HPV (human papillomavirus) infected human epidermal keratinocyte model as well as construction method and application thereof

Abstract

The invention provides an HPV (human papillomavirus) infected human epidermal keratinocyte model and a construction method and application thereof, and relates to the technical field of molecular biology, an HPV genome is integrated to a KRT5 site of genome DNA (deoxyribonucleic acid) of human epidermal keratinocytes to obtain the HPV infected human keratinocyte model, stable integration and expression of an HPV whole genome are successfully realized, and the HPV infected human keratinocyte model is obtained. The position effect caused by random insertion is avoided, and the genetic stability is ensured; the model has the synergistic expression function of HPV early and late genes, maintains the time sequence of virus gene expression, realizes HPV virus particle assembly, and simulates the complete life cycle of HPV. The technical problem that in the prior art, an infection model cannot simulate the complete life cycle of the HPV is solved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to an HPV-infected human epidermal keratinocyte model, its construction method, and its application. Background Technology

[0002] Human papillomaviruses (HPV) can cause benign epidermal lesions (warts and papillomas) and have a risk of cancer. HPV genomic DNA typically exists either extrachromosomally free or integrated into the host chromosome, consisting of approximately 8000 bp of double-stranded circular DNA encoding six early regulatory proteins (E1, E2, E4, E5, E6, and E7) and two structural proteins (L1 and L2). Its life cycle, including infection, replication, and viral assembly, is completed in the host's skin or mucous membrane epithelial cells. Currently, more than 450 HPV types have been identified, classified into five genera (α, β, γ, ν, and μ) based on L1 gene homology. HPV infection exhibits host specificity; different genera show varying tropism for epithelial cells in different locations. α-HPV primarily infects mucous membranes and is called mucosal HPV, while β-HPV primarily infects the skin and is called cutaneous HPV. Furthermore, HPV is classified into low-risk and high-risk types based on its pathogenicity. Epidemiological surveys indicate that low-risk cutaneous HPV types (HPV-1, 2, 3, 4, 7, 10, 12, 15, etc.) are associated with common warts, flat warts, and plantar warts, while high-risk cutaneous HPV types (HPV-5, 8, 14, 17, 20, 36, 38) are closely associated with verrucous epidermal dysplasia. Studies have confirmed that persistent infection with high-risk β-HPV can induce excessive proliferation of keratinocytes and malignant transformation, ultimately leading to squamous cell carcinoma and basal cell carcinoma of the skin.

[0003] Because HPV cannot efficiently infect and replicate in traditional cell culture systems, the commonly used HPV research model is an immortalized keratinocyte cell line (such as HaCaT) in which HPV genes (usually key "early gene regions," such as E6 and E7) are introduced. While these models are easy to operate, their cell sources are mostly cancerous or immortalized cells with unstable genomes, and they cannot fully simulate the layering, differentiation, and complete life cycle of normal epithelial tissue. More importantly, they usually require the use of retroviruses or lentiviruses to randomly integrate HPV gene fragments into the host genome. This random integration cannot control the copy number and integration site, leading to abnormal viral gene expression and failing to accurately reproduce the replication and pathogenesis process of the virus in its natural chromosomal environment. Furthermore, random integration results in uncontrollable viral gene expression and uneven copy numbers, severely affecting the reproducibility of experiments and the reliability of data.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for constructing a human epidermal keratinocyte model infected with HPV, in order to solve the technical problem that existing infection models cannot simulate the complete life cycle of HPV.

[0006] The second objective of this invention is to provide an HPV-infected human epidermal keratinocyte model.

[0007] A third objective of this invention is to provide an HPV-infected human epidermal keratinocyte model prepared by the above-described construction method, or the application of the above-described HPV-infected human epidermal keratinocyte model in screening or evaluating anti-HPV drugs.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for constructing an HPV-infected human epidermal keratinocyte model, comprising integrating the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes to obtain an HPV-infected human keratinocyte model.

[0009] Furthermore, integrating the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes involves introducing a homologous recombinant plasmid containing the HPV genome, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human epidermal keratinocytes. The sgRNA is a specific sgRNA targeting the KRT5 site, and the sequence of the sgRNA is shown in SEQ ID NO.6.

[0010] Furthermore, the homologous recombination plasmid includes the HPV genome, and has an upstream homologous arm upstream of the HPV genome and a downstream homologous arm downstream of the HPV genome; the upstream and downstream homologous arms are used to integrate the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes; The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.8.

[0011] Furthermore, the HPV genome includes HPV1 genome, HPV2 genome, HPV5 genome, HPV8 genome, or HPV16 genome.

[0012] Furthermore, the plasmid expressing Cas9 or Cas12 also contains a selection marker gene; Preferably, the screening marker gene includes a puromycin resistance gene; The plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

[0013] Secondly, the present invention provides an HPV-infected human epidermal keratinocyte model, characterized in that it is prepared by the above-described construction method.

[0014] Furthermore, in the HPV-infected human epidermal keratinocyte model, the HPV genome is in an activated state.

[0015] Furthermore, the activated state includes at least one of early HPV gene initiation expression, HPV genome replication, late gene expression, capsid protein synthesis, and complete HPV viral particle assembly.

[0016] Thirdly, the present invention provides the application of the HPV-infected human epidermal keratinocyte model prepared by the above-described construction method, or the application of the above-described HPV-infected human epidermal keratinocyte model in screening or evaluating anti-HPV drugs.

[0017] Furthermore, the anti-HPV drug includes at least one of the following: neutralizing antibodies targeting capsid proteins, viral entry inhibitors, small molecule capsid binders, small molecule substances targeting key viral replication enzymes, immunomodulators, or therapeutic vaccines.

[0018] This invention provides a method for constructing an HPV-infected human epidermal keratinocyte model, successfully achieving stable integration and expression of the entire HPV genome, avoiding positional effects caused by random insertions, and ensuring genetic stability. The model possesses the co-expression function of early and late HPV genes, maintaining the temporal sequence of viral gene expression, realizing HPV viral particle assembly, and simulating the complete HPV life cycle. This solves the technical problem that existing infection models cannot simulate the complete HPV life cycle. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 The image shows the pKRT5-HPV-Donor expression plasmid provided in Example 1 of this invention; Figure 2 A map of the pLentiV2-Cas9-U6-KRT5-sgRNA expression plasmid provided in Example 1 of this invention; Figure 3 The mRNA expression levels of early and late HPV genes in the HPV-infected human epidermal keratinocyte model provided in Example 2 of this invention; Figure 4 This is an image showing the morphology of viral particles in the HPV1-infected human epidermal keratinocyte model provided in Example 2 of the present invention. Figure 5 This is an image showing the morphology of viral particles in the HPV2-infected human epidermal keratinocyte model provided in Example 2 of the present invention. Figure 6 This is an image showing the morphology of viral particles in the HPV5-infected human epidermal keratinocyte model provided in Example 2 of the present invention. Figure 7 This is an image showing the morphology of viral particles in the HPV8-infected human epidermal keratinocyte model provided in Example 2 of the present invention. Figure 8 This is an image showing the morphology of viral particles in the HPV16-infected human epidermal keratinocyte model provided in Example 2 of the present invention. Figure 9 The results of the sensitivity test of the HPV1-infected human epidermal keratinocyte model against HPV compounds provided in Example 2 of the present invention; Figure 10 The results of the sensitivity test of the HPV2-infected human epidermal keratinocyte model against HPV compounds provided in Example 2 of the present invention; Figure 11 The results of the sensitivity test of the HPV5-infected human epidermal keratinocyte model against HPV compounds provided in Example 2 of the present invention; Figure 12 The results of the sensitivity test of the HPV8-infected human epidermal keratinocyte model against HPV compounds provided in Example 2 of the present invention; Figure 13 The results of the sensitivity test of the HPV16-infected human epidermal keratinocyte model against HPV compounds provided in Example 2 of the present invention. Detailed Implementation

[0021] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0022] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention provides a method for constructing an HPV-infected human epidermal keratinocyte model, comprising integrating the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes to obtain an HPV-infected human keratinocyte model.

[0025] The model successfully achieved stable integration and expression of the entire HPV genome, avoiding positional effects caused by random insertions and ensuring genetic stability. It possesses the ability to co-express early and late-stage HPV genes, maintaining the temporal sequence of viral gene expression, achieving HPV viral particle assembly, and simulating the complete HPV life cycle. This solves the technical problem that existing infection models cannot simulate the complete HPV life cycle. It can be used to study the mechanisms of HPV infection in host cells, investigate skin diseases caused by HPV infection (including viral warts, basal cell carcinoma, squamous cell carcinoma, etc.), and apply to the screening of anti-HPV drugs and the development of detection methods.

[0026] In some specific embodiments, integrating the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes involves introducing a homologous recombinant plasmid containing the HPV genome, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human epidermal keratinocytes; wherein the sgRNA is a specific sgRNA targeting the KRT5 site, and the sequence of the sgRNA is shown in SEQ ID NO.6.

[0027] In some specific embodiments, the homologous recombination plasmid includes an HPV genome, and has an upstream homologous arm upstream of the HPV genome and a downstream homologous arm downstream of the HPV genome; the upstream and downstream homologous arms are used to integrate the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes; the nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.8.

[0028] In some specific implementations, the HPV genome includes HPV1 genome, HPV2 genome, HPV5 genome, HPV8 genome, or HPV16 genome.

[0029] In some specific embodiments, the plasmid expressing Cas9 or Cas12 further contains a selection marker gene; in some specific embodiments, the selection marker gene includes a puromycin resistance gene.

[0030] According to another aspect of the present invention, an HPV-infected human epidermal keratinocyte model is also provided, characterized in that it is prepared by the above-described construction method.

[0031] In some specific implementations, the HPV genome is in an activated state in the HPV-infected human epidermal keratinocyte model.

[0032] The activated state includes at least one of the following: early HPV gene initiation expression, HPV genome replication, late gene expression, capsid protein synthesis, and complete HPV viral particle assembly.

[0033] According to another aspect of the present invention, the application of the HPV-infected human epidermal keratinocyte model prepared by the above-described construction method, or the application of the above-described HPV-infected human epidermal keratinocyte model in screening or evaluating anti-HPV drugs, is also provided.

[0034] In some specific embodiments, the anti-HPV drug includes at least one of the following: a neutralizing antibody targeting the capsid protein, a viral entry inhibitor, a small molecule capsid binder, a small molecule substance targeting a key enzyme in viral replication, an immunomodulator, or a therapeutic vaccine.

[0035] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0036] Example 1: Construction of an engineered human epidermal keratinocyte cell line Using CRISPR / Cas9-mediated homologous recombination technology, the complete genomes of HPV1, HPV2, HPV5, HPV8, and HPV16 were precisely integrated into the KRT5 site of the human epidermal keratinocyte genome, thereby constructing engineered human epidermal keratinocyte cell lines carrying different HPV subtypes. The HPV1, HPV2, HPV5, and HPV8 genomes were synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and each genome contains an early region, a late region, and an upstream regulatory region. The nucleotide sequences of the HPV1, HPV2, HPV5, HPV8, and HPV16 genomes are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The specific steps are as follows: 1. Construction of gene knock-in vector Target selection: The human genome site KRT5 was selected, and sgRNA targeting this site was designed to ensure high cleavage efficiency and low off-target effects. The sgRNA sequence is shown in SEQ ID NO.6.

[0037] Homologous arms: A 550bp left homologous arm (SEQ ID NO.7) and a 550bp right homologous arm (SEQ ID NO.8) were added on both sides of the HPV genome sequence. The homologous arm sequences are homologous to the genome sequences flanking the KRT5 target sequence.

[0038] The left and right homologous arms were cloned into the IRES vector, and then the HPV genome was cloned into the IRES vector containing the homologous arms, resulting in the homologous recombination plasmid IRES vector containing the HPV genome, named pKRT5-IRES-HPV, whose structure is as follows. Figure 1 As shown, the HPV genome is located between two homologous arms at the insertion site. The specific sequences of the homologous recombinant plasmid IRES containing the HPV genome include: the left homologous arm sequence of the target gene, the HPV genome, and the right homologous arm sequence of the target gene.

[0039] The KRT5-sgRNA sequence used for gene editing was cloned into the pLentiV2-Cas9-U6-sgRNA plasmid expression vector to obtain the pLentiV2-Cas9-U6-KRT5-sgRNA expression plasmid, as shown in the diagram. Figure 2 As shown.

[0040] 2. Construction, screening, and genotyping of human epidermal keratinocyte lines carrying the HPV genome. pLentiV2-Cas9-U6-KRT5-sgRNA and pKRT5-IRES-HPV were mixed at a plasmid molar ratio of 1:2, incubated with polyethyleneimine (PEI) transfection reagent, and transfected into normal human epidermal keratinocytes (NHEK). Forty-eight hours after transfection, 1 μg / ml puromycin was added and the cells were cultured for 24 hours. After selection, 80 monoclonal cells were seeded into 96-well plates using a limiting dilution method and cultured for another half month. The selected monoclonal cells were then transferred to 24-well plates for culture, and cellular DNA was extracted for PCR gel electrophoresis and Sanger sequencing identification. The resulting normal human epidermal keratinocyte cell line with KRT5-IRES-HPV knock-in was named NHEK-KRT5-IRES-HPV.

[0041] Example 2: Validation of HPV-infected human epidermal keratinocyte model This embodiment uses the NHEK-KRT5-IRES-HPV constructed in Example 1 as a sample. The mRNA expression levels of early and late HPV genes in the model were detected by qRT-PCR. The results are as follows: Figure 3 As shown, this indicates that viral gene expression was achieved in engineered cell lines after HPV gene knock-in.

[0042] 2. Virus particle assembly and morphology verification: Transmission electron microscopy was used to observe NHEK-KRT5-IRES-HPV sections, and the results are as follows: Figures 4-8 As shown in the figure (scale bar is 500nm), icosahedral virus particles with a diameter of about 55nm were found in keratinocytes, proving that the model can support the complete viral life cycle and successfully achieve viral assembly.

[0043] 3. Model Functional Validation: Drug sensitivity testing was performed using a known anti-HPV compound (podophyllotoxin, Podofilox) at a concentration of 20 μM for 72 hours. The results are as follows: Figures 9-13 As shown, qRT-PCR was used to verify that E6 expression was downregulated or L1 protein expression was reduced in the Podocilox group, demonstrating that the model can be used for drug screening.

[0044] This invention utilizes CRISPR / Cas9 technology to target the KRT5 site on human epidermal keratinocytes. By knocking in the whole genome sequences of different HPV types, an HPV-NHEK cell model is constructed, which is then used to study the mechanism of HPV infection of the skin and to screen anti-HPV drugs.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an HPV-infected human epidermal keratinocyte model, characterized in that, This includes integrating the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes to obtain an HPV-infected human keratinocyte model.

2. The construction method according to claim 1, characterized in that, The KRT5 site for integrating the HPV genome into the genomic DNA of human epidermal keratinocytes involves co-introducing a homologous recombinant plasmid containing the HPV genome, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human epidermal keratinocytes. The sgRNA is a specific sgRNA targeting the KRT5 site, and the sequence of the sgRNA is shown in SEQ ID NO.

6.

3. The construction method according to claim 2, characterized in that, The homologous recombination plasmid includes the HPV genome and has an upstream homologous arm upstream of the HPV genome and a downstream homologous arm downstream of the HPV genome; the upstream and downstream homologous arms are used to integrate the HPV genome into the KRT5 site of the genomic DNA of human epidermal keratinocytes. The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.

8.

4. The construction method according to claim 3, characterized in that, The HPV genome includes HPV1, HPV2, HPV5, HPV8, or HPV16 genomes.

5. The construction method according to claim 2, characterized in that, The plasmids expressing Cas9 or Cas12 also contain selection marker genes; Preferably, the screening marker gene includes a puromycin resistance gene; The plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

6. A human epidermal keratinocyte model infected with HPV, characterized in that, It is prepared by the construction method described in any one of claims 1 to 5.

7. The HPV-infected human epidermal keratinocyte model according to claim 6, characterized in that, In the HPV-infected human epidermal keratinocyte model, the HPV genome was in an activated state.

8. The HPV-infected human epidermal keratinocyte model according to claim 7, characterized in that, The activation state includes at least one of the following: early HPV gene initiation expression, HPV genome replication, late gene expression, capsid protein synthesis, and complete HPV viral particle assembly.

9. The application of the HPV-infected human epidermal keratinocyte model prepared by the construction method according to any one of claims 1 to 5, or the HPV-infected human epidermal keratinocyte model according to any one of claims 6 to 8, in screening or evaluating anti-HPV drugs.

10. The application according to claim 9, characterized in that, The anti-HPV drug includes at least one of the following: neutralizing antibodies targeting capsid proteins, viral entry inhibitors, small molecule capsid binders, small molecule substances targeting key viral replication enzymes, immunomodulators, or therapeutic vaccines.

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