[通过对cDNA文库进行直接免疫筛选鉴定编码牛乳酪蛋白的细菌克隆]

[Identification of bacterial clones that encode cow's caseins by direct immunological screening of the cDNA library].

作者信息

Ivanov V N, Kershulite D R, Baev A A, Akhundova A A, Sulimova G E

出版信息

Mol Biol (Mosk). 1985 Jul-Aug;19(4):955-63.

Abstract

A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.

摘要

利用一种灵敏的免疫测定法，从牛乳腺的rRNA构建的cDNA文库中鉴定携带牛酪蛋白cDNA片段的重组DNA质粒。在硝酸纤维素滤膜上生长的菌落原位裂解，裂解物中的蛋白质印迹到CNBr活化滤纸上。通过与酪蛋白抗血清反应，随后用金黄色葡萄球菌的125I标记蛋白A和放射自显影检测共价结合到CNBr活化纸上或结合到硝酸纤维素滤膜上的抗原。在cDNA文库的5400个细菌克隆中，有4个克隆呈阳性——al、b2、b5、h7。通过免疫印迹法测定了在大肠杆菌中合成的嵌合蛋白前β-内酰胺酶：酪蛋白的分子量。菌落杂交和DNA序列分析表明，克隆b5含有牛κ-酪蛋白的cDNA片段，克隆h7含有β-酪蛋白的cDNA片段。最后一个克隆被命名为pKcasβ-7。