RESUMO
The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine alpha S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 micrograms ml-1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.
Assuntos
Lactoferrina/biossíntese , Lactoferrina/genética , Glândulas Mamárias Animais/metabolismo , Leite/química , Proteínas Recombinantes de Fusão/biossíntese , Animais , Sequência de Bases , Caseínas/genética , Bovinos , Feminino , Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Humanos , Lactação/metabolismo , Masculino , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Complementary DNA libraries representing the capsid protein cistron of the potato virus Y (PVY) isolate 'Chilean', 'Hungarian', MsNr, NsNr, O, and 'Potato US' were synthesized and used as template for polymerase chain reaction (PCR) amplification. An AUG codon for initiating a discrete capsid protein (CP) open reading frame was embedded upstream of the first codon of the CP cistrons. PCR-amplified products of the expected size of 0.8 kilo bases were cloned into the transcription vector pBS(+). The fidelity of each PCR-amplified PVY CP cistron was tested by transcribing recombinant plasmids in vitro and translating the transcripts in two cell free translation systems. Translation analysis of in vitro transcribed PVY CP cistrons consistently yielded a polypeptide co-migrating with authentic CP that was immunoprecipitated by anti PVY 'Chilean' antibodies. The nucleotide sequence of each capsid protein gene was determined by dideoxy sequence analysis. Each capsid protein gene was determined to be 801 nucleotides in length, encoding a deduced protein of 267 amino acids with calculated M(r) ranging from 29,799 to 29,980. The nucleic acid sequence similarity between the six isolates ranged between 89 to 97% and the amino acid similarity between 91 to 99%. The high level of amino acid sequence similarity confirms the classification of these viruses as isolates of PVY.
