SiRNA sequence against HCMV UL86 gene and applications

Abstract

The invention provides a cDNA sequence of siRNA aiming at a cytomegalovirus UL86 gene of human, and the nucleotide sequence is: 5`-GCACGTCAGTTATCATCAACA-3`. According to the design principle of the siRNA, the cDNA sequence of the selected siRNA has a GC content of 42.9 percent, and the selected siRNA is corresponding to 3161-3181 nucleotide sites in UL86 mRNA, siRNA eukaryotic expression vectors aiming at the HCMV UL86 gene are constructed, and plasmids are extracted after transforming Escherichia coli for transfecting AD293 cells, thereby effectively inhibiting the UL86 gene mRNA and the protein expression level, and the sequence can be applied to the preparation of drugs for curing cytomegalovirus infection of human.

Description

technical field [0001] The invention belongs to biotechnology, relates to the technical fields of molecular biology and genetic engineering, and specifically relates to the design and screening of siRNA sequences targeting human cytomegalovirus UL86 gene and its application to inhibit human cytomegalovirus in vivo and in vitro. Background technique [0002] Human cytomegalovirus (HCMV) belongs to the β-herpesvirus subfamily and is widely infected in the population, but most of them are asymptomatic subclinical infections. HCMV infection can cause multi-system severe diseases in immunocompromised populations (such as AIDS, organ transplant patients, etc.), and may be related to the occurrence of various malignant tumors. At the same time, HCMV is also the most important infectious factor leading to congenital malformations and birth defects in newborns. Therefore, the prevention and treatment of HCMV infection has always been one of the hot spots of medical attention at home...

AI Technical Summary

Problems solved by technology

[0006] At present, there are mainly five methods for preparing siRNA: ①Chemical synthesis method: directly synthesize two complementary 21-23nt RNA single strands by chemical methods, and then anneal to form double-stranded siRNA, the disadvantage is that it is expensive; ②In vitro transcription method: Use Oligo DNA as a template to transcribe and synthesize siRNA in vitro. This method has high efficiency, but it is not suitable for a large number of studies; ③Long-fragment dsRNA degraded by RNase III method: After the target mRNA of 200-1000 bp is prepared by in vitro transcription, it is then treated with RNase III III is digested in vitro to obtain a mixture of different siRNAs. This method is not suitable for the study of specific siRNAs, and may also cause non-specific gene silencing; ④ siRNA expression vector method: by transfecting a plasmid or viral vector containing the RNA polymerase III promoter Dye into the host cell, transcribe short hairpin RNA (short hairpin RNA, shRNA), and the shRNA is cleaved into siRNA by Dicer enzyme in the cell to play a role. This method has the advantages of large-scale amplification and long-term research; ⑤PCR Prepared siRNA expression frame method: obtain an expression frame containing RNA polymerase III promoter, DNA template encoding shRNA and RNA polymerase III termination site by PCR method, and then import into cells and transcribe into siRNA, which has the advantage of being simple and fast , but there is a potential risk of gene integration