抗甲型流感病毒短发夹状RNA分子的体外生物合成及功能鉴定

郭述良; 罗永艾

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抗甲型流感病毒短发夹状RNA分子的体外生物合成及功能鉴定

论著 | 更新时间：2026-01-09

- 抗甲型流感病毒短发夹状RNA分子的体外生物合成及功能鉴定
- Biosynthesis and function verification of short hairpin RNA targeting to Influenza A virus
- 陆军军医大学学报 2009年31卷第10期页码：955-958
- 作者机构：
- 作者简介：
- 基金信息：
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  中图分类号： R373.13;R392-33;R392.7
- 纸质出版：2009
- 稿件说明：
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郭述良,罗永艾.抗甲型流感病毒短发夹状RNA分子的体外生物合成及功能鉴定[J].陆军军医大学学报(原第三军医大学学报),2009,31(10):955-958.

　GUO Shu-liang,LUO Yong-ai.Biosynthesis and function verification of short hairpin RNA targeting to Influenza A virus[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(10):955-958.
郭述良,罗永艾.抗甲型流感病毒短发夹状RNA分子的体外生物合成及功能鉴定[J].陆军军医大学学报(原第三军医大学学报),2009,31(10):955-958. DOI：

　GUO Shu-liang,LUO Yong-ai.Biosynthesis and function verification of short hairpin RNA targeting to Influenza A virus[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(10):955-958. DOI：

摘要

目的制备抗甲型流感病毒短发夹状RNA分子（short hairpin RNA

shRNA），并鉴定其功能。方法针对甲型流感病毒(influenza A virus

IAV）核壳蛋白（nucleoprotein

NP）和酸性RNA多聚酶（acidic RNA polymerase

PA）编码mRNA高度保守区序列设计并制备编码shRNA的双链DNA转录模板，分别将其插入pGenSil-1 质粒，构建NP-shRNA和PA-shRNA表达载体，测序正确后，采以T

转录试剂盒制备NP-shRNA和PA-shRNA。将所制备的2种shRNAs分别或联合转入人气道上皮细胞（human bronchial epithelium

HBE），然后感染IAV A/PR8株，通过测定HBE细胞病变效应（cytopathogenic effect

CPE）、培养上清液病毒滴度以及NP、PA mRNA与蛋白表达水平，评估所制备的shRNAs对IAV A/PR8在HBE细胞中复制的抑制效果。结果编码shRNA的2种双链DNA转录模板序列正确。通过体外转录技术制备的NP-shRNA、PA-shRNA每100 μl含量分别为59.4、50.6 nmol，纯度均在2.0以上，浓度和纯度均高。与未转染shRNA的对照组比较，NP-shRNA、PA-shRNA、NP-shRNA＋PA-shRNA转染组细胞内NP mRNA分别减少41.7%、44.1%和68.5%，PA mRNA分别减少43.4%、60.9%和73.7%， NP蛋白表达分别降低92.3%、84.0%和91.7%，CPE显著减轻，培养上清液病毒滴度显著下降。结论成功构建了2种抗IAV短发夹RNA分子，所制备的NP-shRNA、PA-shRNA对IAV A/PR8株在HBE细胞中复制有显著的抑制效果。

Abstract

Objective To investigate the inhibitive effects of biosynthesized short hairpin RNA (shRNA) on the duplication of Influenza A virus (IAV) in human bronchial epithelium (HBE) cells. Methods NP-shRNA and PA-shRNA targeting to the highly conservative sequences

of IAV nucleoprotein (NP) and acidic RNA polymerase (PA) were designed and biosynthesized by in vitro transcription

and then were inserted into the plasmid pGenSil-1. After sequencing analysis

the recombinant plasmids NP-shRNA and/or PA-shRNA were transduced into HBE cells followed by infected with IAV A/PR/8/34 H1N1 (A/PR8) virus. The cytopathogenic effect (CPE) of the HBE cells

hemagglutination assay (HA) and 50% tissue culture infective dose (TCID

) titer of A/PR8 in the culture supernatants were determined respectively. The mRNA and protein expressions of NP and PA were detected by RT-PCR and Western blotting respectively

and the results were compared in order to evaluate the inhibitive efficiency of NP-shRNA and/or PA-shRNA to A/PR8 duplication in HBE cells. Results The content of NP-shRNA and PA-shRNA biosynthesized by in vitro transcription were respectively 59.4 nmol and 50.6 nmol in each 100 μl. The purity was more than 2.0 and the sequences were verified to be correct after sequencing analysis. The CPE of HBE cells and the virus titer in the culture supernatants of cells transfected with NP-shRNA

PA-shRNA or NP-shRNA+PA-shRNA were markedly lower than those of the control groups. In 3 h after 1 TCID

A/PR8 infection

the mRNA level of NP in NP-shRNA group

the mRNA level of PA in PA-shRNA group and those in NP-shRNA＋PA-shRNA group were 41.7%

43.4%

68.5% and 73.7% respectively

lower than those of the control group. At 48 h after 1 TCID

A/PR8 infection

NP synthesis were 92.3%

84.0% and 91.7% respectively of those of the control group. Conclusion Anti-IAV NP-shRNA and PA-shRNA are successfully prepared by in vitro transcription. Both of those markedly inhibit the reproduction of IAV A/PR8 and produce a favorable protective effect on HBE cells.

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