基于In-fusion技术的高通量HIV表型耐药检测系统的建立及应用研究 : = Construction and application of an In-fusion-based high-throughput system to detect HIV phenotypic resistance
目的构建基于In-fusion技术的高通量新型HIV表型耐药检测系统,用于临床表型耐药检测。方法扩增LacZ基因,插入并替换原骨架质粒pLWJ-SV40-Luc中耐药检测片段构建新的骨架质粒pLWJ-SV40-Luc-LacZ,利用In-fusion定向克隆技术,将耐药检测片段插入到改造后的骨架质粒中构建耐药检测载体(RTV),并与膜蛋白表达质粒VSV-G共转染293T细胞产生HIV假病毒,在药物干预下,通过检测感染细胞中Luc+表达量来反应其对药物的敏感性。结果成功引入蓝白斑筛选改造出新的骨架质粒pLWJ-SV40-Luc-LacZ。36例临床检测样本成功扩增出30例,利用In-fusion无缝连接技术成功构建26例RTV,其中21株产生的假病毒具有活性。药物敏感性实验结果显示所得的假病毒表型耐药检测结果与基因型耐药解释结果完全一致。结论成功构建出基于In-fusion技术的高通量新型HIV表型耐药检测系统,可应用于临床耐药检测,以指导临床抗病毒治疗。.
The aim of this study is to develop an In-fusion-based high-throughput HIV novel phenotypic resistance detection system and evaluate its application in the clinical detection of HIV-1 drug resistance. The new HIV back-bone vector pLWJ-SV40-Luc-LacZ was constructed by inserting amplified LacZ gene into pLWJ-SV40-Luc. Using the In-fusion cloning technology, resistance test vectors(RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc-LacZ and replacing the LacZ fragment. The pseudovirus stocks used for drug susceptibility test were produced by co-transfecting 293 T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein(VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells. A total of 36 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 30 samples(83.3 %) and 26 RTVs were successfully constructed by In-fusion cloning, of which 21 RTV had active infection. The phenotypic profiles of the pseudoviruses from 21 clinical samples completely matched the observed genotypes. The results demonstrate that an In-fusion-based high-throughput HIV novel phenotypic resistance detection system was successfully developed, and this testing system appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains and provides information for clinical therapy..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2019-04-15 2019 |
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| Erschienen: |
2019-04-15 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2019 |
|---|---|
| Enthalten in: |
Zhong guo ren shou gong huan bing xue bao - (2019), 04 vom: 15. Apr., Seite 285-291 Original Letters: Enthalten in 中国人兽共患病学报 (DE-600)2995104-5 (DE-600)2995104-5 福建省福州市 |
| Reihe: |
China Academic Journals (CAJ), E, 医药卫生科技 = Medicine & Public Health |
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| Sprache: |
Chinesisch |
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| Weiterer Titel: |
Construction and application of an In-fusion-based high-throughput system to detect HIV phenotypic resistance |
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| Beteiligte Personen: |
吴守丽 [VerfasserIn] |
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| Links: |
oversea.cnki.net [lizenzpflichtig] |
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| Anmerkungen: |
Author info:WU Shou-li;LIU Feng;YAN Ping-ping;WANG Zheng-hua;XIE Mei-rong;YAN Yan-sheng;Fujian Provincial Center for Disease Control and Prevention/Fujian Provincial Key Laboratory of Zoonosis Research;School of Public Health, Fujian Medical University |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
CAJ630570221 |
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| 245 | 1 | 0 | |a 基于In-fusion技术的高通量HIV表型耐药检测系统的建立及应用研究 |b = Construction and application of an In-fusion-based high-throughput system to detect HIV phenotypic resistance |
| 246 | 3 | 1 | |a Construction and application of an In-fusion-based high-throughput system to detect HIV phenotypic resistance |
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| 500 | |a Author info:WU Shou-li;LIU Feng;YAN Ping-ping;WANG Zheng-hua;XIE Mei-rong;YAN Yan-sheng;Fujian Provincial Center for Disease Control and Prevention/Fujian Provincial Key Laboratory of Zoonosis Research;School of Public Health, Fujian Medical University | ||
| 520 | |a 目的构建基于In-fusion技术的高通量新型HIV表型耐药检测系统,用于临床表型耐药检测。方法扩增LacZ基因,插入并替换原骨架质粒pLWJ-SV40-Luc中耐药检测片段构建新的骨架质粒pLWJ-SV40-Luc-LacZ,利用In-fusion定向克隆技术,将耐药检测片段插入到改造后的骨架质粒中构建耐药检测载体(RTV),并与膜蛋白表达质粒VSV-G共转染293T细胞产生HIV假病毒,在药物干预下,通过检测感染细胞中Luc+表达量来反应其对药物的敏感性。结果成功引入蓝白斑筛选改造出新的骨架质粒pLWJ-SV40-Luc-LacZ。36例临床检测样本成功扩增出30例,利用In-fusion无缝连接技术成功构建26例RTV,其中21株产生的假病毒具有活性。药物敏感性实验结果显示所得的假病毒表型耐药检测结果与基因型耐药解释结果完全一致。结论成功构建出基于In-fusion技术的高通量新型HIV表型耐药检测系统,可应用于临床耐药检测,以指导临床抗病毒治疗。 | ||
| 520 | |a The aim of this study is to develop an In-fusion-based high-throughput HIV novel phenotypic resistance detection system and evaluate its application in the clinical detection of HIV-1 drug resistance. The new HIV back-bone vector pLWJ-SV40-Luc-LacZ was constructed by inserting amplified LacZ gene into pLWJ-SV40-Luc. Using the In-fusion cloning technology, resistance test vectors(RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc-LacZ and replacing the LacZ fragment. The pseudovirus stocks used for drug susceptibility test were produced by co-transfecting 293 T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein(VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells. A total of 36 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 30 samples(83.3 %) and 26 RTVs were successfully constructed by In-fusion cloning, of which 21 RTV had active infection. The phenotypic profiles of the pseudoviruses from 21 clinical samples completely matched the observed genotypes. The results demonstrate that an In-fusion-based high-throughput HIV novel phenotypic resistance detection system was successfully developed, and this testing system appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains and provides information for clinical therapy. | ||
| 610 | 2 | 4 | |a 福建省疾病预防控制中心(福建省人兽共患病研究重点实验室) |
| 610 | 2 | 4 | |a 福建医科大学公共卫生学院 |
| 650 | 4 | |a 传染病 | |
| 650 | 4 | |a 内科学 | |
| 650 | 4 | |a 医药、卫生 | |
| 650 | 4 | |a Infectious Disease | |
| 650 | 4 | |a Endocrine and Systemic Disease | |
| 650 | 4 | |a 医药卫生科技 | |
| 650 | 4 | |a Medicine & Public Health | |
| 650 | 4 | |a 人类免疫缺陷病毒 | |
| 650 | 4 | |a 假病毒 | |
| 650 | 4 | |a 表型耐药性检测 | |
| 650 | 4 | |a 基因型耐药性检测 | |
| 650 | 4 | |a human immunodeficiency virus | |
| 650 | 4 | |a pseudoviruse | |
| 650 | 4 | |a phenotypic resistance assay | |
| 650 | 4 | |a genotypic resistance assay | |
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| 700 | 0 | |a 颜苹苹 |4 oth | |
| 700 | 0 | |a 王征桦 |4 oth | |
| 700 | 0 | |a 谢美榕 |4 oth | |
| 700 | 0 | |a 严延生 |4 oth | |
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