构建δ-睡眠肽(DSIP)蛋白与GFP的融合基因表达载体,高效表达和纯化GFP-DSIP融合蛋白.通过SOE-PCR拼接DSIP全长编码基因,并使得DSIP上游具有肠激酶识别位点,经双酶切定向克隆至表达载体pET-28a,构建重组载体pET-28a-DSIP,通过PCR扩增GFP全长编码基因,经双酶切定向克隆至pET-28a-DSIP,构建原核重组表达载体pET-28a-GFP-DSIP,通过双酶切和测序鉴定后,导入E.coli BL21宿主菌中,IPTG诱导表达融合蛋白,采用镍亲和层析和分子筛凝胶层析获得高纯度蛋白,SDS-PAGE分析鉴定.经测序鉴定成功构建了原核重组表达载体pET-28a-GFP-DSIP,在1PTG诱导下获得可溶性的绿色荧光蛋白与睡眠肽的融合蛋白,经Ni-NTA亲和层析纯化成功获得高纯度的融合蛋白.成功构建了DSIP与GFP融合基因的重组表达载体,确定了GFP-DSIP融合蛋白诱导表达的最佳条件,获得了较高纯度的融合蛋白,为进一步研究DSIP蛋白的生物学功能奠定了基础.%The expression vector of delta sleep inducing peptide ( DSIP) and fusion gene of GFP to highly efficiently express and purify CFP-DSIP fusion protein was constructed. The whole length encoding gene of DSIP was joined through SOE-PCR and made the upstream of DSIP possessing an enterokinase recognition site and directionally cloned into an expression vector pET-28a by double enzyme digestion to construct a recombinant expression vector pET-28a-DSIP. The whole length of CFP encoding gene was amplified with PCR, and directionally cloned into pET-28a-DSIP by double enzyme digestion to construct procaryotic recombinant expression vector of pET-28a-GFP-DS!P. After double enzyme digestion and sequence test identification it was induced into host strain E. Coli BL21; and IPTC induced expressed the fusion protein. And obtained high purity protein adopting Ni-NTA affinity chromatography and molecular sieve gel chromatography, and then analyzed and identified by SDS-PAGE. The results showed that the procaryotic expression vector of pET-28a-GFP-DSIP was successfully constructed identified by sequencing; under the induction of IPTG obtained a fusion protein of soluble green fluorescent protein and sleep inducing peptide. And successfully obtained high purity fusion protein adopting Ni-NTA affinity chromatography purification. The recombinant prokaryotic expression vector of fusion gene of DSIP and GFP was successfully constructed, the optimal conditions for induced expression of GFP-DSIP fusion protein was confirmed, and obtained fairly high purity fusion protein. It laid a foundation for further studies on the biological function of DSIP protein.
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