用一种改进的粘粒文库筛选方法克隆人α-乳清白蛋白基因  

Cloning of Human Alpha-Lactalbumin Gene by a Modified Method for a Cosmid Library

作　　者：曹更生[1] 李宁[2] 赵要风[2] 冯继东[2] 

机构地区：[1]河南大学生物工程系,开封475001 [2]中国农业大学农业生物技术国家重点实验室,北京100094

出　　处：《农业生物技术学报》2000年第2期169-172,共4页Journal of Agricultural Biotechnology

摘　　要：本文所介绍的粘粒文库筛选方法，是以PCR技术为基础[1]，并针对粘粒克隆扩增的不均一性而设计的。从扩增一次的粘粒文库中（载体pWE15）取1～2×106个粘粒克隆，将其分成96份，经PCR筛选，从中找出阳性管；将阳性管中所有克隆按每板1～1.5×103个克隆的方式铺极培养（约200个平板），再筛选鉴定含有阳性克隆的菌落平板，最后将阳性菌落平板进行菌落原位杂交，成功地筛选到2个人的α-乳清白蛋白基因阳性克隆（45～55kb）；特异引物直接对粘粒阳性克隆进行部分序列测定及鉴定分析，结果进一步证明该阳性克隆含有不完全的人α-乳清白蛋白基因，并绘出了该阳性克隆约10kb的片段物理图谱。A modified method for a cosmid librare is based on the technique of PCR assay whose primers define a particular sequence and is required to design a strategy according to the characteristic of a cosmid librmp of which some clones may grow more rapidly while others more slowly. 1～2×106 clones were taken from a mono-amplified cosmid library and subdivided into 96 portions,which lengthwise or breadthwise merged into 20 tubes. And then the positive tube was determined by PCR assay. All ofclones in the Positive tube were spread out on agarose plates, and the Positive plate containing the Positive clone was screened and identified. When the desired plate was identified, two positive clones were successfully obtained by plaque in situ hybridization. One of two positive clones was sequenced and analyzed by Peculiar primers directIy. The results of sequencing and restriction analysis indicated that the positive clone contains the part of human alpha-lactalbumin gene. The physical mny of about 10 kb fragment of the positive clone was made.

关 键 词：人α-乳清白蛋白基因 聚合酶链式反应 粘粒文库 

分 类 号：Q512.1[生物学—生物化学] Q785