烟草曲茎病毒复制蛋白基因的原核表达和免疫定位

利用PCR方法从烟草曲茎病毒 (TbCSV)Y35分离物的病株中获得复制蛋白 (Rep)基因 ,将其克隆到原核表达载体pGEX 4T 1上获得重组质粒pGEX Y35Rep。重组质粒导入大肠杆菌BL2 1(DE3)pLysS中 ,IPTG诱导表达后发现部分Rep融合蛋白以可溶性形式表达。利用GST Sepharose 4B亲和层析柱纯化了Rep的融合蛋白 ,免疫家兔获得Rep蛋白的抗体。对TbCSV侵染烟草中Rep蛋白的亚细胞分布研究发现 ,Rep蛋白主要分布于含有细胞核的组份中。利用免疫胶体金技术对感病烟草中Rep蛋白进行了定位 ,发现Rep蛋白存在于细胞核内     

t—PA昆虫细胞表达及特性分析

With the RT-PCR technique, human tissue-type plasminogen activator (t-PA) cDNA was amplified from human melanoma cells. The sequence was proved to be the same as those reported by foreign researchers. The t-PA cDNA containing whole reading frame was inserted into the transfer vector pBacPAK8. The constructed pBac-tPA and the linear BacPAK6 virus DNA were cotransfected with Tn-5B-1 insect cells by lipofectin-mediated transfection method. Eleven pure recombinant viruses were isolated by plaque assay, and one of them, nominated BactPA3, was selected by PCR identification and biological activity comparison. The t-PA activity expressed in serum-containing media reach the highest level at 72 h postinfection. The activity was 3.04 x 10(3) IU/ml, e.g. 1.8 x 10(4) IU/10(6) cells. The highest expression level in serum-free media was almost the same, but needed more time (at 132 h postinfection). SDS-PAGE fibrin autography showed that the molecular weight of the expressed t-PA was about 68 kDa. Its stimulation by fibrinogen, affinity with fibrin and inactivation in plasma were almost the same as the native t-PA purified from human melanoma cell culture. The half-life of t-PA expressed in serum-free media was seven minutes.     

Functional analysis of the human tissue-type plasminogen activator protein: the light chain

A pBR322::Rous sarcoma virus(RSV)-based shuttle vector was used to insert fused genes, composed of the amino-terminal portion of the bacterial chloramphenicol-acetyltransferase gene (cat) and the entire coding region for the C-terminally derived light (L) chain of human tissue-type plasminogen activator (t-PA) cDNA. Cotransfection of rat 3Y1 cells with pRSVneo DNA and pRSVcat/t-PA DNA yielded stably integrated G418-resistant transfectants which contain unrearranged copies of pRSVcat/t-PA DNA. These transfectants synthesize cat/t-PA L-chain mRNA, apparently correctly initiated and terminated. With the help of an enzyme-linked immunosorbent assay (ELISA), it is demonstrated that these cells produce human t-PA antigen. Furthermore, pRSVcat/t-PA L-chain cDNA-containing rat 3Y1 cells synthesize a plasminogen-dependent amidolytic activity which is suppressed by specific anti-human t-PA antibodies. This activity cannot be stimulated by fibrin, a property displayed by native t-PA. It is concluded that the t-PA L-chain cDNA contains the complete genetic information for the plasminogen activator activity.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.     

A zinc finger protein Tsip1 controls Cucumber mosaic virus infection by interacting with the replication complex on vacuolar membranes of the tobacco plant

? In Cucumber mosaic virus (CMV) RNA replication, replicase-associated protein CMV 1a and RNA-dependent RNA polymerase protein CMV 2a are essential for formation of an active virus replicase complex on vacuolar membranes. ? To identify plant host factors involved in CMV replication, a yeast two-hybrid system was used with CMV 1a protein as bait. One of the candidate genes encoded Tsi1-interacting protein 1 (Tsip1), a zinc (Zn) finger protein. Tsip1 strongly interacted with CMV 2a protein, too. ? Formation of a Tsip1 complex involving CMV 1a or CMV 2a was confirmed in vitro and in planta. When 35S::Tsip1 tobacco (Nicotiana tabacum) plants were inoculated with CMV-Kor, disease symptom development was delayed and the accumulation of CMV RNAs and coat protein was decreased in both the infected local leaves and the uninfected upper leaves, compared with the wild type, whereas Tsip1-RNAi plants showed modestly but consistently increased CMV susceptibility. In a CMV replication assay, CMV RNA concentrations were reduced in the 35S::Tsip1 transgenic protoplasts compared with wild-type (WT) protoplasts. ? These results indicate that Tsip1 might directly control CMV multiplication in tobacco plants by formation of a complex with CMV 1a and CMV 2a.     

Expression of recombinant trichosanthin,a ribosome-inactivating protein,in transgenic tobacco

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.     

Expression of active hBMP2 in transgenic tobacco plants

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and “Kozak” sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.     

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.     

Analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis B virus

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBS antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HbsAg gene under a single 35 S promoter was 0.0001-0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002-0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants.     

杆状病毒p35基因诱导烟草产生广谱抗病机理分析

来源于昆虫病毒和动物的抗细胞凋亡基因能够诱导植物对生物或者非生物胁迫产生抗性.但其抗性机理有不同甚至相反的报道.本研究将来源于苜蓿银纹夜蛾核多角体病毒的p35基因转化烟草,T1代转化烟草Western blotting检测P35蛋白的表达,转化烟草接种烟草花叶病毒(Tobacco mosaic virus,TMV)抗病效果增强.进一步的抗病机理研究表明,转化和野生型烟草感染TMV后诱导过氧化氢积累无明显区别,野生型烟草感染24 h后出现DNA Laddering而转化烟草则没有;Western blotting结果显示PR-1蛋白表达没有显著差异.但接种另外一种病原真菌核盘茵(Sclerotiniasclerotiorum)后的RT-PCR分析结果表明,表达P35蛋白的烟草可增强感染核盘菌后PR-1基因的转录.而且表达时间提前.以上结果说明p35基因介导的广谱抗病反应的机理与接种的不同病原有关,对不同病原物的抗病机理存在差异,除抑制细胞凋亡外,还可能通过激活PR基因的表达提高对病原物的抗病能力.     

Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection.

Tobacco genes encoding the PR-1a protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-1a gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. No such induction was found with upstream sequences of 643 base pairs or shorter of the PR-1a gene. When the PR-1a upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35S core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic virus- and salicylate-responsive elements between positions -643 and -689 in the PR-1a promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.     

Replacement of finger and growth factor domains of tissue plasminogen activator with plasminogen kringle 1. Biochemical and pharmacological characterization of a novel chimera containing a high affinity fibrin-binding domain linked to a heterologous protein.

A novel triple-kringle plasminogen activator protein, PK1 delta FE1X, has been produced which is a genetic chimera between the fibrin binding kringle 1 domain of plasminogen and the two kringles and serine protease domains of naturally occurring wild-type tissue plasminogen activator (wt t-PA). This chimera also contains a modification to prevent high mannose type N-linked glycosylation on kringle 1 of t-PA. PK1 delta FE1X is biochemically and fibrinolytically similar to wt t-PA in vitro but retains the decreased plasma clearance rate characteristic of other t-PA variants which lack fibronectin finger-like and epidermal growth factor domains. The serine protease domain of PK1 delta FE1X exhibits the amidolytic activity characteristic of wt t-PA. In an indirect coupled plasminogen activator assay, the specific activity of PK1 delta FE1X is approximately 1.4 times greater than that of wt t-PA. In a fibrin film-binding assay, greater binding to untreated fibrin is observed with wt t-PA than with PK1 delta FE1X. However, following limited plasmin digestion of the fibrin film, PK1 delta FE1X binding increases to the level observed with wt t-PA. The incremental binding to plasmin-digested fibrin observed with PK1 delta FE1X is eliminated if plasmin digestion of the fibrin film is followed by carboxypeptidase B treatment. This result suggests that plasminogen kringle 1 binds plasmin-digested fibrin even after recombination with a heterologous protein. The fibrinolytic activity of PK1 delta FE1X in human plasma clot lysis assays was similar to that of wt t-PA at activator concentrations of approximately 1 microgram/ml. At substantially lower concentrations, approximately 0.1 microgram/ml, PK1 delta FE1X was only slightly less active than wt t-PA. Pharmacokinetic analysis showed that wt t-PA activity is cleared approximately 15 times as rapidly as PK1 delta FE1X following intravenous bolus injection. In a rabbit jugular vein clot lysis model, intravenous bolus injection of 0.06 mg/kg of PK1 delta FE1X showed greater thrombolytic potency than a similar administration of 0.5 mg/kg of wt t-PA. Thus it appears that in vitro exon shuffling techniques can be used to generate novel fibrinolytic agents which biochemically and pharmacologically represent the combination of individual domains of naturally occurring proteins.     

Processing of preproricin in transgenic tobacco

The plant protein toxin ricin has found widespread application as a potential therapeutic agent for many human diseases and in disease-model systems such as those involving apoptosis. Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, specifically in transgenic tobacco carrying the preproricin gene under the control the cauliflower mosaic virus 35S promoter. Production of modified ricin for altered controllable activity and/or fusion therapeutics to target delivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco. Here, recombinant ricin from transgenic tobacco was purified using lectin affinity chromatography and characterized using various biochemical and biophysical techniques. Coomassie blue staining of an SDS-PAGE gel of lactose-agarose purified material identified predominant proteins of 30 and 35 kDa molecular weight. Western analysis using anti-ricin a- and b-chain antibodies confirmed the expression and purification of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin. High-resolution gel filtration chromatography characterized the lactose binding complex as a 66-kDa native molecular weight protein which could be separated into 30- and 35-kDa proteins upon incubation with the reducing agent dithiothreitol. N-terminal sequencing of the recombinant ricin a-chain revealed that an equimolar ratio of two alternately processed peptides was present, which varied by an additional amino acid derived from the signal peptide. Similar analysis of ricin b-chain again identified two forms of this polypeptide as well; however, full-length ricin b-chain and b-chain missing the first alanine residue were present at 11:1 molar ratios. Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the media of the cultures, indicating that cell cultures provide a safe and simple means to produce properly processed recombinant ricin.