Inhibition of AAV-mediated RNAi to SARS-CoV-2 S Gene Expression

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

Abstract

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

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.

Figures/Tables 4

References 15

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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'

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'