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包含部分牛催乳素序列的cDNA克隆的构建与鉴定

Construction and characterization of a cDNA clone containing a portion of the bovine prolactin sequence.

作者信息

Nilson J H, Thomason A R, Horowitz S, Sasavage N L, Blenis J, Albers R, Salser W, Rottman F M

出版信息

Nucleic Acids Res. 1980 Apr 11;8(7):1561-73. doi: 10.1093/nar/8.7.1561.

Abstract

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.

摘要

来自牛脑垂体前叶的含Poly(A)RNA已被用作酶促合成双链cDNA的模板。通过寡聚(dG)-寡聚(dC)加尾技术,将所得双链cDNA插入pBR322的Pst I位点,随后克隆到大肠杆菌chi 1776中。通过与部分纯化的催乳素cDNA进行菌落杂交,鉴定出含有与催乳素mRNA互补序列的克隆。对一个催乳素阳性克隆的250个碱基对序列进行了广泛表征,结果表明其包含真实牛催乳素第119 - 192位氨基酸的编码信息。来自该克隆的重组DNA与重氮化氨基纤维素共价连接,并用于从mRNA混合物中纯化催乳素mRNA。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2349/324017/670372b225dc/nar00424-0127-a.jpg

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