小鼠FcγR Ⅱb胞外区重组蛋白的原核表达及对小鼠SLE模型的治疗作用研究

Prokaryotic expression of the extracellular portion of mouse FcγR Ⅱb and the functions of the ex- pressed proteins in a mouse model of SLE

目的构建小鼠Fcγ receptor Ⅱb（FcγR Ⅱb ）胞外区蛋白原核表达载体pET-FcγRⅡb，诱导表达并纯化，观察其在系统性红斑狼疮（SLE）疾病中的预防与治疗作用。方法PCR扩增获得小鼠FcγR Ⅱb 基因，并将其定向克隆至原核表达载体pET-32a（＋），构建原核表达重组质粒小鼠pET-FcγR Ⅱb ，经PCR、限制性内切酶鉴定及测序分析正确后，转化大肠杆菌BL21（DE3），IFFG进行诱导蛋白表达，确定最佳诱导条件，经Western blot鉴定正确后，纯化目的蛋白。ELISA法检测纯化蛋白与小鼠血清中免疫复合物的结合能力。将MRL／lpr SLE小鼠预防组（周龄12周左右）和治疗组（周龄19周左右）各40只随机分成蛋白液60μl（4．8μg）、120μl组（9．6μg）、180μl（14．4μg）组以及对照PBS组4组，每组10只，通过尾静脉注射将蛋白液注射进入MRL／lpr SLE小鼠体内，连续注射4周，每周1次。注射完成后，观察1周并收集血清，用ELISA法对预防组与治疗组SLE小鼠的可溶性FcγR Ⅱb的含量以及小鼠抗双链DNA抗体含量进行检测。结果扩增出了小鼠FcγR Ⅱb基因，成功构建原核表达重组质粒小鼠pET—FcγRⅡb，IPTG诱导表达并纯化出重组蛋白。重组蛋白与小鼠血清具有结合力；预防组和治疗组蛋白组（含60μl、120μl和180μl组）分别与预防对照组和治疗对照组相比可溶性FcγRⅡb含量增加（P〈0．05），蛋白组（含60μl、120μl和180μl组）两两比较，差异有统计学意义（P均〈0．05）；小鼠抗双链DNA抗体含量检测结果显示预防组和治疗组蛋白组（含60μl、120μl和180μl组）分别与预防和治疗对照组相比，小鼠抗双链DNA抗体含量降低（P〈0．05）；预防和治疗蛋白组（含60μl、120μl和180μl组）分别进行两两比较后发现随着蛋白注射剂量的增加，小鼠抗双链DNA抗体含量逐渐降低（P〈0．05）。结论获得重组小鼠FcγR Ⅱb胞外区可溶性蛋白，初步证实其对SLE模型鼠具有一定的预防和治疗作用。

Objective To clone and express the extracellular portion of mouse Fcγ receptor Ⅱb （FcγR Ⅱb） and to analyze the functions of the expressed proteins in a mouse model of systemic lupus erythematosus （SLE）. Methods The gene fragment encoding FcγR Ⅱb was amplified by PCR, and then inserted into the prokaryotic expression vector pET-32a（ ＋ ） to construct the recombinant expression plasmid pET-FcγR Ⅱb. The expression plasmid was identified with restriction enzymes and then sent to the Shanghai Bio-Engineering Co. LTD for further sequencing analysis. The transformed Escherichia coli （E. coli） BL21 （ DE3 ） strains carrying the recombinant expression plasmid pET-FcγR Ⅱb were induced by isopropyl β-D-1- thiogalactoside （ IPTG）. The expressed fusion proteins were analyzed by Western blot assay and purified with purification kits. The immune complex （IC） -binding ability of FcγR Ⅱb was measured by ELISA. MRL/lpr mice with SLE in both the prevention （12 weeks old, n=40） and the treatment groups （19 weeks old, n= 40） were randomly divided into four groups including 60 μl （4.8 μg） treatment group, 120 μl （9.6 μg） treatment group, 180 μl （ 14.4 μg） treatment group and PBS treatment group with 10 mouse in each group.The MRL/Ipr mice with SLE were injected with the fusion protein through tail vein once a week for four consecutive weeks. Serum samples were collected from each mouse after one week of observation. The levels of FcγR Ⅱb in soluble form in mice form both the prevention and treatment groups as well as the levels of anti- double stranded DNA antibodies were detected by ELISA. Results The gene encoding FcγR Ⅱb was amplified and the recombinant expression plasmid pET-FcγR Ⅱb was successfully constructed. The recombinant proteins were expressed in the prokaryotic expression system, and then successfully purified. The recombinant proteins could bind to IC. Compared with the corresponding PBS control group, the levels of FcγR Ⅱb in soluble form were increased in mice from both prevention and treatment groups after treating with various concentrations of the recombinant protein （P〈0.05）. Significant differences in the levels of FcγR Ⅱb were found among mice of the same age after treating with different concentrations of the recombinant protein （P〈 0.05）. Compared with the corresponding PBS control group, the levels of anti-double stranded DNA antibodies were decreased in mice from both prevention and treatment groups after treating with various concentrations of the recombinant protein （ P〈0. 05 ）. The levels of anti-double stranded DNA antibodies were gradually decreased along with the increasing dosage of protein （P〈0.05）. Conclusion The extracellular portion of murine FcγR Ⅱb in soluble form was successfully expressed. The recombinant proteins played a certain role in the prevention and treatment of SLE in a mouse model.