结核病(tuberculosis，TB)流行病快速筛查常采用结核菌素纯蛋白衍生物(pure protein derivative，PPD)皮试，但特异性低，不良反应大。ESAT-6和CFP-10是结核分枝杆菌的分泌蛋白，但卡介苗(Bacillus Calmette-Guerin，BCG)中缺失了相关基因。本研究表达纯化了结核分枝杆菌的分泌蛋白ESAT6和CFP10的融合蛋白(ESAT6 and CFP10 fusion protein，EC)，并制备成其壳聚糖纳米粒(ESAT6 and CFP10 fusion protein nanoparticals，EC-NPs)，进一步制备成透明质酸可溶性微针贴片(microneedle patch，MNP)。MNP-EC-NPs载药量达到每针0。03 μg，每片可达到1。92 μg，常温下保存期可达6个月，可有效透过表皮。志愿者皮试结果显示MNP-EC-NPs可有效区分BCG接种者，在结核病患者皮肤有效显示阳性反应，无显著不良反应，实验经武汉市肺科医院伦理委员会批准[武肺伦理2022(2)]。本研究建立了一种结核病皮试方法，采用ESAT6-CFP10融合蛋白替代PPD，并采用可溶性微针剂型，提高了结核病皮试诊断的特异性，为结核流行病筛查提供了新的技术方案。
Preparation of soluble microneedle patch with fusion protein nanoparticles secreted by Mycobacterium tuberculosis and application of tuberculosis skin test
Rapid epidemiological screening for tuberculosis(TB)usually uses tuberculin pure protein derivative(PPD)skin test,which has limitations such as low specificity and high side effects.ESAT-6 and CFP-10 are secreted proteins of Mycobacterium tuberculosis,but the related genes are missing from Bacillus Calmette-Guerin(BCG).In this study,the fusion protein ESAT6-CFP10(EC)was expressed and purified,and prepared into chitosan nanoparticles(EC-NPs),which were loaded into the microneedle patch and carried out the preliminary test of tuberculosis skin test.The drug loading capacity of MNP-EC-NPs(microneedle patch,MNP)can reach 0.03 μg per needle and 1.92 μg per patch.The shelf life of MNP-EC-NPs can reach 6 months at room temperature,and it can effectively penetrate the epidermis.Volunteer skin test results showed that MNP-EC-NPs can effectively distinguish between BCG vaccinators,and can effectively show positive reaction in the skin of tuberculosis patients without significant side effects.The experiment was approved by the Ethics Committee of Wuhan Pulmonary Hospital[2022(2)].In this study,a TB skin test method was established,using ESAT6-CFP10 fusion protein instead of PPD,and using soluble microneedle dosage form,which improved the specificity of TB skin test diagnosis and provided a new technical scheme for TB epidemic screening.
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