大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接  

Separate expression of ABCA1 and ligation mediated by Ssp DnaE intein in Escherichia coli BL21(DE3)

作　　者：朱甫祥[1] 缪静[1] 屈慧鸽[1] 迟晓艳[1] 

机构地区：[1]鲁东大学生命科学学院,烟台264025

出　　处：《微生物学报》2009年第12期1601-1606,共6页Acta Microbiologica Sinica

基　　金：山东省自然科学基金(Y2005D14);烟台市科技计划项目(2008152);教育部留学回国人员科研启动基金(20071108);鲁东大学学科建设经费~~

摘　　要：【目的】利用SspDnaEintein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用SspDnaEintein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PAGE分析显示预期大小的ABCA1剪接蛋白条带,并进一步为His-Tag抗体进行的Western blotting证实。【结论】结果表明SspDnaEintein可有效催化ABCA1的连接,为进一步研究利用双腺相关病毒(AAV)载体转运ABCA1基因,克服单个AAV载体容量限制进行由ABCA1基因突变所致Tangier病的基因治疗奠定了基础。[ Objective] By exploring Ssp DnaE intein-catalyzed protein trans-splicing we investigated the ligation of expression product of ATP-binding cassette transporter A1 （ABCA1） gene in E. coli. [ Methods] The ABCA1 cDNA was broken into two halves of N-part and C-part before Cys978 codon which meets the splicing required conserved residue, and then fused to 123 and 36 amino acid-containing N terminal and C terminal coding sequences of Ssp DnaE intein naturally occurring trans-splicing intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pET-28a（ ＋ ）. After transformation into E. coli BL2t （DE3） cells followed by induction the expression of recombinant proteins and the ligation of ABCA1 were observed. [ Results] Through IPTG induction for expression of recombinant protein it displayed an obvious protein band as predicted size of ABCA1 on SDS-PAGE gel. Western blotting using His-Tag specific antibody confirmed that this protein band is tram-spliced ABCA1. [ Conclusion] The data demonstrated that Ssp DnaE intein can efficiently catalyze the ligation of ABCA1 providing an evidence for our ongoing study on ABCA1 gene transfer by a dual AAV vector system to circumvent AAV volume limitation in gene therapy of Tangier disease which resulted from ABCA1 gene mutations.

关 键 词：DnaE INTEIN 蛋白质反式剪接 ABCA1 连接 

分 类 号：Q78[生物学—分子生物学]