海分枝杆菌菌壁蛋白组特性及对Wistar大鼠致病免疫学特征研究

Study on the character of mycobacterium marinum protein and immunology to wistar rats

目的探讨海分枝杆菌菌壁蛋白组特性及对Wistar大鼠致病免疫学特征,提高对海分枝杆菌致病机制的认识。方法 （1）将临床分离的海分枝杆菌经过培养7∽10 d后,挑取培养的菌落,加入到0.9%氯化钠液体中,离心、PBS冲洗、超声破碎制备菌壁蛋白。将制备好的样品蛋白进行双向电泳,凝胶图像分析。（2）应用McFarland比浊法,制备菌液,估计所配制的细菌剂量浓度。选取雄性Wistar大鼠45只,数字随机法分成3组：实验组A和B、对照组,每组15只。取菌液0.1 ml（浓度：3×108cfu/ml）经尾静脉注射感染A组,腹腔注射感染B组,对照组经尾静脉注射0.9%氯化钠0.1 ml液体。感染第4周后抽取尾静脉血液1 ml,采用ELISA法分别检测3组大鼠血清中sIL-2R、TNF-α和IFN-γ表达。结果 （1）海分枝杆菌菌壁蛋白分子质量处于10∽100 kD,PI值主要分布于4.5∽6.0之间,在pH 4.5∽5.5之间蛋白点分布比较密集,主要集中在偏酸性侧。（2）实验组A和B大鼠血清中sIL-2R、TNF-α和IFN-γ表达无统计学差异（实验组A和B之间,t=1.39∽1.91,P=0.065∽0.174）,分别与对照组比较均有统计学差异（A组与对照组比较,t=5.98∽24.08,P〈0.05;B组与对照组比较,t=3.79∽27.62,P〈0.05）。结论海分枝杆菌菌壁致病性蛋白在10∽100 kD之间,Wistar大鼠全身感染4周后可引起白细胞介素表达增强。

Objective To explore the character of Mycobacteriurn marinum（M, marinum）germ wall protein and the immunology to Wistar rats, as well as improve the understanding of the pathogenic mechanism of M. marinum. Methods （1）The colonys of the clinical isolates of M. marinum were picked up and cultured for 7 to 10 days, added to 0.9,4 sodium chloride liquid, centrifuged and washed with PBS fluid, then extracted bacterial wall protein broken by ultrasound. We analyzed the M. marinum protein by two-dimensional electrophoresis and gel-image. （2） Germ liquid was made by McFarland nephelometry. Forty-five male Wistar rats were selected, and divided into three groups by numeral random, including experiment group A and B, and control group. Every group has fifteen rats. Group A were infected with 0.1 ml germ-fluid （germ density 3x108 cfu/ml） via tail vein, while Group B by intraperitoneal injection, but the control group only accepted 0.1 ml normal saline via tail vein. Took 1 ml blood from tail vein after infected four weeks, and assayed the expression of slL-2R, TNF-ct and IFN-7 respectively in serum by ELISA. Results （1）The molecular weight of M. marinum＇s germ wall-held proteins was between 10-100 kD, the Isoelectric Point（P1）was between 4.5-6.0. The protein points concentrate at pH 4.5-5.5, mainly in acid side. （2）The expressions of slL-2R, TNF-α and IFN-T had no statistical difference between group A and B（tA.R=l.39-1.91, P=0.065-0.174）, but all of them had significant difference compared with the control group respectively（tA=5.98-24.08, P〈0.05; tB=3.79-27.62, P〈0.05）. Conclusion The pathogenic germ wall-held proteins of M. marinum are about 10-100 kD, and the expression of interleukins will increase after infected four weeks in Wistar rats.