萤火虫Luc基因的克隆及生物信息学分析

Cloning and bioinformatics analysis of Luc gene of firefly

[目的]克隆野生型虫荧光素酶(luciferase)基因(Luc)全长序列并对其进行生物信息学分析。[方法]以虫荧光素酶报告栽体pXP2 DNA为模板,应用PCR技术,获得萤火虫Luc基因目标条带并克隆到pGEM-T-Easy载体,利用CD search、ProtParam、ProtScale和SOMPA等11种预测软件对其保守区域、一级结构、理化性质、空间结构及活性区域等进行预测分析。[结果]该酶由551个氨基酸残基组成,属于可溶性亲水蛋白。其二级结构主要由无规卷曲和α-螺旋组成。三级结构以6q2m.1的A链为模板进行同源建模,其活性区域由125个氨基酸残基构成,面积和体积分别为2 483.879A^2和1 834.454A^3。在该蛋白质的活性区域之外,筛选出38个具备引入二硫键条件的潜在位点。[结论]成功克隆了Luc基因,并对野生型虫荧光素酶进行了生物信息学分析预测,为进一步过定点突变提高虫荧光素酶的热稳定性奠定了理论基础。

[Objective]To clone the Luc gene of luciferase,and analyze the bioinformation.[Method]Using luciferase reporter pXP2 DNA as the template,the objective PCR band of Luc was successfully amplified using PCR technique and then it was cloned to pGEM-T-Easy vector. the conservative region,primary structure,physical and chemical properties,hydrophobicity,structure,active region were analyzed by nine prediction software,such as CD search,ProtParam,Protscale and SOMPA and so on.[Result]The enzyme was composed of 551 amino acid residues,and belongs to soluble hydrophilic protein. The secondary structure was mainly composed of random curl and α-helix. The tertiary structure model was constructed with PDB ID 6 q2 m.1 A-chain as the template. the active region was consisted of 125 amino acid residues,and the area and volume were2 483. 879 A^2 and 1 834. 454 A^3. Sixty-three potential sites,which could be added by disulfide bond,were selected from the protein sequence.[Conclusion]Luc gene of wild-type luciferase was cloned and bioinformatics analysis was carried out to which lay a theoretical foundation for the improving thermostability of luciferase by site-specific mutagenesis.