粘质沙雷氏菌几丁质酶(ChiC)基因克隆及其生物信息学分析

Gene Cloning and Bioinformatics Analysis of a Chitinase C(ChiC) Gene from Serratia marcescens

从粘质沙雷氏菌(Serratia marcescensATCC14041)中克隆出几丁质酶基因(ChiC),将回收纯化的PCR产物与载体pMD18-T连接,构建成的重组质粒命名为pMD-Ch iC,将重组质粒转化到受体E.coliDH5α中进行克隆,经BamHI和NheI双酶切验证、核酸序列测定证实,重组质粒pMD-Ch iC含有几丁质酶C基因(ChiC)。利用生物信息学的方法,推测该粘质沙雷氏菌ChiC基因编码的蛋白质由480个氨基酸组成。预测该蛋白的等电点为5.63,分子量约为52kD。针对粘质沙雷氏菌中的几个几丁质酶基因做了进化树,进而验证了粘质沙雷氏菌(Serratia marcescens)几丁质酶(ChiC)在沙雷氏菌几丁质酶中的分类;同时对粘质沙雷氏菌几丁质酶C(ChiC)蛋白的高级结构作出了预测,得到其编码的属于18家族的蛋白质高级结构图谱。

Chitinase gene Chic from Serratia macescens ATCC14041 was cloned. The PCR product was inserted into the pMD18 -T Vector （named pMD -ChiC）. The recombinant plasmid was transformed into E. coli DH5ct. By # sequenced and cleaved with Barn HI/Nhe Ⅰ, it shows that recombinant plasmid contains ChiC. Bioinformatics analysis shows that the length of complete coding sequence was 1 443 bp, and the protein of the Chic had a 480 - residue precursor. The deduced isoelectric point was 5.63, and the molecular weight was about 52 kD. Chitinase cladogram of Serratia sp. was made, and the classification of Chitinase C in Serratia sp. was validated. Meanwhile the tertiary structure sketch map of the chitinase ChiC from S. marcescens was forecasted,and the senior structure sketch of the protein which belongs to 18 family was made.