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Identification of Bacterial Clones Encoding Bovine Caseins by Direct Immunological Screening of the cDNA Library

Ivanov, Vladimir N.; Kershulite, Dangerute R.; Bayev, Aleksej A.; Akhundova, Aida A.; Sulimova, Galina E.; Judinkova, Elena S.; Gorodetsky, Stanislav I.

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by ^125I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-β-lactamase: casein proteins synthesized in
Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine χ-casein, clone 3-H7 contained a cDNA fragment of β-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of α_si-casein.

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Academic Units
Center for Radiological Research
Publisher
Elsevier Science
Published Here
September 11, 2015
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