RESUMO
The gene coding for the major outer capsid protein (VP7) of simian rotavirus SA-11 has been expressed in a baculovirus-insect cell system. The resulting protein is 35 kDa and is primarily associated with the endoplasmic reticulum. Neutralizing SA-11 polyclonal antiserum and VP7 monospecific antiserum reacted specifically with the expressed gene product. Antiserum derived against the recombinant VP7 protein neutralized SA-11 rotavirus infectivity in a fluorescent focus assay.
Assuntos
Baculoviridae/genética , Proteínas do Capsídeo , Capsídeo/genética , Genes Virais , Vetores Genéticos , Rotavirus/genética , Proteínas Estruturais Virais/genética , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Sequência de Bases , Capsídeo/imunologia , Clonagem Molecular , DNA Viral/química , Cobaias , Dados de Sequência Molecular , Mariposas/genética , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Mapeamento por Restrição , Rotavirus/imunologia , Integração ViralRESUMO
The major inner capsid protein (VP6) of SA-11 simian rotavirus has been expressed in Escherichia coli using a cloned cDNA derived from SA-11 double-stranded RNA segment 6. The cloned gene was fused to the N-terminal coding sequence of lacZ resulting in the synthesis of a 44-kDa protein. Several smaller polypeptides were also observed, resulting predominantly from transcription and translation within the gene 6 coding sequence. The recombinant VP6 proved to be antigenic by immunoblot analysis using polyclonal serum against SA-11 rotavirus and by Western-blot analysis using monospecific serum derived from purified viral VP6.
Assuntos
Antígenos Virais , Proteínas do Capsídeo , Capsídeo/biossíntese , Capsídeo/genética , Clonagem Molecular , DNA Viral/biossíntese , Escherichia coli/genética , Rotavirus/genética , Animais , Sequência de Bases , Western Blotting , DNA Viral/genética , Genes Virais , Haplorrinos/microbiologia , Óperon Lac , Dados de Sequência Molecular , Testes de Precipitina , Sondas RNA , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
Human pulmonary surfactant proteolipid SP-B arises by proteolytic processing of a 42,000-dalton precursor. The active proteolipid SP-B is one of two small hydrophobic proteins identified in surfactant that impart surface-active properties to surfactant phospholipids. We report the isolation and characterization of complete SP-B cDNA from a human lung cDNA library. The cDNA was used to isolate the gene encoding the SP-B precursor from a lambda EMBL3 library of human embryonic kidney DNA. The entire SP-B gene was sequenced and is approximately 9.5 kb long, with 11 exons and 10 introns including a large 823-nucleotide 3' untranslated exon. The sequence derived from the exons differs from the cDNA sequence at 3 positions out of 2001, only one of which is in the translated region. Direct RNA sequencing indicated that the 5' untranslated region is only 14 nucleotides long. A number of putative regulatory elements were found upstream of the SP-B gene, including a GC box and several putative cAMP and glucocorticoid receptor binding sites. Several Alu repeats and a region of potential Z-DNA formation were found in the introns. Southern blotting of human genomic DNA probed with SP-B cDNA indicated the presence of only one SP-B gene in the human genome, and the gene was localized to chromosome 2.
Assuntos
DNA/isolamento & purificação , Genes , Proteolipídeos/genética , Surfactantes Pulmonares/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/análise , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proteínas Associadas a Surfactantes Pulmonares , Homologia de Sequência do Ácido NucleicoRESUMO
Human pulmonary surfactant proteolipid of Mr = 5,000, now termed surfactant protein C (SP-C), is produced by proteolytic processing of an Mr = 22,000 precursor. The active hydrophobic peptide imparts surface active properties to pulmonary surfactant phospholipids. We have determined the entire nucleotide sequence of two distinct genes encoding SP-C from a genomic library prepared from human leukocytes. SP-C genes were encoded by approximately 3.0 kilobase pairs of DNA containing six exons and five introns. In both genes, the active hydrophobic region of the polypeptide was located in the second exon that encodes a peptide of 53 amino acids. The entire nucleotide sequences of the two classes of SP-C genes differed by only 1%. Two cDNAs encoding SP-C were distinguished on the basis of an 18-nucleotide deletion at the beginning of the fifth exon; no such deletion was detected within the two classes of SP-C genes. Comparison of the 3'-untranslated regions of SP-C cDNA clones and the two classes of genomic clones demonstrated that cDNAs with and without the 18-base pair deletion could be derived from both of the genes. This 18-base pair deletion occurs in nucleotide sequences compatible with two distinct RNA splice sites. One additional cDNA clone showed the addition of an 8-base pair insert at the end of exon 5, which was also compatible with two distinct splice sites. Both classes of SP-C genes were represented by cDNAs, demonstrating that both classes of genes are actively transcribed. The two SP-C genes were readily distinguished on the basis of their nucleotide sequences and restriction fragment analyses of their flanking DNA. Two distinct classes of human SP-C genes are transcribed, and the heterogeneity in the SP-C RNAs appears to result from differential splicing.
