IDENTIFICATION OF CLONES THAT ENCODE CHICKEN TROPOMYOSIN BY DIRECT IMMUNOLOGICAL SCREENING OF A CDNA EXPRESSION LIBRARY
IDENTIFICATION OF CLONES THAT ENCODE CHICKEN TROPOMYOSIN BY DIRECT IMMUNOLOGICAL SCREENING OF A CDNA EXPRESSION LIBRARY
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DOI：
10.1073/pnas.80.1.31
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
HUGHES, SH
中科院分区：
文献类型：
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作者：
HELFMAN, DM;FERAMISCO, JR;HUGHES, SH
.apprx 的 c[互补]DNA 文库。利用质粒表达载体pUC8，从鸡平滑肌mRNA中制备了9000个成员。在 cDNA 制备过程中的不同阶段添加 Sal I 和 EcoRI 连接子产生了一群分子，其中大多数在 cDNA 末端具有对应于模板 mRNA 5'' 末端的 EcoRI 连接子，在对应于 mRNA 3'' 末端的末端具有 Sal I 连接子。然后将 cDNA 分子插入 EcoRI-Sal I 切割质粒载体 pUC8，该载体包含来自 EcoRI 位点上游 lacZ 基因的转录和翻译起始序列。向 cDNA 中顺序添加接头可确保大部分 cDNA 以正确的表达方向插入到 pUC8 中。将菌落复制铺板到硝基纤维素纤维上并用氯仿蒸气原位裂解。通过首先将滤膜与兔抗原肌球蛋白抗体一起孵育，然后与 125I 标记的山羊抗兔 IgG 一起孵育，筛选文库中产生与鸡原肌球蛋白免疫相关的产物的菌落。检测到两个菌落与抗血清发生特异性反应。对来自两个克隆的质粒进行部分序列分析；两种质粒均含有编码原肌球蛋白的 cDNA。这些方案对于从大型 cDNA 表达文库中鉴定低水平表达的基因的 cDNA 克隆可能有用。
A c[complementary]DNA librabry of .apprx. 9,000 members was prepared from chicken smooth muscle mRNA by using the plasmid expression vector pUC8. Addition of Sal I and EcoRI linkers at different stages during the preparation of the cDNA resulted in a population of molecules, most of which had EcoRI linkers at the end of the cDNA that corresponded to the 5'' end of the template mRNA and Sal I linkers at the end that corresponded to the 3'' end of the mRNA. The cDNA molecules then were inserted into a EcoRI-Sal I-cut plasmid vector, pUC8, that contains the transcriptional and translational start sequences from the lacZ gene upstream of the EcoRI site. The sequential addition of the linkers to the cDNA ensured that most of the cDNA were inserted into pUC8 in the proper orientation for expression. The colonies were replica plated onto nitrocelulose fibers and lysed in situ with chloroform vapor. The library was screened for colonies producing products immunologically related to chicken tropomyosin by incubating the filters first with a rabbit antitropomyosin antibody and 2nd with a 125I-labeled goat anti-rabbit IgG. Two colonies were detected that reacted specifically with the antisera. Plasmids from both clones were partially subjected to sequence analysis; both plasmids contained cDNA that encoded tropomyosin. These protocols are potentially useful for the identification of cDNA clones for genes expressed at low levels from large cDNA expression libraries.