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Article(id=1200500176805818839, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0895, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1690128000000, receivedDateStr=2023-07-24, revisedDate=1711555200000, revisedDateStr=2024-03-28, acceptedDate=null, acceptedDateStr=null, onlineDate=1764151146365, onlineDateStr=2025-11-26, pubDate=1718121600000, pubDateStr=2024-06-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764151146365, onlineIssueDateStr=2025-11-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764151146365, creator=13701087609, updateTime=1764151146365, updator=13701087609, issue=Issue{id=1200500165426672625, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='6', pageStart='1509', pageEnd='1896', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764151143651, creator=13701087609, updateTime=1764225143180, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200810542001680840, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200810542001680841, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200500165426672625, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1804, endPage=1811, ext={EN=ArticleExt(id=1200500177724371518, articleId=1200500176805818839, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Preparation of soluble microneedle patch with fusion protein nanoparticles secreted by Mycobacterium tuberculosis and application of tuberculosis skin test, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

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.

, correspAuthors=Jian-hua KANG, Wei WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Fan CHEN, Rong-sheng ZHU, Jing ZHOU, Yue HU, Yun XUE, Jian-hua KANG, Wei WANG), CN=ArticleExt(id=1200500181084009254, articleId=1200500176805818839, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=结核分枝杆菌分泌蛋白融合蛋白纳米粒可溶性微针贴片的制备及结核病皮试的应用, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

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。

, correspAuthors=康建华, 王伟, authorNote=null, correspAuthorsNote=
*康建华, Tel: 86-796-85339220, E-mail: ;
王伟, Tel: 86-769-86229999-3276, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2024, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=5YNx+l55hMzDGsoOLlyX2Q==, magXml=rwuW13RAXPWBFslsQ7LjhA==, pdfUrl=null, pdf=3vkOo3nveHC76P45mWKmXA==, pdfFileSize=2140334, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=DK2+wl1vrNzV1PxycNq8jw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=DjUVuWVBPHPqcEzXpfEX2A==, mapNumber=null, authorCompany=null, fund=null, authors=

#共同第一作者.

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Department of Laboratory Medicine, Dongguan Chang′an Hospital, Dongguan 523843, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1201118425973223685, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, authorId=1201118425708982509, language=CN, stringName=康建华, firstName=建华, middleName=null, lastName=康, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=4, *, address=4.东莞市长安医院检验科, 广东 东莞 523843, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1201118422114463762, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, xref=null, ext=[AuthorCompanyExt(id=1201118422122852371, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, companyId=1201118422114463762, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4. 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A: PCR amplification of ESAT6 and CFP10 genes; B: Overlap-PCR amplification of ESAT6-CFP10; C: Recombinant vector double enzyme digestion identification; D: The expression of ESAT6-CFP10 fusion protein was optimized by orthogonal test. 1, 2 and 3 were the sample supernatant obtained by 0.5 mmol·L<sup>-1</sup> IPTG induced expression at 37, 30 and 26 ℃ for 12 h, respectively; E: 1, 2, 3 and 4 are ESAT6-CFP10 target protein effluents eluted by 50 mmol·L<sup>-1</sup> imidazole; 5 and 6 are ESAT6-CFP10 target protein effluents eluted by 80 mmol·L<sup>-1</sup> imidazole; F: Results of purification of ESAT6-CFP10 target proteins by molecular sieve, 1 and 2 are the protein samples with the first peak value detected by UV spectrophotometer, 3, 4, 5 and 6 are the protein samples with the second peak value detected by UV spectrophotometer; G: Determination of Bradford standard curve of ESAT6-CFP10 target protein; H: SDS-PAGE (left) and Western blot detection of purified fusion protein (right). M: Marker , figureFileSmall=9D1HWCN/XYqu+HLj6V7kLg==, figureFileBig=iSSP3IB6cILf7JHW5aOjUw==, tableContent=null), ArticleFig(id=1201118429559353826, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=EN, label=null, caption=null, figureFileSmall=rFgWSnJS3kWoNAjA8ws2zg==, figureFileBig=p8c00RPzGWCAjWkex7MgWw==, tableContent=null), ArticleFig(id=1201118429689377260, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=CN, label=Figure 2, caption= Preparation of fusion protein nanoparticles. A, B: The effects of different Cs/TPP mass ratio on the size and zeta potential of chitosan nanoparticles; C, D: The effects of pH of different Cs solution on the size and zeta potential of chitosan nanoparticles; E, F: The effects of different concentrations of fusion proteins on the encapsulation efficiency and drug loading of protein chitosan nanoparticles. Cs: Chitosan; TPP: Sodium tripolyphosphate , figureFileSmall=rFgWSnJS3kWoNAjA8ws2zg==, figureFileBig=p8c00RPzGWCAjWkex7MgWw==, tableContent=null), ArticleFig(id=1201118429798429173, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=EN, label=null, caption=null, figureFileSmall=7A88XrhoOqPUG+4t05QSWg==, figureFileBig=7DKHbkY86i8wpO0Ok63dUQ==, tableContent=null), ArticleFig(id=1201118429941035519, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=CN, label=Figure 3, caption= Characterization of ESAT6-CFP10 fusion protein chitosan nanoparticles. A: Appearance of fusion protein chitosan nanoparticle solution; B, C: The particle size and potential diagram of fusion protein chitosan nanoparticles, respectively; D: Transmission electron microscope image of fusion protein chitosan nanoparticles; E: Standard curve of protein; F: Cumulative release rate of fusion protein chitosan nanoparticles , figureFileSmall=7A88XrhoOqPUG+4t05QSWg==, figureFileBig=7DKHbkY86i8wpO0Ok63dUQ==, tableContent=null), ArticleFig(id=1201118430213665299, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=EN, label=null, caption=null, figureFileSmall=GUhofEp/sSJJhhVVj9UAtQ==, figureFileBig=3PPTS8sEpkHMQlgJbi2olQ==, tableContent=null), ArticleFig(id=1201118430364660258, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=CN, label=Figure 4, caption= Preparation and transdermal determination of the microneedle patch. A: Prepared microneedle patch; B: Microneedle array (8×8); C: Microscopic morphology of a single microneedle (40×); D: Microneedle patch loaded with Rhodamine dye; E: Microneedle array was formed after the microneedle patch containing Rhodamine was transdermal on the skin of mice; F: HE staining was used to observe the pores formed by microneedles through the skin of mice. HE: Hematoxylin-eosin , figureFileSmall=GUhofEp/sSJJhhVVj9UAtQ==, figureFileBig=3PPTS8sEpkHMQlgJbi2olQ==, tableContent=null), ArticleFig(id=1201118430612124204, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=EN, label=null, caption=null, figureFileSmall=Q86E1U03WbYtVYWoIXlw/w==, figureFileBig=qhqNCj2dgk9NbIafxdpZPQ==, tableContent=null), ArticleFig(id=1201118430775702071, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=CN, label=Figure 5, caption= Measurement of drug loading and shelf life of drug-loaded microneedle patch (MNP). A: The content of ESAT6-CFP10 fusion protein in 10 microneedles. 1, 2 and 3 represent three independent repeated trials, respectively; B: Relative content of ESAT6-CFP10 fusion protein in the microneedle patch at different time points stored at room temperature , figureFileSmall=Q86E1U03WbYtVYWoIXlw/w==, figureFileBig=qhqNCj2dgk9NbIafxdpZPQ==, tableContent=null), ArticleFig(id=1201118431027360319, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=EN, label=null, caption=null, figureFileSmall=BBaMLs4eyKbgPxjuLrV00g==, figureFileBig=SPEC/eZBco5FQ6d4vXJxHg==, tableContent=null), ArticleFig(id=1201118431216104016, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=CN, label=Figure 6, caption= Skin test of volunteers. A: PPD skin test of BCG vaccination volunteers; B: BCG vaccination volunteers showed negative in MNP-EC-NPs skin test; C: PPD skin test of tuberculosis patients; D: Results of MNP-EC-NPs skin test in TB patients. PPD: Pure protein derivative; BCG: Bacillus Calmette-Guerin; EC-NPs: ESAT6 and CFP10 fusion protein nanoparticals; TB: Tuberculosis , figureFileSmall=BBaMLs4eyKbgPxjuLrV00g==, figureFileBig=SPEC/eZBco5FQ6d4vXJxHg==, tableContent=null), ArticleFig(id=1201118431358710359, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Volunteer testPPDMNP-EC-NPsP value
BCG vaccination / mm5.3 ± 1.80< 0.001
TB patients / mm17.5 ± 5.24.6 ± 0.8< 0.001
), ArticleFig(id=1201118431459373661, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=CN, label=Table 1, caption=

Comparison of redness and induration between PPD injection and MNP-EC-NPs.

, figureFileSmall=null, figureFileBig=null, tableContent=
Volunteer testPPDMNP-EC-NPsP value
BCG vaccination / mm5.3 ± 1.80< 0.001
TB patients / mm17.5 ± 5.24.6 ± 0.8< 0.001
), ArticleFig(id=1201118431585202794, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Experience evaluationPPDMNP-EC-NPsP value
VAS pain5.0 ± 2.31.2 ± 0.42< 0.001
Duration time / day22.0 ± 7.64.0 ± 2.7< 0.001
), ArticleFig(id=1201118431732003439, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200500176805818839, language=CN, label=Table 2, caption=

Analysis of VAS pain and duration time of induration. VAS: Visual analogue scale

, figureFileSmall=null, figureFileBig=null, tableContent=
Experience evaluationPPDMNP-EC-NPsP value
VAS pain5.0 ± 2.31.2 ± 0.42< 0.001
Duration time / day22.0 ± 7.64.0 ± 2.7< 0.001
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结核分枝杆菌分泌蛋白融合蛋白纳米粒可溶性微针贴片的制备及结核病皮试的应用
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陈凡 1, # , 朱荣生 1, # , 周菁 2 , 胡月 1 , 薛云 3 , 康建华 4, * , 王伟 4, *
药学学报 | 研究论文 2024,59(6): 1804-1811
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药学学报 | 研究论文 2024, 59(6): 1804-1811
结核分枝杆菌分泌蛋白融合蛋白纳米粒可溶性微针贴片的制备及结核病皮试的应用
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陈凡1, #, 朱荣生1, #, 周菁2, 胡月1, 薛云3, 康建华4, * , 王伟4, *
作者信息
  • 1.湖北大学生命科学学院, 省部共建生物催化与酶工程国家重点实验室, 湖北 武汉 430062
  • 2.武汉市肺科医院检验科, 湖北 武汉 430030
  • 3.河南科技大学医学技术与工程学院, 河南 洛阳 471003
  • 4.东莞市长安医院检验科, 广东 东莞 523843

通讯作者:

*康建华, Tel: 86-796-85339220, E-mail: ;
王伟, Tel: 86-769-86229999-3276, E-mail:
Preparation of soluble microneedle patch with fusion protein nanoparticles secreted by Mycobacterium tuberculosis and application of tuberculosis skin test
Fan CHEN1, Rong-sheng ZHU1, Jing ZHOU2, Yue HU1, Yun XUE3, Jian-hua KANG4, * , Wei WANG4, *
Affiliations
  • 1. State Key Laboratory of Biocatalysis and Enzyme Engineering, Life Science School of Hubei University, Wuhan 430062, China
  • 2. Clinical Laboratory of Wuhan Pulmonary Hospital, Wuhan 430030, China
  • 3. College of Medical Technology and Engineering, Henan University of Science and Technology, Luoyang 471003, China
  • 4. Department of Laboratory Medicine, Dongguan Chang′an Hospital, Dongguan 523843, China
出版时间: 2024-06-12 doi: 10.16438/j.0513-4870.2023-0895
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摘要
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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。

结核病  /  皮试  /  纳米粒  /  可溶性微针

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.

tuberculosis  /  skin test  /  nanoparticle  /  microneedle patch
陈凡, 朱荣生, 周菁, 胡月, 薛云, 康建华, 王伟. 结核分枝杆菌分泌蛋白融合蛋白纳米粒可溶性微针贴片的制备及结核病皮试的应用. 药学学报, 2024 , 59 (6) : 1804 -1811 . DOI: 10.16438/j.0513-4870.2023-0895
Fan CHEN, Rong-sheng ZHU, Jing ZHOU, Yue HU, Yun XUE, Jian-hua KANG, Wei WANG. Preparation of soluble microneedle patch with fusion protein nanoparticles secreted by Mycobacterium tuberculosis and application of tuberculosis skin test[J]. Acta Pharmaceutica Sinica, 2024 , 59 (6) : 1804 -1811 . DOI: 10.16438/j.0513-4870.2023-0895
正文
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世界卫生组织(WHO) 2022年全球结核病(tuberculosis, TB) 报告表明全球约有1 060万人结核病新发病例, 160万人死于结核病[1]。我国现有活动性肺结核患者600万左右, 在2021年新发病例约86万[2]。WHO结核病终止策略计划在2030年将结核病新发病例减少80%, 死亡病例减少90%[3], 但仅有少量发达国家可达到指标, 其余大部分国家的防治指标远远落后预期[4]。
对结核病的及时诊断和治疗是减除结核病的最有效手段。目前临床诊断结核病常用痰涂片镜检, 但阳性率太低。当采用X射线作为辅助诊断手段时, 非结核性肺病的影响又往往不可忽视[5]。细菌培养是结核病诊断的“金标准”, 但耗时过长, 往往需要4~8周, 延误最佳治疗期[6]。聚合酶链式反应(PCR) 技术过于灵敏, 同时需要在生物安全实验室操作, 限制了该技术的应用[7-9]。结核病在不同阶段用抗体检测的特异性和灵敏度变化较大, 因而WHO不推荐血清学检测方法[10]。目前结核流行病早期筛查方法是结核菌素皮试实验, 主要是纯蛋白衍生物(pure protein derivative, PPD) 皮试, 在发展中国家常用, 但检测特异性低[11, 12]。微型注射针(MicronJet 600TM) 的发明使皮试操作更容易[13], 但该注射器价格高, 诊断特异性也没有提高。近年来, 利用100~1 000 μm长的可溶性材料制备的针状体(微针, microneedle) 制成微针贴片(microneedle patch, MNP) 用于诊断受到关注[14, 15]。用透明质酸(hyaluronic acid, HA) 装载PPD后的人体皮肤试验, 无疼痛、瘙痒等不良反应, 但不能区分卡介苗(Bacillus Calmette-Guerin, BCG) 免疫者[16]。BCG在我国是计划接种项目, PPD皮试在BCG免疫者中也呈阳性反应, 而BCG缺失了ESAT-6和CFP-10蛋白。ESAT-6和CFP-10是结核分枝杆菌早期分泌性低分子量蛋白, 能诱导机体产生T细胞免疫应答, 具有良好的抗原刺激性并能被大多数结核病患者所识别[17]。本研究用可溶性微针作为结核分枝杆菌ESAT6-CFP10融合蛋白的载体, 用作结核病的皮试诊断试剂, 不但提高皮试的特异性, 还可有效减少不良反应, 其自助式检测方式, 更有利于结核病流行病筛查。
材料与方法
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材料与动物  大肠杆菌DH5α和BL21, 含有质粒pET28a、pET28a-CFP10、pET28a-ESAT6的DH5α菌种均为本实验室保存; BamHI (1010S)、HindIII (1060S)、T4 DNA ligase (e0879) (日本TAKARA公司); 琼脂糖凝胶回收试剂盒(28606, 德国Qiagen公司); BCA蛋白定量试剂盒(A53225, 美国Thermo Fisher Scientific公司); Ni Sepharose 6 fast flow (17531806, 美国GE公司); Smartdex G-75 (SEC0125, 常州天地人和生物科技公司); ESAT-6兔多克隆抗体(ab45073, 英国Abcam公司); HRP标记的鼠抗兔单克隆抗体(31464, 美国Invitrogen公司); 低分子量壳聚糖(448869, 美国Sigma公司); 医学透析袋MD16 (131414-1, 美国Spectrum公司); 750~1 000 kDa透明质酸钠(sodium hyaluronate, HA, 美国lifecore公司); 罗丹明B染料(81-88-9, 杭州Aladdin公司), 盐酸利多卡因注射液(2307J07, 上海朝晖药业); 实验所需引物由上海生物工程有限公司合成。昆明小鼠(雌性, 6周龄) 由武汉大学动物实验中心提供, 实验动物合格证编号: 42000600054253。实验按照湖北大学实验动物伦理委员会及国际动物实验的指导原则进行, 所有动物实验均获得了湖北大学动物实验伦理委员会批准(批准号: 20220014)。
仪器  磁力搅拌器(MR Hei-Mix D, HEIDOLPH, Germany); 粒径电位测试仪(zeta sizer ZS90, Malvern, UK); 透射电子显微镜(Tchnai G②20S-TWIN, FEI, USA), 酶标仪(MQX200R, Bio Tek, USA); 真空冷冻干燥仪(FD-1A-50, 无锡沃信)。
ESAT6-CFP10融合蛋白表达质粒的构建  根据CFP10和ESAT6的基因序列分别设计含有linker的两对引物CFP10-F1: 5′-CGGGATCCATGGCAGAGATG AAG-3′; CFP10-R1: 5′-GCTGCCGCCACCGCCGCTTC CGCCACCGCCGCTTCCACCGCCACCGAAGCCCA TTTGCGA-3′; ESAT6-F2: 5′-GGTGGCGGTGGAAGC GGCGGTGGCGGAAGCGGCGGTGGCGGCAGCAT GACAGAGCAGCAG-3′; ESAT6-R2: 5′-CCAAGCTTG TGCGAACATCCCAGT-3′, 两端带有BamHI和HindIII酶切位点, 分别扩增CFP10和ESAT6。用琼脂糖凝胶回收试剂盒将目的条带CFP10-Linker和Linker-ESAT6进行胶回收。将得到的CFP10-Linker和Linker-ESAT6作为双模板, 进行两步法PCR, 鉴定目的条带正确后, 用琼脂糖凝胶回收试剂盒将目的条带CFP10-Linker-ESAT6进行胶回收。将CFP10-Linker-ESAT6和pET28a空载经双酶切后回收, 用T4 DNA ligase连接后测序, 构建pET28a-ESAT6-CFP10质粒。
ESAT6-CFP10融合蛋白的表达  将质粒pET28a-CFP10-ESAT6转化到BL21中, 异丙基-β-D-硫代半乳糖苷(IPTG) 诱导表达后破菌, 用Ni琼脂糖凝胶4 Fast Flow填料纯化, 将纯化好的蛋白置于截留分子量为20 kDa的超滤离心管中, 4 ℃ 3 000 r·min-1离心40 min, 用SDS-PAGE电泳检测, 进一步用Smartdex G-75凝胶纯化, SDS-PAGE验证蛋白纯度, 并用Bradford法测定蛋白浓度。
Western blot  将纯化后的ESAT6-CFP10融合蛋白转移到PVDF膜上, 封闭后分别加入ESAT-6兔多克隆抗体和HRP标记的鼠抗兔单克隆抗体, 用DAB显色, 检测融合蛋白免疫原性。
壳聚糖纳米粒的制备  在一定体积的1 mg·mL-1的壳聚糖(Cs) 工作液中, 加入ESAT6-CFP10融合蛋白溶液, 在磁力搅拌器的作用下(500 r·min-1), 将壳聚糖和ESAT6-CFP10融合蛋白混合10 min后, 继续搅拌并逐滴加入一定体积的0.5 mg·mL-1的三聚磷酸钠(sodium tripolyphosphate, TPP) 溶液, 室温条件下持续搅拌90 min, 得到蓝色乳光的ESAT6-CFP10融合蛋白壳聚糖纳米粒。
壳聚糖纳米粒的表征  用粒径电位测试仪测定样品的粒径大小、分散系数(polymer dispersity index, PDI) 及zeta电位大小, 同时用透射电子显微镜观测纳米粒形态。将制备好的融合蛋白壳聚糖纳米粒于4 ℃ 15 000 r·min-1离心30 min, 分别收集上清和沉淀, 沉淀用去离子水重悬于真空冷冻干燥仪干燥成粉末, 并称取纳米粒质量, 通过Bradford测定总蛋白的浓度和上清剩余蛋白的浓度, 以空白壳聚糖纳米粒的上清液作为内参。根据公式(1) 和(2) 计算融合蛋白壳聚糖纳米粒的包封率和载药量。
$\quad 包封率 (\text{EE}\%) = [总蛋白浓度-(上清蛋白浓度-空\\白纳米粒上清浓度)]/总蛋白浓度×100\%$
$\quad 载药量 (\text{DL}\%) = [总蛋白浓度-(上清蛋白浓度-空\\白纳米粒上清浓度)]×总体积/蛋白壳聚糖纳米粒质\\量×100\%$
体外释放实验  将得到的融合蛋白壳聚糖纳米粒和空白壳聚糖纳米粒分别称取500 mg, 用1 mL蒸馏水重悬壳聚糖纳米粒于两个透析袋中。将透析袋置于50 mL的pH7.4磷酸盐缓冲液(phosphate buffer solution, PBS) 中, 于37 ℃恒温条件下释放, 每到0、0.5、1、1.5、2、4、6、8、12、24、48 h取出1 mL透析袋外层的释放介质, 置于4 ℃储存, 用于下一步分析。在取释放介质的同时, 用pH 7.4 PBS将取出的体积补足。将得到的各个时间点的释放介质, 通过Bradford工作液测定各个时间点的蛋白浓度。根据累计释放率的公式(3) 计算累计释放率, 并绘制累计释放曲线。
$\omega \mathrm{i}=C_{\mathrm{i}} V_1+\sum C_{\mathrm{i}-1} \cdot V_2 /\left(C_{\text {总 }}-C_{\text {消耗 }}\right) \cdot V_0 \times 100 \%$
其中, V1表示释放介质的总体积, 即50 mL; V2为每次取出的体积为1 mL; C总表示透析袋内ESAT6-CFP10蛋白的原浓度, C消耗表示游离蛋白的浓度; V0为透析袋内总体积, 即1 mL。
可溶性微针的制备  根据文献制备微针贴片[18, 19]。配制8%透明质酸凝胶, 分别加入融合蛋白壳聚糖纳米粒(ESAT6 and CFP10 fusion protein nanoparticals, EC-NPs) 和罗丹明染料, 倒入聚二甲基硅氧烷模型中, 干燥后制成微针贴片, 显微镜下观测微针形态。用盐酸利多卡因注射液麻醉小鼠, 背部去毛后进行透皮实验。
载药量与保存期测试  分别制备了6张装载EC-NPs的微针贴片, 每张贴片上随机剪取10根微针, 放入装有1 mL PBS的离心管中, 4 ℃混匀24 h后, 通过Bradford法测定总蛋白的浓度。该实验重复3次, 计算出单针微针载有ESAT6-CFP10融合蛋白的含量。为了检测载药微针贴片的保存期, 同一批次制备21张载药微针贴片, 室温避光保存。随机选取3张, 每张贴片上剪取10根微针, 测定ESAT6-CFP10融合蛋白的平均含量, 首次测定值作为基准含量(100%), 随后每隔1个月随机测定3张载药贴片, 连续检测6个月, 通过ESAT6-CFP10融合蛋白的平均含量的变化, 判断载药微针贴片在室温下的保存时间。
载药微针贴片功能验证  按临床试验标准招募24名志愿者, 其中BCG接种健康受试者12名, 结核病患者(痰检阳性, 临床初次确诊) 12名, 试验经武汉市肺科医院伦理委员会批准[武肺伦理2022 (2)], 志愿者签订知情同意书。将载有EC-NPs的微针贴片压入受试者小臂皮肤中, 48 h后观测皮试结果。志愿者在接受皮试后, 用疼痛数字评分法(visual analogue scale, VAS) 进行评分, 记录形成红肿硬块的大小, 出现时间和持续时间, 记录各组数据进行统计学分析。
数据分析  实验数据用GraphPad Prism 6软件(GraphPad Software, USA) 进行统计学数据分析。数据以平均值± SEM表示, P < 0.05为具有显著性差异。BCG免疫健康对照组和结核病患者组之间的比较采用Mann-Whitney U检验。
结果与分析
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1 ESAT6-CFP10融合蛋白的表达纯化
从保存质粒中分别扩增得到CFP10和ESAT6的目的基因片段, 其中CFP10的大小为300 bp, ESAT6的大小为288 bp (图 1A)。将两个目的基因进行胶回收。通过Overlap-PCR将CFP10-Linker和Linker-ESAT6连在一起, 形成融合CFP10-Linker-ESAT6, 其大小为630 bp (图 1B)。与marker相比, 目的条带在750 bp到500 bp之间, 即图中的目的条带呈现单一条带, 且条带大小正确, 将融合目的基因进行胶回收, 构建重组质粒, 测序证实基因成功融合。运用BamHI和HindIII将ESAT6-CFP10和非线性载体进行双酶切(图 1C), 确证重组载体pET28a- ESAT6-CFP10成功构建。分别将重组载体转化至DH5α和BL21中。
为了优化目的蛋白的表达量, 进行了四因素三水平的正交试验。正交试验的结果如图 1D所示, 可知目的蛋白的大小为28 kDa, 其中加入终浓度为0.5 mmol·L-1 IPTG, 在26 ℃的LB培养基中低温诱导表达12 h, 融合蛋白(ESAT6 and CFP10 fusion protein, EC) 的表达量最高。pET28a的载体上N端和C端分别带有His标签, 所以选用Ni柱进行纯化, 分别用含50和80 mmol·L-1的咪唑进行梯度洗脱, 通过跑SDS-PAGE检测结果, 如图 1E所示, 当咪唑浓度在50 mmol·L-1时, 洗脱下来的杂蛋白较少, 目的蛋白更纯。为得到更纯的目的蛋白, Ni柱纯化后并浓缩融合蛋白, 使用分子筛进行进一步纯化, 收集蛋白并进行SDS-PAGE电泳, 得到较为纯的融合蛋白(图 1F), 并用Bradford法建立了ESAT6-CFP10融合蛋白的标准曲线(图 1G)。同时, Western blot证实了所表达纯化的ESAT6-CFP10融合蛋白具有生物学活性, 可以用作特异性抗原皮试制剂(图 1H)。
2 ESAT6-CFP10融合蛋白壳聚糖纳米粒的制备条件
随着壳聚糖和TPP的质量比的增大, 形成的壳聚糖纳米粒的粒径呈现减小的趋势。当壳聚糖和TPP的质量比为3∶1和4∶1时, 溶液中出现沉淀。随着Cs/TPP的质量比的增大, zeta电位呈现先增大后减小的趋势(图 2A、B)。当Cs/TPP质量比为5∶1, zeta电位最大。当Cs/TPP质量比为5∶1时, 随着pH的增大, 纳米粒的粒径也增大, 而zeta电位逐渐降低。最后选择pH为4.5作为实验条件(图 2C、D)。保持pH = 4.5条件不变, 当Cs/TPP质量比为5∶1时, 加入相同体积不同浓度的融合蛋白制备融合蛋白壳聚糖纳米粒。随着EC蛋白浓度的增加, 包封率和载药量同时出现先增大后减小的趋势, 在蛋白质量浓度为0.4 mg·mL-1时, 包封率和载药量均较好(图 2E、F)。
3 ESAT6-CFP10融合蛋白壳聚糖纳米粒的性质鉴定
根据单因素试验确定用9 mL的1.0 mg·mL-1 pH 4.5的Cs溶液, 3.6 mL的0.5 mg·mL-1的TPP溶液, 2 mL的0.4 mg·mL-1的融合蛋白溶液, 制备融合蛋白壳聚糖纳米粒溶液带蓝色乳光(图 3A)。测得融合蛋白壳聚糖纳米粒的平均粒径为114.1 ± 1.791 nm, 纳米粒PDI为0.241 ± 0.054, 分散性良好(图 3B)。纳米粒的zeta电位为19.4 ± 1.715 mV, 测得的电位值> 15 mV (图 3C), 说明融合蛋白壳聚糖纳米粒较为稳定, 不宜聚集[20]。离心后用去离子水重悬, 用透射电子显微镜拍摄, 可以看到融合蛋白壳聚糖纳米粒呈圆形, 纳米粒整体较为均一(图 3D)。建立Bradford工作液的标准曲线(图 3E), 根据公式测得累计释放率, 在前5 h融合蛋白壳聚糖纳米粒释放得非常迅速, 在5~20 h释放趋于稳定, 24 h释放率可达90%以上(图 3F)。
4 微针贴片透皮检测
将融合蛋白壳聚糖纳米粒溶解至HA中, 制备含有融合蛋白壳聚糖纳米粒可溶性微针贴片(图 4A)。微针贴片为8×8的正方形矩阵, 呈圆锥形, 底部半径为250 μm, 高为500 μm (图 4B、C)。用同样方法制备含罗丹明染料的微针贴片(图 4D), 压入脱毛后的小鼠皮肤中, 10 min后去掉贴片, 可观察到小鼠皮肤上有清晰的含有染料的针孔, 证明制备的微针贴片具有很好的透皮能力(图 4E)。进一步将透皮后的小鼠皮肤制成组织切片, HE (hematoxylin-eosin) 染色后镜检观察, 可发现微针透皮后形成了明显的微孔, 前端到达真皮层, 进一步说明微针可以穿过皮肤组织递送抗原成分(图 4F)。
5 载药微针贴片
根据EC-NPs的释放特性, 测定了微针在4 ℃释放24 h后的ESAT6-CFP10融合蛋白的含量。通过3次重复试验, 测得单针载有ESAT6-CFP10融合蛋白的量约为0.03 μg (图 5A)。即8×8阵列的微针贴片可装载ESAT6-CFP10融合蛋白约1.92 μg。将载药微针贴片置于室温保存, 连续检测6个月, 发现ESAT6-CFP10融合蛋白只有微量降解, 总含量仍保持在93%左右(图 5B)。说明载药微针贴片在不需要冷链保存的条件下, 仍有很好的稳定性。
6 志愿者皮试
皮试显示4×4阵列的贴片即可产生明显的反应, 即ESAT6-CFP10融合蛋白载药量为0.48 μg时, 可观察到明显的阳性反应。PPD注射皮试时, 在BCG免疫健康者皮肤上可形成5.3 ± 3.1 mm的红肿硬块(图 6A, 表 1), 而MNP-EC-NPs在BCG免疫健康者皮肤上无任何反应, 证明了ESAT6-CFP10融合蛋白的特异性比PPD要好(图 6B, 表 1)。当结核病患者用PPD注射皮试时, 可形成17.5 ± 5.2 mm大小的红肿硬块(图 6C), 且持续时间长达22天左右(表 2)。而MNP-EC-NPs用于结核病患者皮试可形成4.6 ± 0.8 mm大小的红肿硬块(图 6D, 表 1), 持续时间只有4天左右(表 2)。此外, VAS评分显示, PPD注射皮试的不适感, 明显高于MNP-EC-NPs皮试(表 2)。说明MNP-EC-NPs皮试不仅特异性强于PPD注射, 并且不良反应少, 皮肤恢复快。
讨论
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近几年来全球结核病的发病率和死亡率下降缓慢甚至出现上升的趋势, 迫切需要有效的结核病流行病筛选方法。由于PPD是多种蛋白质的混合物, 导致其特异性低, 不良反应较多。一系列结核分枝杆菌的分泌蛋白被用作早期筛查TB的皮试检测候选试剂, 包括PE13、PE5、MPB70、TB10.4和TB27.4等, 这些皮试均已证实对人体安全[21, 22]。将ESAT-6和CFP-10按1∶1 (C-Tb) 混合用于皮试检测TB已进入Ш期临床试验[23], C-Tb皮试能够很好地区别BCG免疫者和TB患者, 特异性比TST高, 但不能消除皮试部位疼痛和瘙痒等不良反应[24]。
在一些报道中, ESAT-6和CFP-10的融合蛋白被用作免疫制剂及血清学诊断制剂[25, 26]。作为皮试制剂, 需要保证药品的有效性和安全性。本研究发现, 利用His标签进行亲和层析并不能得到完全纯化的融合蛋白, 因此, 进一步利用凝胶过滤层析得到了纯化的ESAT6-CFP10融合蛋白。为了改善微针贴片的储存条件, 使用离子交联法制备成ESAT6-CFP10融合蛋白壳聚糖纳米粒, 24 h释放率达到90%以上, 而迟发型超敏反应观察时间一般在48 h后, 因而满足结核病皮试的需要。同时, 装载有EC-NPs的微针贴片在常温环境下保存期可长达6个月, 从而减少了冷链运输保存的成本。
通过小鼠实验和志愿者测试, 证明了制备的微针贴片具有很好的透皮能力。而ESAT6-CFP10的有效性和特异性已在小鼠皮肤上得到证实, 采用常规注射途径, 0.5 μg ESAT6-CFP10融合蛋白在小鼠皮肤上可形成13.7 ± 5.7 mm的硬块[27]。本研究在志愿者皮试中进一步确定了ESAT6-CFP10融合蛋白作为皮试抗原的特异性和有效性。在BCG免疫者皮试中, MNP-EC-NPs皮试呈阴性反应, 特异性明显高于常规PPD注射皮试。而对于结核病患者, 虽然MNP-EC-NPs皮试与PPD注射皮试均呈阳性反应, 但由于微针皮试疼痛感低, 形成的红肿硬块小, 持续时间短, 因而相对于注射皮试有明显的优势。常规皮试需要用到注射器和针头, 通常需要专业人员操作, 不利于偏远地区或医疗资源不足地方的流行病筛查。而微针技术具有无疼、自助式、不需冷链运输保存等优点, 很好地解决了上述局限性。
本研究建立了一种新的结核病皮试技术, 为进一步在人体皮肤开展大规模结核病皮试奠定了基础。但是, 由于一些非结核分枝杆菌中也含有ESAT-6和CFP-10, 可能导致肺部感染受试者会形成假阳性结果[28]; 结核病潜伏感染者体内也会有ESAT-6和CFP-10抗体, 因而用ESAT6-CFP10融合蛋白作为抗原, 不能区分潜伏感染与活动性结核病患者[29, 30]。因此, 还需要开展大量的工作, 筛选出更好的结核病皮试抗原。
作者贡献: 陈凡是本文的主要撰写者, 搜集资料并分析, 撰写草稿; 朱荣生、周菁是实验的参与者; 胡月负责实验材料的准备; 薛云负责本文思路的提出; 康建华和王伟指导并修改论文。所有作者阅读并认可终稿。
利益冲突: 本文所有作者声明不存在利益冲突关系。
基金
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  • 国家自然科学基金资助项目(32072899)
  • 武汉市肺科医院临床研究项目(YNZZ202207)
  • 广东省高水平医院临床研究项目(20203357006)
文献
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文章信息
doi: 10.16438/j.0513-4870.2023-0895
  • 接收时间：2023-07-24
  • 首发时间：2025-11-26
  • 出版时间：2024-06-12
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  • 收稿日期：2023-07-24
  • 修回日期：2024-03-28
基金
国家自然科学基金资助项目(32072899)
武汉市肺科医院临床研究项目(YNZZ202207)
广东省高水平医院临床研究项目(20203357006)
作者信息
    1.湖北大学生命科学学院, 省部共建生物催化与酶工程国家重点实验室, 湖北 武汉 430062
    2.武汉市肺科医院检验科, 湖北 武汉 430030
    3.河南科技大学医学技术与工程学院, 河南 洛阳 471003
    4.东莞市长安医院检验科, 广东 东莞 523843

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*康建华, Tel: 86-796-85339220, E-mail: ;
王伟, Tel: 86-769-86229999-3276, E-mail:
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Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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