA为横板．经反转录合成甜菜碱醛脱氢酶(BADH)基因第一链cDNA。在人工合成的两端引物引导下,通过多聚酶链式反应(PCR)。扩增获得双链cDNA。把重组有BADH基因的pucl9转化至E．Coli DH5a菌株,亚克隆后测定了基因的全序列。所得到的BADH基因全长序列为1491bp,编码497个氨基酸。与文献报道的相比较,核苷酸序列同源性99．8％．氨基酸序列同源性达99．6％。在此基础上,构建了BADH基因的高等植物表达载体．     

结核分枝杆菌furA基因真核表达质粒的构建及表达

以BamH Ⅰ和Hind Ⅲ双酶切pRSET-furA,获得结核分枝杆苗铁调控基因furA,将其克隆入真核表达载体pcDNA3．1（-）,构建了重组质粒pcDNA-furA,经酶切鉴定正确后,将重组质粒以阳离子聚合物转染CHO细胞。经RT—PCR分析表明,furA可在CHO细胞中转录;用间接免疫荧光检测,表达有FurA蛋白的细胞着染。以上结果表明,通过构建结核分枝杆菌furA基因的真核表达载体pcDNA-furA,使知以基因可以在CHO细胞中表达。     

用于丝状真菌的苯菌灵标记基因benA表达载体的构建

为了构建适用于丝状真菌遗传转化的表达载体,在质粒pAN52-1的高效组成型三磷酸甘油醛脱氢酶基因启动子PgpdA和色氨酸C基因终止子TtrpC之间加入内切酶NotⅠ和ClaⅠ两个位点,构成质粒pAN52-2;把质粒pAN52-2中从启动子PgpdA到终止子TtrpC的基因片段通过PstⅠ和XbaⅠ酶切位点导入到pUC19载体上,并且应用PCR的方法在启动子Pg-pdA的前端引入HindⅢ酶切位点,构成质粒pUCPT-2;通过PCR,在苯菌灵抗性基因benA两端分别加入内切酶NotⅠ和ClaⅠ酶切位点,并克隆到pGEM-T载体上,得到质粒pGEM-Ben;用NotⅠ分别酶切质粒pUCPT-2与pGEM-Ben,连接苯菌灵抗性基因benA到pUCPT-2上,得到了pUCPT-Ben载体。经PCR、酶切和核苷酸序列检测,证实了PgpdA-benA-TtrpC表达元件连接成功,即得到了苯菌灵抗性基因高效表达载体。     

酮古龙酸菌山梨酮脱氢酶的原核表达及活性鉴定

目的:克隆酮古龙酸菌Y25的山梨酮脱氢酶基因sndh2,在大肠杆菌中进行表达,并检测表达产物的活性。方法:以酮古龙酸菌Y25基因组DNA为模板,PCR扩增sndh2基因,连接到pET22b表达载体后转入大肠杆菌BL21(DE3)中,经IPTG诱导表达;对菌体裂解液进行SDS-PAGE分析;以D-木糖为底物,采用非变性聚丙烯酰胺凝胶电泳后活性染色及DCIP检测法鉴定表达产物的脱氢酶活性。结果:扩增得到1290 bp的山梨酮脱氢酶基因;构建了表达质粒pET22b-sndh2,SDS-PAGE结果显示获得相对分子质量为43.1×103的可溶性表达产物;非变性聚丙烯酰胺凝胶电泳胶上出现的蓝黑色条带及DCIP检测液颜色的变化说明表达产物在以D-木糖为底物时表现出脱氢酶活性。结论:在大肠杆菌中表达的山梨酮脱氢酶具有生物活性。     

2,5-DKG还原酶Ⅱ基因的克隆和在大肠杆菌中的表达*

参照文献上的2,5-二酮基-D-葡萄糖酸（简称2,5-DKG）还原酶II基因序列,合成两个引物序列并在两端加上EcoRI和BamHI两个酶切位点,抽提棒状杆菌SCB3058菌株的染色体为模板进行PCR反应,克隆得到2,5-DKG还原酶II基因,酶切验证与预期的结果相符合。将此片段克隆到pGEM-T载体上保存．将2,5-DKG还原酶II基因用EcoRI和BamHI内切酶切下,连接到pBV220载体上,构建成表达载体。42℃诱导不能得到稳定的蛋白表达条带和酶活力,测序发现基因的3’末端的原PCR引物外少合了一     

番茄1-氨基环丙烷羧酸(ACC)合成酶基因的反义RNA-核酶嵌合DNA序列的构建

根据番茄ACC合成酶基因（LE—ACC2）DNA序列,以番茄(LycopersiconesculentumMill)果实的总DNA为模板,利用PCR技术扩增得到预期大小的该基因编码区内部分DNA序列,插入到质粒载体pGEM—3zf(＋)的BamHⅠ和HindⅢ位点之间后转化E．coliDH—5α,可选出重组子pRE,经酶切,PCR及DNA序列分析证明克隆成功;将pRE上的目的DNA序列以反义方式构建到我室已合成并克隆的含核酶DNA序列的重组质粒pRⅠ的BamHⅠ和HindⅢ之间,构成含有反义RNA-核酶嵌合DNA序列的重组质粒pREⅠ,经酶切及序列分析,结果与预期一致.     

成熟志贺毒素全长与截短A亚单位在E.coli中的表达及纯化

应用PCR技术从Ⅰ型痢疾志贺菌(Shigella dysenteriaetype I)中,扩增出约895 bp的成熟ShT-A基因片段,克隆至pGEM-T载体中,经蓝白斑筛选、PCR和双酶切鉴定正确后,命名为pGEM-TA2。测序结果表明,ShT-A与GenBank中ShT-A序列完全一致。用BamHⅠ和KpnⅠ双酶切克隆质粒,得到为895 bp成熟ShT-A与pQE30原核重组表达载体连接,构建原核重组表达质粒。经IPTG诱导,SDS-PAGE电泳观察,没有目的蛋白表达。DNAsis软件分析ShT-A基因序列,发现513 bp处有HindⅢ位点,故以ShT-A上游引物的BamHⅠ和HindⅢ从pGEM-TA2切出一个513 bp的截短片段,重新插入pQE30表达载体,得到pQE30-A513重组表达质粒,转化E.coliM15,经IPTG诱导,SDS-PAGE电泳观察,在20 ku处出现1条特异性的表达蛋白带,与截短ShT-A513分子量相符,表达量约占菌体总蛋白的37.4%,表达形式为包涵体。为抗体的制备提供了必要的物质基础。     

Expression analysis of the aquaporins during zebrafish embryonic development

The aquaporins are integral membrane proteins from a larger family of major intrinsic protein (MIP) that form pores in the membrane of cells. These proteins selectively transport water and other small uncharged solutes across cell plasma membranes. The organization of water within cells and tissues is fundamental to life, and the aquaporins play an important role in serving as the plumbing system for cells. As many as thirteen mammalian AQPs have been characterized, which have been shown to be vital for the regulation of water homeostasis in most tissues, such as renal water balance and brain-fluid homeostasis. However, complete expression patterns of most of the aquaporins in lower vertebrate at embryo stages has not been elucidated. Currently, we systematically described the temporal-spatial expression pattern of nine zebrafish aquaporins, using whole amount in situ hybridization. The results of whole mount in situ hybridization revealed that members of aquaporins family displayed diverse expression pattern, each of aquaporins has its unique distribution in different cell types and tissues, suggesting that they might play distinct roles in the embryonic development. Overall, current study will provide new insight into the expression of vertebrate quaporins and an important basis for the functional analysis of aquaporins in zebrafish development.     

Developmental expression patterns of fosl genes in Xenopus tropicalis

Fos-like antigens (Fosl) including Fosl1 and Fosl2 exclusively heterodimerize with Jun members to form AP-1 complex, thereby participating in various cellular progresses including cell cycle regulation. However, expression patterns of these two genes during embryonic development remains largely unknown. In the present study, both temporal and spatial expression patterns of fosl1 and fosl2 were examined during embryonic development of Xenopus tropicalis. Real-time quantitative PCR results showed that the expression of the two genes was increased from stage 2 to stage 42. However, expression level of fosl1 is much higher than that of fosl2 at stage 42. Whole-mount in situ hybridization showed that fosl1 was expressed in eyes, branchial arch, notochord, otic vesicle, and liver. However, fosl2 was expressed in lung primordium from stage 34 to stage 38, in addition to the moderate expression in eyes and branchial arch at stage 42. Thus, the developmental expression patterns of these two fosl genes is different in Xenopus embryos. These results provide a basis for further functional study of these two genes.     

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

普通烟草八氢番茄红素脱氢酶基因的原核表达及表达谱分析

基于NCBI数据库中本氏烟(Nicotiana benthamiana)的烟草八氢番茄红素脱氢酶PDS基因(ABE99707)的核苷酸序列,设计并合成特异性引物,以烟草栽培品种红花大金元叶片总RNA为模板,通过PCR方法获得了烟草NtPDS基因的cDNA片段。序列分析表明,该基因编码区为1749 bp,编码582个氨基酸,推测该蛋白等电点为7.53,理论分子量为65.04 kD。通过构建融合表达载体pET-32a-NtPDS,并转化大肠杆菌BL21(DE3),在37℃下经1 mmol/L IPTG诱导4 h表达后,产生了以可溶性蛋白形式存在的NtPDS融合蛋白,并通过Western blotting验证融合蛋白获得表达。利用半定量RT-PCR技术进行组织表达模式分析发现,该基因在烟草的叶片、花和茎中均有表达,在根中没有表达。该结果为进一步研究烟草八氢番茄红素脱氢酶NtPDS的活性和生物学功能奠定了基础。     

Characterization and optimization of heterologous expression in Escherichia coli of the chitinase encoded by the chiA gene of Bacillus halodurans C-125

The diversity of the biotechnological applications of chitinolytic enzymes requires different enzyme-producing strains with different properties suitable for each process. In this work the chitinase encoded by the chiA gene of Bacillus halodurans has been studied. The protein shows a modular structure characterized by the catalytic domain of glycosyl hydrolases family 18 (GH18), fibronectin type III domain (FnIIID) and a carbohydrate-binding module family 5 (CBM5). The expression of the gene in Escherichia coli has made it possible to demonstrate the functionality of the protein which is active in the temperature range of 5–55 °C and pH values of 5.5–8.5 while maintaining a high stability under suboptimal conditions. The enzyme hydrolyzes colloidal chitin and different p-NP(GlcNAc)_n (n = 1–3) by an “-exo” type mechanism according to the information deduced from its sequence. The production of the protein was optimized by constructing recombinant strains, and the effect of the expression vector used, the cell density of the culture, the concentration of inducer and the induction time were studied. Based on its spectrum of activity, stability and mechanism of action, it arises as an enzyme of potential interest for production of N-acetyglucosamine or conversion of chitin into biologically active chito-oligosaccharides.     

Structural and functional characterization of Tomato SUMO1 gene

Small ubiquitin-related modifier (SUMO) genes regulate various functions of target proteins through post-translational modification. The SUMO proteins have a similar 3-dimensional structure as that of ubiquitin proteins and occur through a cascade of enzymatic reactions. In the present study we have cloned a new SUMO gene from Tomato (Solanum lycopersicum L.), cv Saudi-1, named SlS-SUMO1 gene by PCR using specific primers. This gene has SUMO member's features such as C-terminal diglycine (GG) motif as processing site by ULP (ubiquitin-like SUMO protease) and has SUMO consensus ΨKXE/D sequence. Phylogenetic analysis showed that SlS-SUMO1 gene is highly conserved and homologous to Potatoes Ca-SUMO1 and Ca-SUMO2 genes based on sequence similarity. Expression protein of SlS-SUMO1 gene found to be localized in the nucleus, cytoplasm, and nuclear envelop or nuclear pore complex. SUMO conjugating enzyme SCE1a with SlS-SUMO1 protein co-expressed and co-localized in nucleus and formed nuclear subdomains. This study reported that the SlS-SUMO1 gene is a member of SUMO family and its SUMO protein processing using GG motif and activate and transport to nucleus through Sumoylation system in the plant cell.     

基于抗菌导向的乳铁蛋白异源表达研究进展

乳铁蛋白是哺乳动物天然免疫系统和获得性免疫系统中的重要防御成分,具广泛生物学功能,包括调节体内铁平衡、广谱抗菌、抗炎症、抑制肿瘤生长、增强机体免疫力等,在医药、食品、饲料领域有重要应用价值。目前乳铁蛋白规模化生产技术瓶颈是提取成本高,利用基因重组技术构建高效表达系统是突破这一瓶颈的重要途径。基于抗菌导向的乳铁蛋白及其衍生分子在大肠杆菌、酵母、昆虫、哺乳动物和植物中表达的研究进展进行了综述。     

内毒素结合肽的改良、原核表达及纯化

目的：对人内毒素结合肽(endotoxin binding peptide,EBP)进行改良,获得突变体mEBP(mutate of endotoxin binding peptide,mEBP)基因并进行原核表达,纯化获得高纯度的mEBP。方法：应用PCR定点诱变方法获得mEBP基因,构建PinpointXa-3/mEBP融合表达载体,在BL21(DE3) pLysS宿主菌进行表达,溶菌酶裂解法提取包涵体,融合蛋白经PinpointTM Xa 纯化后,由factorXa酶切,获得目的肽,RP-HPLC纯化获得高纯度的mEBP。结果 应用PCR定点诱变技术完成了EBP第5.18位谷氨酰胺 赖氨酸的定点突变,且通过原核表达,色谱纯化获得了高纯度的mEBP。结论 成功获得高纯度的mEBP,为下一步的抗内毒素功能检测奠定基础。