乙型肝炎病毒X抗原结合蛋白1的原核表达及亚细胞定位

Subcellular location and prokaryotic expression of a novel binding protein 1 of HBxAg

目的应用酵母双杂交及生物信息学技术筛选乙型肝炎病毒X抗原结合蛋白1(HBXBP1)并进行克隆,探讨其在大肠埃希菌BL21中表达及亚细胞定位。方法应用反转录PCR(RT-PCR)技术,以HepG2细胞mRNA为模板,扩增获得HBXBP1基因片段,连接到pGEM-T载体,双酶切鉴定及测序正确后克隆至原核表达载体pET-32a(+)中,转化大肠埃希菌BL21,通过IPTG诱导表达以获得HBXBP1融合蛋白,并经SDS-PAGE及Western blot分析证实融合蛋白的特异性;构建HBXBP1基因的绿色荧光蛋白表达质粒pEGFP-C1-HBXBP1,转染HepG2细胞,24h后于荧光显微镜下观察表达蛋白的亚细胞定位。结果 RT-PCR扩增获得921bp的HBXBP1基因片段,插入原核表达载体pET-32a(+)中,转化大肠埃希菌BL21,经IPTG诱导成功获得约56kD的重组蛋白,经Western blot分析证实其具有良好的特异性;成功构建HBXBP1基因的绿色荧光蛋白表达质粒pEGFP-C1-HBXBP1,其表达蛋白定位于细胞质。结论 pET-32a(+)-HBXBP1原核表达载体在大肠埃希菌BL21中诱导表达HBXBP1融合蛋白;HBXBP1基因可表达HBXBP1蛋白且定位于细胞质,为研究HBXBP1蛋白的免疫原性和生物学特性奠定了一定的理论基础。

Objective To clone the human gene of hepatitis B virus X antigen binding protein 1 （HBXBP1）, which was screened with yeast two-hybrid system and bioinformatics techniques,and to construct prokaryotic expression vector of pET-32a（＋）-HBXBP1, and to discuss the expression of recombinant protein in E.coli BL21 and subcellular location. Methods The DNA fragment of HBXBP1 with about 921 bp was amplified by reverse transcription PCR （RT-PCR）, taking the mRNA extracted from HepG2 cells as the template, and cloned into pGEM-T vector. After identified by restriction enzyme digestion and sequenced, the correct target DNA fragment was inserted into inducible prokaryotic expression vector pET-32a（＋） and then transformed into competent E.coli BL21. After analyzed by restriction enzyme digestion and PCR, the positive transformed clones were identified and induced with IPTG to obtain fusion protein. The HBXBP1 fusion protein was analyzed by Western blot hybridization. Green fluorescent protein （GFP） expression vector pEGFP-HBXBP1 was constructed and transfected into HepG2 cells and the subcellular location of the proteins expressed by HBXBP1 were analyzed through green fluorescent microscopy after 24 hours. Results The 921 bp DNA fragment of HBXBP1 was amplified by RT-PCR. The recombinant expression vector pET-32a（＋）-HBXBP1 was constructed successfully. After transformated with pET-32a（＋）-HBXBP1 and inducted with IPTG, the recombinant target protein with about 56 kD was obtained, which was consistent with our anticipation and was specific varified by Westem blot assay. The pEGFP-HBXBP1 vector was successfully cloned, HBXBP1 protein was expressed in cells and subcellularly located in cytoplasm. Conclusions recombinant prokaryotic expression vector pET／｜32a（＋）／｜HBXBP1 was constructed, and the HBXBP1 gene was cloned successfully. The HBXBP1 fusion protein could be expressed in prokaryotic expression system of [WTBX]E.coli[WTBZ]. HBXBP1 gene could express HBXBP1 protein which locates in cytoplasm subcellularly. These results lays a foundation for studying on the immunogenicity and biological characteristics of HBXBP1 protein.