重组Kinectin蛋白的表达、纯化及应用研究

Expression,purification and the application research of the recombinantion Kinectin protein

目的优化重组kinectin蛋白的诱导表达及纯化条件;探讨用其致敏的树突状细胞(DC)对自体T淋巴细胞增殖能力的影响。方法以pMAL-C2为载体构建重组质粒,并转入TB1宿主菌。从IPTG加入时机、诱导温度、诱导时间及IPTG浓度方面对工程菌的诱导条件进行优化;过Amylose-resin亲和层析柱对诱导产物进行分离纯化。用获取的MBP-kinectin融合蛋白致敏从人外周血中分离培养的DC,并用ELISA检测细胞上清液中的IL-12、IFN-γ含量;再用MTT法检测致敏DC刺激自体T淋巴细胞增殖的能力。结果 工程菌在37℃振荡培养2个小时后加入终浓度为0.5mmol/L的IPTG,降低温度到34℃继续诱导3个小时,可明显提高诱导效率;所得MBP-kinectin与DC共培养上清液中IL-12、IFN-γ含量分别为(615.32±7.93)pg/ml,(544.28±7.17)pg/ml,用其刺激的T淋巴细胞增殖指数为53.14±0.62,均高于麦芽糖结合蛋白(MBP)组和空白对照组,且差异有统计学意义。结论pMAL-C2载体可用于构建kinectin重组质粒;通过对诱导条件的优化,可获得较高的诱导效率;所得MBP-kinectin融合蛋白能致敏DC并具有很强的刺激自体T淋巴细胞增殖的能力。

Objective Optimize the method to express and purify the recombinant kinectin protein. Explore the effect to the proliferation activity of auto-T-lymphocyte co-incubation with dendritic cell（DC） sensitized by the recombination protein. Methods Constructed the recombination plasmid based on the pMAL-C2. Then transfected it into the expression host （E. coli TB1） and optimized the induction condition, include the opportunity to add the IPTG, temperature,induction time and the concentration of IPTG. Then purified the induced production throught the Amylose-resin column to obtain the MBP-kinectin protein, the protein was used to sensitized the DC induced and expanded from normal human peripheral blood mononuclear cells（PBMCs）, and detected the content of IL-12, IFN-γin the supernatant fluid. Then used the MTT method to detect the proliferation activity of auto-T-lymphocyte stimulated by thesensitized DC. Results After the engineering bacteria cultivated in 37℃ for 2 hours, IPTG was added to the concentration of 0.5 mmol/L, then reduced the temperature to 34℃ and continues to cultivate for 3 hours can help to improve the production of the dissoluble interest protein; The concentration of IL-12,IFN-γin the supernatant of the MBP-kinectin and DC co-culture fluid are （615.32 ± 7.93） pg/ml, （544.28 ± 7.17） pg/ml respectively, and the proliferation index of auto-T-lymphocyte stimulated by the sensitized DC is 53. 14±0. 62. All the above index are higher than the MBP group and the blank group,and the difference are statistical significance. Conclusion pMAL-C2 can be used to recombinate and express the gene segment of kinectin in vitro. Higher induction efficiency can be obtained after optimized the induction conditions. The recombination protein can sensitize DC and then strongly stimulate the proliferation activity of auto-T-lymphocyte.