重组结核分枝杆菌 ESAT6-CFP10蛋白与 PPD 皮肤试验的动物实验比较

A comparative evaluation of recombinant Mycobacterium tuberculosis ESAT6-CFP10 and PPD as the antigen ci reagents for skin test in guinea pigs

目的：动物实验中评价重组结核分枝杆菌ESAT6-CFP10蛋白（以下简称：重组ESAT6-CFP10蛋白）皮肤试验情况，同时与PPD（结核菌素纯蛋白衍生物）皮肤试验进行比较。方法以不同分枝杆菌分别致敏豚鼠，致敏成功后每只豚鼠皮内分别注射重组ESAT6-CFP10蛋白和PPD。用双盲法记录注射后48 h局部皮肤反应的纵径和横径，计算其均值。以结核分枝杆菌活菌感染豚鼠，在感染后不同时间内选取一定只数的豚鼠，皮内分别注射重组ESAT6-CFP10蛋白和PPD，记录注射后24 h局部皮肤反应的纵径和横径，计算其均值，并统计两种皮试反应的阳转率情况。结果在结核分枝杆菌活菌、结核分枝杆菌死菌和卡介苗致敏豚鼠中，PPD皮肤试验均为阳性，局部硬结直径分别为（11．4±0．9） mm、（11．8±1．1） mm和（13．2±0．8） mm。在结核分枝杆菌活菌致敏豚鼠中，重组ESAT6-CFP10蛋白皮肤试验为阳性，局部硬结直径为（13．7±5．7） mm；在结核分枝杆菌死菌和卡介苗致敏豚鼠中，重组ESAT6-CFP10蛋白皮肤试验均为阴性，局部硬结直径为0。结核分枝杆菌H37 Rv感染豚鼠后，第9天开始随机选取相应只数的豚鼠进行重组 ESAT6-CFP10蛋白和PPD的皮肤试验。第2周时24只豚鼠全部进行皮试试验，ESAT6-CFP10蛋白全部阳转，阳转率为24/24，硬结反应平均值为（19．9±3．0） mm；而PPD的阳转率仅为15/24，硬结反应平均值为（6．1±5．5） mm。第4周时PPD开始全部阳转，阳转率为33/，硬结反应平均值为（12．7±2．5） mm。结论重组ESAT6-CFP10蛋白皮肤试验能够鉴别卡介苗接种和结核分枝杆菌死菌、活菌感染，并且较PPD能够早期进行诊断。

Objective To comparatively evaluate the effects of a recombinant Mtb protein ESAT 6-CFP10 （ rESAT6-CFP10 ） and a purified protein derivative （ PPD ） as skin test reagents in guinea pigs . Methods Guinea pigs were sensitized with different Mycobacteria species .After sensitization , all guinea pigs were intradermally injected with rESAT6-CFP10 and PPD.At 48 h after the injection, the size of ery-thema at injection sites was measured by using a double-blind method .For guinea pigs sensitized with viable Mtb, the size of erythema at injection sites were measured at 24 h after the injection .The positive conversion rates of skin test with rESAT 6-CFP10 and PPD were calculated .Results The results of PPD skin test were positive in all guinea pigs sensitized with viable Mtb , killed Mtb and BCG with erythema diameters of （11.4 ±0.9） mm, （11.8±1.1） mm and （13.2±0.8） mm, respectively.Positive skin test with rESAT6-CFP10 was only observed in guinea pigs infected by viable Mtb-showing erythema diameters of （13.7±5.7） mm. The skin test with rESAT6-CFP10 was negative in guinea pigs sensitized by killed Mtb-and vaccinated by BCG.The skin tests by using rESAT6-CFP10 and PPD were performed on randomly selected guinea pigs at ninth day after infection by Mycobacterium tuberculosis H37Rv.At the 2nd week, totally 24 selected guinea pigs showed positive skin test results with rESAT6-CFP10 （24/24） with erythema diameters of （19.9± 3.0） mm, while only 15 out of 24 had positive PPD skin test with erythema diameters of （6.1±5.5） mm. At the 4th week, all guinea pigs showed positive PPD skin test （3/3） with erythema diameters of （12.7± 2.5） mm.Conclusion The skin test by using recombinant ESAT 6-CFP10 protein can effectively distin-guish viable Mtb infection from BCG vaccination and killed Mtb sensitization , which is a more suitable anti-gen than PPD for the early diagnosis of Mtb infection .