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At least 595 records · Page 33Linked to original sources
PubMed2005

[Construction of DNMT1 siRNA stable expressing vector and evaluation of its silenced efficiency in blocking gene expression].

OBJECTIVE: To construct the specific stable expression and high efficiency small interfering RNA(siRNA) expression vector that can block DNMT1 gene function. METHODS: Using vector-based RNA interference technique, the authors constructed a vector to transcribe functional short interfering RNA (RNAi). After transfection by lipofectmine (TM) reagent, the treated cells were selected by G418. The expression levels of RNA and protein of DNMT1 were analyzed by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting. The status of methylation of E-cadherin was analyzed by methylation-specific PCR(MSP). RESULTS: The expression level of endogenous DNMT1 mRNA in transfected SMMC-7721 cell lines with DNMT1 RNAi construct was 43% less than that in control cell 7721-pSU cell lines. The protein level in the former was about 10% less than that in the latter. The efficiency of the siRNA of DNMT1 was found to be higher than 90%. Demethylation of promoter of E-cadherin was obtained due to the inhibition of DNMT1. CONCLUSION: DNMT1 siRNA stable expressing vector was obtained by gene-recombined technology. There was no complete sameness between the levels of protein and RNA in gene silenced cell lines. The efficiency of the siRNA should be confirmed by Western-blotting.

Blotting, Western↗
PubMed1999

Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector.

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.

3T3 Cells↗
PubMed1992

Expression pattern of SR alpha promoter in human embryonal carcinoma and transgenic tissues in mice.

Human embryonal carcinoma is thought to be a counterpart of mouse embryonal carcinoma, which has provided useful information for studying early molecular events in murine embryogenesis. A major practical problem in the use of human embryonal carcinoma for molecular pathological studies is the lack of an efficient expression system for foreign genes. The SR alpha promoter is a fusion promoter containing the SV 40 early promoter and the R segment and part of the U5 sequence of the long terminal repeat derived from human T-cell leukemia virus type I. We analyzed the expression pattern of the SR alpha promoter in human and mouse embryonal carcinoma lines and transgenic mouse tissues. Efficient and stable expression was detected in all cell lines tested, and tissues from all mice of four independent transgenic lines carrying the SR alpha-CAT vector showed a detectable level of CAT expression. These data suggest that the SR alpha promoter is useful for studies of both human embryonal carcinoma and transgenic mouse tissue. Using this expression system, we are now able to label human embryonal carcinomas with various genes, for example beta galactosidase, and follow their fate at the single-cell level in nude mice, where xenotransplanted human embryonal carcinoma expresses differentiation capability.

Animals↗
PubMed1998

High-level expression of the thermoalkalophilic lipase from Bacillus thermocatenulatus in Escherichia coli.

An efficient expression system for the previously only weakly expressed thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was developed for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYT-EXP1 (pT1) expression vector downstream of the temperature-inducible lambda promoter PL. Three different expression vectors were constructed: (i) pT1-BTL2 containing the mature lipase gene, (ii) pT1-preBTL2 containing the prelipase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fused to the signal peptide of the OmpA protein, the major outer membrane protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expression levels of 7000-9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 660,000 soluble lipase U/g cells was produced, whereas, with E. coli DH5 alpha and BL321, production levels of 30,000 U/g cells were achieved. However, most of the lipase remained insoluble but active after cell breakage because of the unprocessed OmpA signal peptide. A simple cholate extraction followed by proteinase K cleavage and ultrafiltration allowed the isolation of 1.15 x 10(6) units of 90% pure mature lipase/wet cells.

Bacillus↗
PubMed1999

Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.

Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.

Animals↗
PubMed1993

Encapsidation of a recombinant LuIII parvovirus genome by H1 virus and the fibrotropic or lymphotropic strains of minute virus of mice.

We previously constructed a recombinant LuIII parvovirus genome lacking viral coding sequences and used it to generate luciferase-transducing virions, by cotransfection of cells with a helper plasmid expressing LuIII viral proteins. Here, we describe similar cotransfections using alternative, replication-defective helpers encoding the non-structural and capsid proteins of parvovirus H1, or of either the fibrotropic or lymphotropic parvovirus strain of minute virus of mice [MVM(p) or MVM(i)]. Each cotransfection generated transducing virus which directed luciferase expression after infection of HeLa cells. The transducing activity of virus produced using either LuIII or H1 helper plasmids could be specifically neutralized by antiserum raised against the corresponding infectious virus. When the recombinant LuIII parvovirus was pseudotyped with MVM(p) or MVM(i), the resulting virions efficiently expressed luciferase after infection in human or murine cells known to be permissive for both MVM strains. The MVM(p) pseudotyped virus also expressed this reporter efficiently when infected into the murine A9 fibroblast line. In contrast, the recombinant virus generated with an MVM(i) helper gave luciferase expression that was barely detectable after infection of A9 cells which are highly restrictive for MVM(i) productive infection. These results support the notion that the allotropic determinant of these MVM strains functions through their capsid proteins. Pseudotyping of recombinant parvovirus genomes should be useful in controlling their host range as vectors, and in studying mechanisms influencing the permissiveness of parvovirus infections.

Capsid↗
PubMed1994

Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts.

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

Arabidopsis↗
PubMed1992

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.

Amino Acid Sequence↗
PubMed2004

[Expression of truncated 56 kDa antigen gene of Orientia tsutsugamushi in different vectors].

OBJECTIVE: To construct recombinant plasmids containing the truncated gene of the major surface antigen sta56 of Orientia tsutsugamushi (Ot.) Karp strain for expression antigen in E. coli so as to compare the expression efficiency in different systems. METHODS: From the recombinant plasmid TOPO-sta56 containing sta56 of Orientia tsutsugamushi Karp strain, several truncated genes of sta56 with different length were amplified and subcloned into the expression vectors pPROEX HTb and pET30a. These genes were expressed in E. coli DH5alpha and BL21(DE3) respectively when induced by IPTG. The expressed recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. RESULTS: Six recombinant plasmids containing truncated sta56 genes of different length were constructed as follow: pHTbOt957, pHTbOt498, pHTbOt342 and pETOt957, pETOt498, pETOt342. The recombinant sta56 proteins were highly expressed as 6 x His fusion proteins in E. coli DH5alpha and BL21(DE3) respectively. The fusion proteins showed as different bands of different molecular weight respectively when analyzed with SDS-PAGE. Western blot demonstrated that the recombinant proteins were recognized by the positive serum of Ot. patients. CONCLUSION: The sta56 gene of Orientia tsutsugamushi Karp strain could be highly expressed in E. coli and its expression showed better efficiency in pET30a than in pPROEX HTb. The recombinant sta56 antigen with immunoreactivity could be used as diagnostic reagent for Ot. infection.

Animals↗
PubMed2002

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter.

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.

Animals↗
PubMed1983

Expression in Saccharomyces cerevisiae of human interferon-alpha directed by the TRP1 5' region.

The complete 5' flanking region of the yeast TRP1 gene encoding N-(5'-phosphoribosyl)- anthranilate isomerase, a nonabundant protein, has been cloned and the nucleotide sequence data has been extended from -102 to -440. The CT block--CAAG structure common to all efficiently expressed yeast genes is altered in the 5' region of TRP1 and a sequence postulated to be involved in general amino acid regulation is absent. There are two possible TATA boxes at -224 and -262. TRP1, in common with HIS3, HIS4 and TRP5 has a region of dyad symmetry upstream of the coding sequence which may play a role in initiation of transcription. The relative efficiency of gene expression directed by the complete 5' TRP1 region was assessed by comparison with that of PGK by inserting a cDNA for a human interferon-alpha downstream of their respective 5' regions. The respective interferon yields indicate that their in vivo expression capabilities are a function of their 5' regions.

Aldose-Ketose Isomerases↗
PubMed2005

Large-scale protein expression for proteome research.

Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.

Amino Acid Sequence↗
PubMed2006

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system.

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.

Angiogenesis Inhibitors↗
PubMed1998

High-level expression of bovine beta-lactoglobulin gene in transgenic mice.

To study the expression of the bovine beta-lactoglobulin (BLG) gene we isolated the BLG gene from a genomic library and introduced it into murine germline. Bovine BLG gene including 2.8 kbp of 5' and 1.9 kbp of 3' flanking region was expressed efficiently and mammary gland-specifically in transgenic mice. Expression levels of BLG in milk exceeded 1 mg ml-1 in all four mouse lines analyzed. However, in two mouse lines originating from female founders BLG expression levels varied from less than 0.02 mg ml-1 up to 1 mg ml-1. In both lines originating from male founders all analyzed female mice excreted bovine BLG into their milk at a high and constant level of 1-2 mg ml-1. BLG expression was stable within individual mice in two successive lactations and the amount of BLG in the milk of mice correlated with the level of BLG mRNA in the mammary tissue. Methylation analyses of HpaII sites revealed that transgene copies were on average more methylated in mice which excreted low levels of BLG into their milk. Each mouse line had its own methylation pattern and, in addition, each mouse had more or less identical methylation patterns in mammary gland, brain and kidney DNA. Genomic sequencing of the BLG gene indicated that the promoter region (bases -162 to +391 with respect to the transcription start site) was heavily methylated except for distinct CpG sites that were only partially methylated both in transgenic mice and lactating cattle.

Animals↗
PubMed2001

Recombinant antithrombin: production and role in cardiovascular disorder.

Plasma-derived antithrombin (AT) concentrates have been used for the management of hereditary and acquired deficiencies since the early 1980s. Recombinant versions of other blood factors and their derivatives are increasingly becoming available, providing a safe and abundant supply of these important therapeutics. However, the complexity of the AT molecule and the large doses often required for supplementation treatments preclude the use of traditional cell culture bioreactors for recombinant production. The development of a very efficient expression system has been necessary for the cost-efficient recombinant production of AT. Transgenic production, with its ability to yield high levels of heterologous protein and its scale-up flexibility, is an attractive alternative to plasma fractionation. Purification of recombinant AT from the milk of transgenic dairy goats has been developed to provide a homogeneous, well-defined, and abundant supply of this factor. This article describes the production of recombinant AT and aspects of clinical applications of this molecule to cardiovascular disorders.

Animals↗
PubMed2004

Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation.

Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

Angiogenesis Inhibitors↗
PubMed2005

Expression and characterization of rubella virus glycoprotein E1 in yeast cells.

OBJECTIVES: To express E1 glycoprotein of rubella virus (RuV) strain JR23 in yeast and develop a diagnostic assay using expressed E1 protein as coating antigen in comparison with other diagnostic assays. METHODS: cDNA of E1 open reading frame of RuV was PCR amplified using plasmid pMD18-T-E1 as template and cloned into plasmid pBluscriptII SK+. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pBluscriptII SK(+)-E1 plasmid DNA was digested by restriction endonuclease EcoR I and Xba I, and a fragment of 1.5 kb was isolated and cloned into a yeast expression pGAPZ(alpha)A, resulting in pGAPZ(alpha)A-E1. After confirmation by sequencing, pGAPZ(alpha)A- E1 was transformed into yeast GS115 cells with LiCl method. E1 protein expression in GS115 was analyzed by SDS-PAGE and Western blot. An indirect ELISA was developed using the recombinant E1 protein as coating antigen for detecting RuV E1 antibodies in 90 serum samples. To compare the specificity, sensitivity and reproducibility of the assay with other methods, the same serum samples were also assayed by RuV culture medium as coating antigen (Jingmei kit and German RECI kit). Statistical analyses were performed to compare the differences among these methods and to determine which coating antigen source, the recombinant E1 protein or RuV-infected culture medium, is more suitable for the assay. RESULTS: A fragment of 1.5 kb, corresponding to the full open reading frame of E1, was PCR amplified and cloned in yeast expression vector. The clone was confirmed by restriction digestion, PCR and sequencing. E1 as a secretive protein was successfully expressed by GS115. Its molecular weight was about 58 kDa. SDS-PAGE showed that the recombinant protein was expressed efficiently and constantly in Pichia pastoris GS115 cells. The expression level reached a peak 48 h after culturing and stabilized thereafter. E1 protein was detected in both supernatant and cells. Western blot showed that the secretive E1 protein in the supernatants could react with both the anti-RuV-positive serum and a monoclonal antibody against E1. However, E1 protein derived from cells could only react with the anti-RuV-positive serum, polyclonal antibody, but not the monoclonal antibody. Compared with the German RECI kit, the sensitivity, specificity, and accordance rate of the assay using recombinant E1 protein as coating antigen were 67.11, 71.43 and 67.78%, respectively, while those of the assay using RuV-infected culture medium as coating antigen were 50, 78.57 and 54.44%, respectively. Compared with the German RECI kit, the sensitivity, specificity, and accordance rate of the ELISA assay using the Jingmei kit were 84.71, 71.43 and 82.22%, respectively. The data indicated that recombinant E1 protein derived from the yeast expression system can serve as a better source than RuV-infected cell medium as coating antigen for detecting antibodies against RuV in the indirect ELISA assay. Statistical analysis of the data generated from two independent experiments using recombinant E1 protein as coating antigen indicated that the assay was very consistent with no statistically significant difference between the two experiments (p > 0.05). 76 out of 90 serum samples were detected positive using the German RECI kit, while 68, 55 and 41 samples were positive using the Jingmei kit, recombinant E1 and RuV-infected cell medium, respectively. Statistical analyses indicated that the positive rates were significantly different among all four assays (p < 0.05) except for one pair (German RECI kit and the Jingmei kit: p > 0.05). Comparing the positive rates obtained from the assay using recombinant E1 and that using RuV-infected cell medium, it seems that the recombinant E1 protein is a better source than RuV culture medium as coating antigen in the indirect ELISA assay for detection of RuV antibody. CONCLUSIONS: The recombinant yeast expression vector of RuV E1 glycoprotein was constructed successfully. The E1 protein as a secretive protein was successfully expressed by GS115 and maintained its antigenicity very well. As coating antigen, the recombinant E1 protein served a better source than RuV culture medium in the indirect ELISA method for the detection of RuV antibody.

Antibodies, Viral↗
PubMed2003

Mice humanised for the EGF receptor display hypomorphic phenotypes in skin, bone and heart.

Mice lacking the epidermal growth factor receptor (EGFR) develop epithelial defects and a neurodegenerative disease and die within the first month of birth. By employing a conditional knock-in approach using the human EGFR cDNA mice humanised for EGFR (hEGFRKI/KI) were generated. Homozygous hEGFRKI/KI mice are viable and live up to six months. However, these mice are growth retarded and show skin and hair defects similar to Egfr-/- mutants. Interestingly, the neurodegeneration is fully rescued in hEGFRKI/KI mice, however, they develop a severe heart hypertrophy with semilunar valve abnormalities. Moreover, hEGFRKI/KI mice display accelerated chondrocyte and osteoblast differentiation, a phenotype that is also present in Egfr-/- mice and has not been previously described. The severity of the phenotypes correlates with the expression levels of the hEGFRKI allele, which is not efficiently expressed in epithelial and bone cells, but is expressed at similar and even higher levels as the endogenous Egfr in brain and heart. These results demonstrate that mice humanised for EGFR display tissue-specific hypomorphic phenotypes and describe a novel function for EGFR in bone development.

Animals↗