CAJ | 학술논문

目的：通过原核细胞表达和纯化9R-GFP-PHD重组蛋白，并利用纯化后的9R-GFP-PHD蛋白来检测与磷脂酰肌醇4，5-二磷酸（PIP2）和 IP3的结合能力以及细胞膜上 PIP2的动态变化。方法通过分子克隆技术将磷脂酶 C-δ1的普列克同源域（PHD）、荧光蛋白 GFP及细胞穿膜多肽9R融合以构建相应蛋白表达载体。利用原核表达体系 BL21大肠埃希菌和镍柱进行重组蛋白的表达和纯化，其中 GFP和9R-GFP为9R-GFP-PHD的对照蛋白。获取重组蛋白后，通过同位素实验和液体闪烁计数器，分别检测和比较 GFP、9R-GFP和9R-GFP-PHD与[3H]标记的 PIP2和 IP3的结合能力以及之间的竞争性抑制。另外，利用MDCK细胞检测和比较孵育的 GFP、9R-GFP和9R-GFP-PHD后，3种重组蛋白在细胞内的分布情况。通过图片相减处理和荧光实时定量分析 MDCK细胞膜上的9R-GFP-PHD在 ATP刺激后分布的动态变化，从而间接反映细胞膜上 PIP2的水解变化。结果通过原核表达和镍柱纯化体系顺利获得了GFP、9R-GFP和9R-GFP-PHD重组蛋白。体外结合实验证实9R-GFP-PHD与PIP2和IP3均有很强的结合能力，而且IP3能竞争性抑制9R-GFP-PHD与PIP2的结合。在孵育了3种荧光融合蛋白后，荧光共聚焦显微镜观察发现9R-GFP 主要分布在细胞质中，而9R-GFP-PHD特异性分布于细胞膜上。荧光实时定量分析显示，ATP刺激通过P2y受体激活磷脂酶C以水解 PIP2，从而使 MDCK细胞膜上的9R-GFP-PHD减少20％左右。结论本研究中构建的9R-GFP-PHD利用了细胞穿膜多肽、PHD与 PIP2和 IP3结合特性及 GFP 的荧光可视和定量分析特性，有效地检测细胞膜上PIP2的动态变化，将为今后进一步研究细胞内钙动员提供新的工具蛋白。
목적：통과원핵세포표체화순화9R-GFP-PHD중조단백，병이용순화후적9R-GFP-PHD단백래검측여린지선기순4，5-이린산（PIP2）화 IP3적결합능력이급세포막상 PIP2적동태변화。방법통과분자극륭기술장린지매 C-δ1적보렬극동원역（PHD）、형광단백 GFP급세포천막다태9R융합이구건상응단백표체재체。이용원핵표체체계 BL21대장애희균화얼주진행중조단백적표체화순화，기중 GFP화9R-GFP위9R-GFP-PHD적대조단백。획취중조단백후，통과동위소실험화액체섬삭계수기，분별검측화비교 GFP、9R-GFP화9R-GFP-PHD여[3H]표기적 PIP2화 IP3적결합능력이급지간적경쟁성억제。령외，이용MDCK세포검측화비교부육적 GFP、9R-GFP화9R-GFP-PHD후，3충중조단백재세포내적분포정황。통과도편상감처리화형광실시정량분석 MDCK세포막상적9R-GFP-PHD재 ATP자격후분포적동태변화，종이간접반영세포막상 PIP2적수해변화。결과통과원핵표체화얼주순화체계순리획득료GFP、9R-GFP화9R-GFP-PHD중조단백。체외결합실험증실9R-GFP-PHD여PIP2화IP3균유흔강적결합능력，이차IP3능경쟁성억제9R-GFP-PHD여PIP2적결합。재부육료3충형광융합단백후，형광공취초현미경관찰발현9R-GFP 주요분포재세포질중，이9R-GFP-PHD특이성분포우세포막상。형광실시정량분석현시，ATP자격통과P2y수체격활린지매C이수해 PIP2，종이사 MDCK세포막상적9R-GFP-PHD감소20％좌우。결론본연구중구건적9R-GFP-PHD이용료세포천막다태、PHD여 PIP2화 IP3결합특성급 GFP 적형광가시화정량분석특성，유효지검측세포막상PIP2적동태변화，장위금후진일보연구세포내개동원제공신적공구단백。
Objective To utilize the purified 9R-GFP-PHD protein to detect the combining capacity with PIP2/IP3 and the dy-namic change of PIP2 on cellular membrane by the expression of the prokaryotic cell and the purificatiojn of 9R-GFP-PHD recombi-nant protein.Methods The molecular cloning technique was adoted to conduct the fusion of pleckstrin homology domain(PHD)of phospholipase C-δ1,fluorescent protein GFP and cell-penetrating peptide 9R for constructing the corresponding protein expression vector.The expression and purification of recombinant protein were conducted by using the prokaryotic system BL2 1 Escherichia coli and nickel column,in which GFP and 9R-GFP were the control protein of 9R-GFP-PHD.After obtaining the recombinant pro-tein,the binding affinities of GFP,9R-GFP and 9R-GFP-PHD with[3H]labeled PIP2 or IP3 were measured and compared by the iso-tope experiment and the liquid scintillation counter.In addition,the MDCK cells were utilized to detect and compare the distribution situation of 3 kinds of recombinant protein in cells after incubating GFP,9R-GFP and 9R-GFP-PHD.The image subtraction pro-cessing and the real-time fluorescent quantitative analysis was used to examine the dynamic change of distribution of 9R-GFP-PHD in the MDCK cell membrane after ATP stimulation,thus indirectly reflected the hydrolysis change of PIP2 in the cell membrane. Results GFP,9R-GFP and 9R-GFP-PHD recombinant proteins were smoothly obtained by the prokaryotic expression and nickel column purification system.The in vitro binding experiments showed that 9R-GFP-PHD had high binding affinity with PIP2 and IP3.After incubation of three kinds of fluorescent fusion protein,the confocal fluorescent microscopic observation found that 9R-GFP was mostly distributed in plasma,while 9R-GFP-PHD was specifically distributed on cellular membrane.The real-time fluores-cence quantitative analysis showed that ATP stimulation activated phospholipase C for hydrolyzing PIP2 by P2y receptor,thus 9R-GFP-PHD on the MDCK membrane was deceased by about 20%.Conclusion The constructed 9R-GFP-PHD utilizes the cell-pene-trating peptide,PHD,PIP2/IP3 binding features,fluorescent visuality and the quantitative analytic characteristics of GFP to effec-tively detect the dynamic change of PIP2 on cellular membrane,which provides a new tool protein for further study of intracellular calcium mobilization.