AAV介导的RNAi对SARS-CoV-2 S基因表达的抑制作用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0596

摘要/Abstract

摘要：

旨在研究AAV介导shRNA在体内、体外特异性抑制SARS-CoV-2 S基因的表达,为抑制SARS-CoV-2感染提供参考。设计了7条靶向SARS-CoV-2 S基因的shRNA,分别构建了shRNA1-7的质粒表达载体,与S基因表达载体共同转染体外培养细胞,通过qRT-PCR和Western blot分析其对S基因表达的抑制作用,筛选出抑制效率最高的shRNA1;构建包装表达shRNA1的重组腺相关病毒（rAAV）载体,将rAAV-shRNA1与S基因表达质粒共同转导293T细胞,S基因表达的抑制率为65.6%;两者通过滴鼻或肌肉注射方式共同转导小鼠,qRT-PCR检测其在体内对S基因表达的抑制率分别为36.8%和90.3%。

关键词: 新型冠状病毒, 刺突蛋白, RNA干扰, 腺相关病毒

Abstract:

The purpose of this study is to investigate the inhibitory effect of AAV-delivered shRNA on the expression of SARS-CoV-2 S gene in vitro and in vivo,and to provide reference for further research on the inhibition of SARS-CoV-2 infection. Seven shRNAs targeting SARS-CoV-2 S were designed. The expression vectors of shRNA and S gene were co-transfected into cultured cells,and the inhibition effect on S gene expression was analyzed by qRT-PCR and Western blot,and shRNA1 presented the highest inhibition. The rAAV vector of expressing shRNA1 was constructed. When rAAV-shRNA1 and S gene expression plasmid were co-transfected into 293T cells,the inhibition rate of S gene expression was 65.6%. Mice were injected nasally or intramuscularly with rAAV-shRNA1 and S gene expression plasmid,and the inhibition rate of the S gene expression was 36.8% and 90.3%,respectively.

Key words: SARS-CoV-2, spike protein, RNA interference, adeno-associated virus

刘晓玫, 王东鑫, 张春, 魏双施. AAV介导的RNAi对SARS-CoV-2 S基因表达的抑制作用[J]. 生物技术通报, 2022, 38(3): 188-193.

LIU Xiao-mei, WANG Dong-xin, ZHANG Chun, WEI Shuang-shi. Inhibition of AAV-mediated RNAi to SARS-CoV-2 S Gene Expression[J]. Biotechnology Bulletin, 2022, 38(3): 188-193.

图/表 4

表1 shRNA 寡核苷酸序列

Table 1 Oligonucleotide sequence of shRNA

shRNA编号shRNA No.	寡核苷酸类型Oligo type	寡核苷酸序列Oligo sequence
shRNA1	顶链Top strand	5'- CACCGCTTCCACTGAGAAGTCTAACTTCAAGAGAGTTAGACTTCTCAGTGGAAGC -3'
	底链Bottom	5'- AAAAGCTTCCACTGAGAAGTCTAACTCTCTTGAAGTTAGACTTCTCAGTGGAAGC -3'
shRNA2	顶链Top strand	5'- CACCGCACACGCCTATTAATTTAGTTTCAAGAGAACTAAATTAATAGGCGTGTGC -3'
	底链Bottom	5'- AAAAGCACACGCCTATTAATTTAGTTCTCTTGAAACTAAATTAATAGGCGTGTGC -3'
shRNA3	顶链Top strand	5'- CACCGGTGATTCTTCTTCAGGTTGGTTCAAGAGACCAACCTGAAGAAGAATCACC -3'
	底链Bottom	5'- AAAAGGTGATTCTTCTTCAGGTTGGTCTCTTGAACCAACCTGAAGAAGAATCACC -3'
shRNA4	顶链Top strand	5'- CACCGCTGGTGCTGCAGCTTATTATTTCAAGAGAATAATAAGCTGCAGCACCAGC -3'
	底链Bottom	5'- AAAAGCTGGTGCTGCAGCTTATTATTCTCTTGAAATAATAAGCTGCAGCACCAGC -3'
shRNA5	顶链Top strand	5'- CACCGCAACTGTGTTGCTGATTATTTTCAAGAGAAATAATCAGCAACACAGTTGC -3'
	底链Bottom	5'- AAAAGCAACTGTGTTGCTGATTATTTCTCTTGAAAATAATCAGCAACACAGTTGC -3'
shRNA6	顶链Top strand	5'- CACCGCCGGTAGCACACCTTGTAATTTCAAGAGAATTACAAGGTGTGCTACCGGC -3'
	底链Bottom	5'- AAAAGCCGGTAGCACACCTTGTAATTCTCTTGAAATTACAAGGTGTGCTACCGGC -3'
shRNA7	顶链Top strand	5'- CACCGGTGTTGGTTACCAACCATACTTCAAGAGAGTATGGTTGGTAACCAACACC -3'
	底链Bottom	5'- AAAAGGTGTTGGTTACCAACCATACTCTCTTGAAGTATGGTTGGTAACCAACACC -3'

图1 不同shRNA对S基因表达的抑制作用分析比较 A：定量RT-PCR检测;B：Western blot检测。“-”表示只转染pFastBacDual-ITR-S-T2A-EGFP质粒,“NC”表示阴性对照质粒pAAV-U6。**P<0.01, ***P<0.001, ns表示无统计学差异

Fig. 1 Analysis and comparison of the inhibitory effects of different shRNAs on the expression of S gene A：Quantitative RT-PCR detection. B：Western blot detection. “-” refers to that only the pFastBacDual-ITR-S-T2A-EGFP plasmid was used to transfect,and “NC” refers to the negative control plasmid pAAV-U6. **P<0.01, ***P<0.001, and "ns" means no statistically significant difference

图2 rAAV介导shRNA对S基因表达的体外抑制作用分析

Fig. 2 Inhibition analysis of rAAV-mediated shRNA on S gene expression in vitro

图3 rAAV介导shRNA对S基因表达的体内抑制作用检测 A：肺脏组织S基因表达的qRT-PCR检测;B：肌肉组织S基因表达的qRT-PCR检测。 *P<0.05, **P<0.01, ***P<0.001

Fig. 3 Detection of the in vivo inhibitory effect of rAAV-mediated shRNA on S gene expression A：qRT-PCR detection of S gene expression in lung tissue. B：qRT-PCR detection of S gene expression in muscle tissue. *P<0.05, **P<0.01, ***P<0.001

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