Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A two part purity testing regimen for genetically engineered live viral vaccines is described using a human adenovirus 5: rabies glycoprotein gene recombinant as a model vaccine. Initially, restriction endonuclease analysis of the recombinant viral genome verified the integrity of the recombinant construct and identified the vector genome. The second stage employed the polymerase chain reaction to facilitate a more detailed study of the target rabies glycoprotein cassette. The size of the target region was predicted from known nucleic acid sequence information and compared to that obtained after electrophoresis with molecular weight standards. Digestion of the polymerase chain reaction product with a second restriction endonuclease cleaved the target into a number of small fragments. Resolution of the fragments by gel electrophoresis allowed analysis of the target region alone, verifying its identity and integrity.
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PMID:Approaches for genetic purity testing of live recombinant viral vaccines using a human adenovirus:rabies model. 147 4

The genetic stability of a live human adenovirus 5: rabies glycoprotein recombinant vaccine has been assessed upon 20 serial passages in a permissive cell line of human origin. Restriction endonuclease analysis and the polymerase chain reaction were used to examine the integrity of the expression cassette for the rabies glycoprotein and the viral vector at the site of insertion of the cassette. It was found that the restriction endonuclease profile was identical for each sample assayed. A more detailed analysis of the expression cassette following amplification by the polymerase chain reaction revealed no changes in the size and number of fragments originating from the coding sequence for the glycoprotein nor the signals controlling the expression of the protein product. The amplified product obtained from the 10th and 20th passages was subjected to nucleotide sequencing. Additionally, 20 plaques isolated from the 20th passage of the virus expressed the rabies glycoprotein as demonstrated by fluorescent antibody staining with glycoprotein specific monoclonal antibodies. These results suggest that the recombinant vaccine maintains the integrity of the heterologous sequences upon passage in tissue culture.
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PMID:In vitro assessments of the genetic stability of a live recombinant human adenovirus vaccine against rabies. 764 30

Rabies persists in Ontario wildlife in two predominant species: the red fox (Vulpes vulpes) and the striped skunk (Mephitis mephitis). A protocol applying reverse transcription/polymerase chain reaction (RT/PCR) and restriction endonuclease analysis (REA) to the rabies virus nucleoprotein gene was previously reported by Nadin-Davis et al. (Journal of General Virology 74, 829-837, 1993) to be useful for discrimination of rabies virus variants in Ontario. Four main types, which showed no host species specificity but which did exhibit different geographical distributions, were identified. Between 1989 and 1992 an area north and west of the city of North Bay experienced unusual and substantial rabies activity. In this report we describe the use of these molecular techniques to investigate the epidemiology of this recent rabies outbreak in central Ontario. It is shown that two of the four previously identified variants had invaded this region from the south and east, but in addition viruses very closely related to arctic isolates of rabies virus were found. The nucleoprotein and glycoprotein genes of this arctic type were sequenced and compared to those of its more southerly neighbours.
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PMID:A molecular epidemiological study of rabies virus in central Ontario and western Quebec. 793 Nov 45

An infectious recombinant human adenovirus which carries the rabies glycoprotein gene and accompanying SV40 control elements can be given orally to skunks to immunize them against rabies. We have looked for adenovirus in the feces and oral fluids of animals that have been given this recombinant and have obtained 111 virus positive samples from 16 test animals. DNA from these virus isolates was examined for possible mutations. One possible insertion mutation was detected by SmaI restriction endonuclease analysis of genomic DNA. Further analysis by HaeIII restriction and nucleotide sequencing of polymerase chain reaction products encompassing the whole SV40-rabies insert revealed that this isolate contained an insert of the 72 base pair sequence found in the SV40 promoter region. A second mutation, in which 54 base pairs were deleted from within the rabies glycoprotein gene, was also detected in two independent isolates from one skunk.
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PMID:Characterization of a human adenovirus 5: rabies glycoprotein recombinant vaccine reisolated from orally vaccinated skunks. 882 55

A simple method for the rapid detection of rabies virus was developed employing a single-tube reverse transcription polymerase chain reaction (RT-PCR). The method utilized a single buffer system for both RT and PCR and was performed without interruption as a single thermal cycling programme. Two primer sets within the genes coding for rabies nucleoprotein and glycoprotein were used to amplify a 533 bp and a 406 bp amplicon, respectively. The amplified products were detected with a challenge virus strain (CVS) of rabies. There was no amplified product with uninfected mouse or dog brain. The method was applied to detect rabies virus in 10 mouse inoculation test (MIT)-positive and three MIT-negative brain tissue samples. The amplified product was found only in the MIT-positive samples. The amplified product was confirmed by restriction endonuclease analysis using Hinf1. The results from RT-PCR correlated well with the results from MIT. This indicates that the single-tube RT-PCR may be a useful method for detecting rabies virus in brain tissue samples from suspected cases of rabies.
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PMID:Preliminary report on a single-tube, non-interrupted reverse transcription-polymerase chain reaction for the detection of rabies virus in brain tissue. 1133 52

Despite the recognized stability of rabies virus, differences among isolates from different species have been found. This work was carried out with the aim to identify antigenic and genomic differences in Brazilian rabies virus isolates and to verify whether such alterations would bear any relationship with the different hosts for the virus in nature. For that, 79 Brazilian rabies viruses isolated from different host species and from distinct regions within Brazil were submitted to antigenic characterization with a panel of 11 monoclonal antibodies (Mabs) directed to lyssavirus antigens and to genomic analyses by the reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the N gene followed by restriction endonuclease analysis (REA). In addition, the nucleotide sequences of part of the N gene (225 bp) of seven isolates, taken as representative of the majority of the viruses under study, were determined. The analyses with the Mabs and RT-PCR/REA allowed the identification of two major groups of variants, the first formed by most isolates of cattle and bats and the second formed by viruses of dog origin. Partial sequencing of the N gene confirmed the similarity among isolates from cattle origin and those of vampire bats. However, viruses from non-haematophagous bats exhibited consistent differences from those of vampire bat isolates. Such findings suggest that the variants have evolved fairly stable modifications, which are not altered after passage in a dead-end host of a distinct species. No association could be established between antigenic or genomic alterations and geographic distribution of the isolates, which suggests that evolution of the virus has been directed to adaptation to the host species.
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PMID:Studies on antigenic and genomic properties of Brazilian rabies virus isolates. 1586 75

Safe and effective vaccination is important for rabies prevention. Here, genetically engineered rabies vaccine CAV2-deltaE3-Rgp was developed and characterized. The recombinant genome pPoly2-CAV2-deltaE3-Rgp carrying the rabies glycoprotein (Rgp) cDNA was generated by a series of strictly gene cloning steps and infectious recombinant virus CAV2-deltaE3-Rgp was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. To efficiently construct cloned recombinant canine adenovirus type 2 genome pPoly2-CAV2-deltaE3-Rgp bearing exogenous Rgp gene, The Rgp gene was first subcloned from the clone vector pMD18-T into the eukaryon expression vector pVAX1. The Rgp expression cassette was then subcloned into the shuttle vector pVAXdeltaE3 and subsequently into the canine adenovirus type 2 backbone vector pPoly2-CAV2. To indirectly confirm pPoly2-CAV2-deltaE3-Rgp, conventional restriction endonuclease digestion was performed. CAV2-deltaE3-Rgp can generate typical CPE of CAV-2. CAV2-deltaE3-Rgp was tested by restriction endonuclease digestion, PCR, DNA sequencing. As a result, The Rgp expression cassette was successfully integrated into the target region of the CAV2 genome. It is confirmed by RT-PCR, Western blot that CAV2-deltaE3-Rgp can express Rgp antigen in MDCK cell. This recombinant virus, CAV2-deltaE3-Rgp, was intramuscularly injected into dogs. All vaccinated dogs produced effective antibodies against CAV and RV after three inoculations. This recombinant virus would be prospective in immunizing dogs against CAV and RV.
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PMID:[Construction and identification of recombinant canine adenovirus type 2 expressing exogenous rabies glycoprotein (Rgp)]. 1755 45

The purpose of this study was to express the matrix protein of rabies virus in baculovirus expression system and prepare its polyclonal antibody. Using the total RNA of RABV strain BD06 as a template, RT-PCR technique was utilized to amplify the sequence of M gene, which were then inserted into shuttle vector pFastbac I to construct the recombinant vector pFastbac I-M. After identification using the double restriction endonuclease cleavage method, the recombinant vector pFastbac I-M were transformed into the competent E. coli DH10 Bac to construct the recombinant expression vector Bacmid-M, which were transfected into Sf9 cells mediated by lipofectamine 2000 to obtain the recombinant baculovirus AcMNPV-M. The mice anti-His monoclonal antibody, rabbit anti-RV positive serum and canine anti-RV positive serum were used in Western Blot assays to identify the expression and reactogenicity of the recombinant. The recombinant M protein were purified under denaturing conditions using the nickel iron affinity chromatography column, then used to immunize the New Zealand White rabbit to prepare its polyclonal antibody. Western Blot assay and FAVN assay were used to validate the polyclonal antibody. Our results showed that the M protein of RABV were successfully expressed in baculovirus expression system,of which molecular weight was of about 25kD;the recombinant M protein has a good reactogenicity and immunogenicity; the rabbit polyclonal antibody prepared by purification of M protein could react with the M protein of RABV strain BD06,SRV 9,CVS-24,ERA,PV2061 and aG. Undoubtedly, the successfully preparation of both recombinant M protein and its polyclonal antibody support a material foundation for further study on the properties of M protein of RABV.
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PMID:[Expression and Purification of M Protein of RV in Baculovirus and Preparation of Its Polyclonal Antibody]. 2999 70