| 田丽春,朱情情,刘艾洁,王青青,黄光瑞.携带小鼠白介素-4截短型突变体基因的5型腺相关病毒载体的构建及外源蛋白的体外表达[J].,2019,19(2):211-216 |
| 携带小鼠白介素-4截短型突变体基因的5型腺相关病毒载体的构建及外源蛋白的体外表达 |
| Construction of Recombinant Adeno-associated Virus Encoding Interleukin-4 Antagonistic Mutant and Exogenous Protein Expression in vitro |
| 投稿时间：2018-06-28 修订日期：2018-07-23 |
| DOI：10.13241/j.cnki.pmb.2019.02.003 |
| 中文关键词: 白介素-4 突变体 腺相关病毒 基因治疗 |
| 英文关键词: Interleukin-4 Mutant AAV Gene therapy |
| 基金项目:国家自然科学基金项目（81300016，31500704） |
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| 中文摘要: |
| 摘要 目的：制备携带碳端结构域缺失的小鼠白介素-4（Interleukin-4，IL-4）基因突变体的5型重组腺相关病毒（recombinant adeno- associated virus, rAAV）并在细胞水平检测其介导的外源蛋白表达情况。方法：通过分子克隆技术构建携带小鼠IL-4碳端22个氨基酸缺失的突变体的表达质粒pSNAV-mIL-4ΔC22，三质粒共转染法制备重组的5型AAV病毒，体外感染人支气管上皮样细胞系16HBE和BEAS-2B，并通过Western blot和ELISA检测外源蛋白表达。结果：DNA测序表明构建的小鼠IL-4碳端第118位氨基酸位点处截短的突变体基因表达序列正确无误，制备的重组病毒载体rAAV5-mIL-4ΔC22滴度约为3×1011 vg/mL。rAAV5-GFP感染16HBE和BEAS-2B细胞后36小时开始可见持续稳定的荧光蛋白表达，重组病毒rAAV5-mIL-4ΔC22感染16HBE和BEAS-2B细胞后外源蛋白在培养上清中呈分泌型表达。结论：本研究成功构建了携带小鼠IL-4碳端结构域缺失型突变体的AAV表达质粒并制备了重组病毒rAAV5-mIL-4ΔC22，该病毒可有效转染16HBE和BEAS-2B细胞并介导外源基因分泌表达截短型小鼠IL-4突变体蛋白。 |
| 英文摘要: |
| ABSTRACT Objective: To prepare recombinant adeno-associated virus harboring truncated murine interleukin-4 mutant gene and detect the exogenous protein expression in vitro. Methods: An mIL-4 antagonistic mutant DNA expression plasmid pSNAV-mIL-4ΔC22 was constructed through gene clone technology, a recombinant virus vector rAAV5-mIL-4ΔC22 was prepared by three plasmids co-transfection, human bronchoid epithelioid cell line 16HBE and BEAS-2B were transfected with recombinant AAV5 vectors, then the expression of foreign protein was detected by Western blot and ELISA. Results: The mutant gene mIL-4ΔC22 was verified by DNA se- quencing and the titer of virus vector rAAV5-mIL-4ΔC22 is about 3×1011 vg/mL. 16HBE and BEAS-2B expressed GFP lastly and sta- bly when 36 hours after receiving rAAV-GFP trensfection. Conclusion: Our study constructed recombinant vector rAAV5-mIL-4ΔC22 encoding murine IL-4 antagonistic mutant protein deleting the C-terminus region, the virus vector effectively transfected 16HBE and BEAS-2B, and mediated exogenous protein secretory expression in vitro. |
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