SgRNA combined with immunogene to inhibit high risk HPV expression, gene knockout vector thereof, and application of sgRNA and vector

Abstract

The invention provides an sgRNA combined with immunogene to inhibit high risk HPV expression, a gene knockout vector thereof, and an application of the sgRNA and the vector. The sgRNA sequences of a human papilloma virus 16 type gene suitable for CRISPR-Cas9 targeting editing and a human PD-1 gene are selected, and the plasmid vector of the sgRNA and a CRISPR-Cas9 nuclease gene expression vector are transferred to HPV16+ human cervical carcinoma cells and HPV16+ transplantation tumor-bearing mice, and can obviously inhibit HPV16 expression and inhibit tumor growth. The gene expression vector prepared in the invention has the advantages of simple method steps, good sgRNA targeting property, high CRISPR-Cas9 knockout efficiency, accurate targeting splicing of high risk HPV16 type and PD-1 genes, efficient reduction of the expression of the high risk HPV16 type gene, and obvious inhibition of the tumor growth through combined application.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biomedicine, and relates to a design method for CRISPR / Cas9 to specifically modify multiple gene targets of high-risk HPV16 and PD-1, and to prevent human cervical cancer by knocking out the oncogene expression of high-risk HPV16. And the combined immune gene therapy strategy was used to enhance the oncogene targeting splicing of high-risk HPV16 to treat xenograft tumors in HPV16+ mice. Background technique [0002] In recent years, genome editing tools have been widely used in the field of biomedicine, and clustered regularly interspaced short palindromic repeats (CRISPR) technology has become a hot spot in genome editing. CRISPR is a sequence naturally present in bacterial DNA that binds to CRISPR-associated (Cas) nucleases that function as guide RNAs to protect the bacterial genome from attack by target-specific sequences detected in invasive phages. CRISPR / Cas9 technology was rated as o...

AI Technical Summary

Problems solved by technology

[0006] There are problems in the eradication of high-risk HPV infection in normal human cervical epithelium and the treatment of advanced cervical cancer: (1) Drugs cannot effectively eliminate high-risk HPV infected in cells; (2) The efficiency of other methods to specifically knock out HPVE6 and E7 genes Low, the effect of reducing HPV gene expression is poor; (3) There are few therapeutic drugs targeting human cervical cancer, the effect is limited, and the side effects are more obvious; (4) The strategy of using multiple therapeutic methods to treat cervical cancer needs to improve the therapeutic effect

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