鼠疫F1-V融合蛋白在大肠杆菌中的高效表达及鉴定

High-level expression of Yersinia pestis F1-V fusion protein in Escherichia coli and its identification

目的通过构建pET-11c-F1-V融合表达质粒,从而获得具有免疫原性的融合蛋白。方法克隆鼠疫耶尔森氏菌(Yersinia pestis)的F1基因和V基因,然后将F1基因和V基因分别连接到pGEM-T载体上,经测序正确后再将F1基因和V基因连接到pET-11c原核表达载体上,构建pET-11c-F1-V融合表达质粒,并转化到BL21(DE3)大肠杆菌中,进行PCR及双酶切鉴定,筛选阳性克隆,通过IPTG诱导F1-V表达,经SDS-PAGE检测表达产物,通过Western-Blot检测重组蛋白的免疫原性。结果测序结果显示F1基因的碱基序列与Gene-bank(X 61996)完全一致,V基因的碱基序列与Gene-bank(M 26405)相比在564 bp处有一同义突变;SDS-PAGE在Mr约58 000处出现一条蛋白条带,经薄层扫描分析目的蛋白条带约占全菌体蛋白的25％左右,主要以可溶性蛋白形式存在;Western-Blot显示重组蛋白具有免疫原性。结论成功地构建了pET-11c-F1-V融合表达质粒,并且在大肠杆菌中获得了高效表达,表达的蛋白具有免疫原性。

Objective To construct pET-llc-Fl-V fusion expression plasmid in order to get fusion protein with immunogenicity. Methods Fl gene and V gene of Yersinia pestis were cloned and inserted into pGEM-T vector. After sequence correction, the recombinant expression plasmid pET-11c-F1-V was constructed by inserting the DNA fiagment of Yersinia pestis F1 gene and V gene into pET-11c and transformed into E. coli BL21 (DE3) cell. The positive clones were selected by PCR and enzymes digest and, the expression product of Fl gene induced by IPTG was detected by SDS-PAGE, while the expression protein immunogenicity was detected by Western-blot. Results Sequence analysis revealed that DNA sequence of Fl gene was as same as that of Gene-bank record (X 61996). DNA sequence of V gene was mutanted at the site of 546 bp, but encoded same ammo acid. An expression band about Mr 58 000 was found by SDS-PAGE. The aim protein, most of it was in soluble form, was about 25% of total cell protein. While the immunogenicity of expression protein was detected by Western-blot. Conclusion pET-llc-Fl-V is successfully constructed and its high-level expression in Escherichia. coli is attained. The aim protein has immunogenicity .