黄海黄杆菌YS-9412-130低温碱性蛋白酶的rn基因克隆和序列测定

摘要

黄海黄杆菌YS-9412-130具有稳定的分泌低温碱性蛋白酶的能力。将其染色体DNA用Sau 3AⅠ部分酶切后，低熔点琼脂糖回收2-10Kb的DNA片段，用Klenow大片段酶半补齐，与用SalⅠ酶切且半补齐的质粒pUC19连接后，转化E.Coli.JM109，构建基因文库。用酪蛋白平板法和酶联免疫吸附（ELISA）的活性筛选方法从基因文库中筛选到一株产海洋低温碱性酶的阳性克隆（命名为pHH1）。序列测定分析表明，此重组质粒包含有长度为768bp低温碱性蛋白酶基因的完整的开放读码框架（ORF）和上游基因调控序列。此片段编码由256个氨基酸组成的酶，计算分子量为33 000Dal。通过Southern杂交证实了此片段来自于黄海黄杆菌YS-9412-130基因组DNA。%F. yellowsea YS-9412-130 stably secreted low-temperature alkaline protease. For the preparation of gene libraries, genomic DNA from F. yellowesea YS-9412-130 was digested partially with Sau3AⅠ,and the fragments from 2Kb to 10kb were recovered.The cohesive ends partially filled in by klenow fragment of DNA polymeraseⅠ(PolⅠk). DNA of plasmid pUC19 was cleaved with SalⅠand subsequently partially filled in with PolⅠk. The plasmid and the genomic DNAs were mixed and ligated. Then the recombinant plasmid transformed E.coli.JM109. One positive clone was obtained from the libraries with casein flat and ELISA sieve method. The analysis of sequence detection show that the positive clone includs the Open Read Frame of low-temperature alkaline protease gene. The fragment coded the enzyme of 256 amino acid, the molecular weight of which is 33 000Dal. Through Southern hybridization, the fragment is confirmed to come from F.yellowsea YS-9412-130 genomic DNA.