RESUMO
Expressions of several genes in bacteria were carried out by independent promoter. However, in case of eukaryotes ribosome skipping and introduction of IRES are employed as alternative to multiple translation initiation. Foot and mouth disease virus (FMDV) 2A peptide has been widely used for co-expression of multiple genes in eukaryotic, plant and mammalian systems. The 18 amino acid 2A peptide of FMDV facilitates efficient co-translational dissociation of the polyprotein into discrete protein products. To study the role of 2A in multimeric protein production a construct consisting of tandem repeat of 4 units of C- terminal VP1 linked through 2A sequence was made and expressed in E. coli. Along with tetramer protein, trimer, dimer and monomer proteins were produced. Stability studies showed that the tetramer protein was cleaved to smaller monomer on storage. The results provide scope for using FMDV 2A for expressing multiple genes under a single promoter in prokaryotes.
Assuntos
Animais , Clonagem Molecular , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Febre Aftosa/metabolismo , Vírus da Febre Aftosa/metabolismo , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Técnicas Genéticas , Peptídeo Hidrolases/química , Peptídeos/química , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Virais/químicaRESUMO
Economizing the research protocols by using low cost technologies is the need of laboratories of developing world. Screening of recombinant E. coli colonies is the crucial step in gene cloning and expression studies. In the present study, the cost effectiveness of colony lysis method and colony PCR method in the screening of recombinant E. coli colonies was compared. The colony lysis method was 20 two times more cost effective and less time consuming and can be used to screen the recombinant E. coli colonies in large scale instead of colony PCR method.
Assuntos
Técnicas Bacteriológicas/economia , Análise Custo-Benefício , Escherichia coli/genética , Técnicas Genéticas/economia , Reação em Cadeia da Polimerase/economia , Recombinação GenéticaRESUMO
For effective FMD control programme, India needs large quantities of cheaper diagnostics in addition to vaccine. Diagnostic reagents produced through conventional methods may not be able to meet such requirements. Alternatively, rDNA technology using suitable heterologous systems that permit production of recombinant antigens to the most native form may be exploited. Studies conducted in our laboratory have led us to select carboxy terminal part of VP1 for expression and evaluation. The protein, which was purified from E.coli under denaturing conditions, was renatured and its reactivity was compared with the protein expressed in insect cells through recombinant baculovirus. The expressed protein in the insect cell whole lysate reacted more efficiently with antibodies raised against whole virus than the purified and renatured protein produced in E.coli. But for its lower reactivity, protein produced from E.coli was found to be suitable in type detection. In addition, the size of the protein is small (16 kD) and production and purification of it from E.coli may be cost effective. Hence, it may be exploited for FMDV typing.
Assuntos
Animais , Antígenos Virais/genética , Aphthovirus/genética , Sequência de Bases , Capsídeo/genética , Proteínas do Capsídeo , Linhagem Celular , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes/genética , SpodopteraRESUMO
A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.