蜡质芽孢杆菌aiiA基因的克隆及融合表达

设计一对可扩增aiiA基因完整的开放阅读框的简并引物对aiiA1和aiiA2,通过PCR技术对3株蜡质芽孢杆菌(Bc)的aiiA基因进行检测.结果表明,它们均含有aiiA基因.利用pMD18-T克隆载体直接从GP7菌株的PCR产物中克隆了aiiA基因.测序结果表明,该基因(GenBank登录号:AY943831)由753个碱基组成,编码含有250个氨基酸残基的蛋白质.该蛋白质推测的分子质量为28 ku,等电点约4.235.核苷酸序列的BLAST分析结果表明,与之同源性较高的基因均为Bc组aiiA基因(87%-99%).在氨基酸序列多重比较的基础上,应用PHYLIP软件构建了A iiA蛋白的系统发育树.此外,利用原核融合表达载体pMXB10初步研究了A iiA、几丁质结合蛋白(CBD)及Inte in融合蛋白诱导表达的情况.

Cloning and fusion expression of aiiA gene from Bacillus cereus

In order to better understand the molecular genetics of Bacillus cereus(Bc),a pair of degenerate primers(designated aiiA1/aiiA2),which can amplify the entire coding region of aiiA gene,were designed and used to investigate the existence of aiiA gene in 3 Bc strains by PCR.The results showed that all of them contained the aiiA gene.A novel aiiA was cloned from Bc GP7 by PCR with pMD18-T vector and was sequenced.The nucleotide sequence had been registered in GenBank with accession no.AY943831.It consisted of 753 bases which encoded a polypeptide of 250 amino acids residues with a calculated molecular weight of 28 ku and an isoelectric point value of 4.235.BLAST analysis of its nucleotide sequence showed 87%-99% identity with those of the aiiA genes from Bc group.On the basis of the multiple alignment of the amino acids sequences,a phylogenetic tree of AiiA was constructed and analyzed by UPGMA method from PHYLIP 3.63 program.In addition,the fusion protein of AiiA with CBD and Intein was expressed in Escherichia coli ER2566.