CXCL12-α基因编码区的克隆与生物信息学分析

Cloning and Bioinformatics Analysis of Human CXCL12-α Gene Coding Region

目的：克隆人源CXCL12-α的成熟肽编码序列后进行生物信息学分析及其蛋白质结构与功能预测。方法：采用RTPCR方法从人骨髓组织中克隆CXCL12-α基因序列,并将编码其成熟蛋白的核酸片段插入原核表达载体pET-30a（＋）中,转化Escherichia coli后进行酶切鉴定和DNA序列测定。利用在线网络生物信息学相关数据库和分析软件对测序结果进行分析,并对重组蛋白的一、二、三级结构及功能等进行验证和预测。结果：获得了基因序列为263 bp的人源CXCL12-α基因,与GenBank中公布序列一致。双酶切鉴定及DNA测序验证结果正确。生物信息学检索该基因编码蛋白的氨基酸序列与理化参数显示,蛋白无跨膜区,在第29位有一个Ser为蛋白激酶磷酸化位点,第1~21位可能为信号肽区域。ɑ-螺旋,无规则卷曲,延伸链和β-转角数量分别占总二级结构的44.94%、22.47%、21.35%、11.24%。同源建模预测信息可信度为0.68,该蛋白G-factor结构计分总平均为0.33,结构检验表明该蛋白属于正常范围内。二级结构和拓扑结构信息、蛋白质结合位点三维图和预测蛋白可能催化口袋及结合位点、三维结构模拟的可视化结构显示其空间结构稳定。结论：人源CXCL12-α分子克隆成功,网络数据库等生物信息学分析及结合蛋白质结构与功能预测可为深入研究CXCL12-α提供理论指导。

Objective：Cloning human CXCL12α coding sequence of mature peptides for the bioinformatic analysis and prediction of protein structure and function. Method：CXCL12 -α coding sequence from human bone marrow tissue was cloned with RT- PCR. Then its mature protein fragment was ligated into the prokaryotic expression vector pET -30a（ ＋ ） and transformed into Escherichia coil The target gene was determined after restriction enzyme digestion and DNA sequencing. Adpoting the online network relevant databases and bioinformatics software for analysis the target recombinant protein primary structure, secondary structure and tertiary structure and functions for prediction. Result：The results show that the obtained 263 bp gene sequence of human CXCL12 -α gene sequences are consistent to what have published in GenBank,restriction enzyme digestion and DNA sequencing results are correct. Bioinformatics search reveales that the gene encoding the amino acid sequence of physical and chemical parameters, α- helix, random coil, extended chain and [3 - angle account for 44. 94 % ,22. 47 % ,21.35 % and 11.24 % of the secondary structure. CXCL12 -α protein contains a functional domain of which belongs to the chemokine superfamily. Homology modeling prediction information reliability is 0. 68, the protein G - factor structure of the total average score is 0. 33,which verified the protein structure is in the normal range. Secondary structure and topology information, three- dimensional map of protein binding sites and predict protein possible binding sites for catalytic pocket are predicted here. Three - dimen- sional visualization of structural simulation spatial structure is stable. Conclusion：Human CXCL12 - α had been cloned successfully. The experimental results of protein structure and function prediction by bioinformatics databases and analysis provides theoretical guidance for the further study of CXCL12 -α.