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Plasmid, system and preparation for targeted knockout of human papilloma virus (HPV) URR (upstream regulatory region) genes and preparation method of plasmid, system and preparation

A technology of human papillomavirus and gene, applied in other methods of inserting foreign genetic materials, gene therapy, medical preparations of non-active ingredients, etc., can solve the problems of high price, difficult synthesis of high-algebra PAMAM, etc., and achieve low cost , remove viruses and diseased cells, and effectively kill

Active Publication Date: 2019-05-24
ZHUHAI SHU TONG MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the synthesis of high-algebraic PAMAM is difficult and expensive

Method used

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  • Plasmid, system and preparation for targeted knockout of human papilloma virus (HPV) URR (upstream regulatory region) genes and preparation method of plasmid, system and preparation
  • Plasmid, system and preparation for targeted knockout of human papilloma virus (HPV) URR (upstream regulatory region) genes and preparation method of plasmid, system and preparation
  • Plasmid, system and preparation for targeted knockout of human papilloma virus (HPV) URR (upstream regulatory region) genes and preparation method of plasmid, system and preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1, Construction of CRISPR / Cas9 plasmid expression vector targeting HPV16 / 18URR

[0067] (1) The DNA sequences of this example are: HPV16 and HPV18 URR base sequences (NCBI database RefSeq: NC_001526.2 and NC_001357.1). Using CRISPR Design (http: / / crispr.mit.edu / ) online sgRNA design tool, design sgRNA for HPV16 / 18URR and screen the sgRNA with the best effect through cell experiments.

[0068] The selected HPV16 URR sequences are:

[0069] TAATTCATGTATAAAACTAagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO. 1).

[0070] The selected HPV18 URR sequences are:

[0071] AGGGAGTAACCGAAAACGGTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SEQ ID NO. 2).

[0072] Among them, the uppercase letters are the sequence that binds to the target DNA, and the lowercase letters are the scaffold, which is the secondary structure region where the sgRNA functions.

[0073] (2) The expression vector constructio...

Embodiment 2

[0078] Example 2, Induction of apoptosis after CRISPR / Cas9-URR transfection

[0079]The CRISPR / Cas9-URR system targeting HPV16 / 18URR expressed after the CRISPR / Cas9-URR plasmid is transfected into cells can quickly recognize the HPV16 / 18URR sequence and play a cutting role. After cleavage, cells are mainly repaired rapidly through the NHEJ pathway that is not restricted by the cell cycle, thereby introducing small fragment insertions or deletions (indels), resulting in frameshift mutations, and ultimately loss of URR function (driving E6E7 oncogene transcription), HPV16 / 18E6E7 cancer Protein expression is inhibited. On the other hand, since the copy number of URR can be as high as 50 copies, all of them can be recognized by sgRNA and cleaved by Cas9, so that multiple DSBs can be generated. If DSBs are not repaired in time and accumulate, they will induce programmed cell death mechanism and directly eliminate HPV of diseased cells. All of the above can lead to decreased proli...

Embodiment 3

[0088] Example 3, CRISPR / Cas9-URR sgRNA cutting efficiency verification

[0089] After Cas9 functions in cells, the DSBs formed by cleavage can rapidly induce cells to self-NHEJ repair. However, the error-prone repair method of NHEJ is very easy to introduce small fragments of gene insertion or deletion at the breakpoint. (Wild-type) nucleotide sequences are mixed and then annealed and extended to form a special hybrid duplex with a mismatch at the cleavage site (corresponding to the repaired base insertion or deletion). This hybrid duplex can be recognized by T7 Endonuclease I and cut at the mismatch.

[0090] Therefore, after the cells are transfected with CRISPR / Cas9-URR, the total genomic DNA of all the cells is extracted to obtain a mixed product containing both the DNA sequence repaired after cutting and the unprocessed wild-type sequence. After PCR amplification, the mixed PCR product of the DNA sequence repaired after cutting and the wild-type sequence is obtained, a...

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Abstract

The invention relates to a plasmid, system and preparation for targeted knockout of human papilloma virus (HPV) URR (upstream regulatory region) genes and a preparation method of the plasmid, system and preparation. The plasmid comprises sgRNA, Cas9 and an expression vector PX330 which can bind to the HPV URR genes. When transfected into cells, the plasmid specifically induces coding shift mutations of HPV oncogenic elements in corresponding HPV subtype positive cells to loss carcinogenic properties, even too much DSB can lead to cell apoptosis to reduce virus load, remove virus and pathological cells and reverse cancerous transformation, and important clinical application value is achieved. The preparation can effectively kill HPV positive tumor cells and effectively treat HPV positive tumor cells and is simple in preparation and low in cost.

Description

technical field [0001] The invention belongs to the field of CRISPR gene therapy, and specifically relates to a plasmid, system, preparation and preparation method for targeted knockout of human papillomavirus URR gene. Background technique [0002] Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases worldwide. Epidemiological studies have shown that about 80% of women will be infected with HPV at least once in their lifetime, while the probability of men is about 50%. Among them, the persistent infection of low-risk HPV can cause skin, genital and perianal warts, while the persistent infection of high-risk HPV can lead to malignant tumors such as cervical cancer, vulvar cancer, vaginal cancer, penile cancer, and perianal cancer. Increases the chance of HIV infection. In recent years, with the development of society and economy, HPV infection has become a serious social public health problem in China. [0003] At present, a few dev...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N9/22A61K31/715A61K48/00A61K9/06A61K47/34A61P35/00
Inventor 包煜贤
Owner ZHUHAI SHU TONG MEDICAL TECH CO LTD
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