Diagnosis and treatment target for treating HAdV-7 infection and application

Abstract

The invention discloses a diagnosis and treatment target spot for treating HAdV-7 infection and application, and relates to the field of medicine. A diagnosis and treatment target for treating HAdV-7 infection, which is a transferrin receptor TFRC; the invention relates to application of a diagnosis and treatment target for treating HAdV-7 infection, and application to diagnosis and treatment of human type 7 adenovirus HAdV-7 infection. Experimental verification finds that the transferrin receptor TFRC is a symbolic molecule infected by the human type 7 adenovirus HAdV-7, and the virus load of the human type 7 adenovirus HAdV-7 can be effectively reduced by knocking down the transferrin receptor TFRC, so that the transferrin receptor TFRC can be used as a target spot for diagnosing and treating the infection of the human type 7 adenovirus HAdV-7 and can be used for preparing a medicine for treating the infection of the human type 7 adenovirus HAdV-7. Therefore, a wide prospect is provided for diagnosis and treatment of the human type 7 adenovirus HAdV-7.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to diagnostic and therapeutic targets and applications for treating HAdV-7 infection. Background Art

[0002] Human adenovirus (HAdV) infection is a major cause of severe respiratory infections in children. Currently, over 100 genotypes and 52 serotypes of AdV have been identified, divided into seven species groups, HAdV-A through -G. Adenoviruses are primarily transmitted through respiratory droplets, fecal-oral routes, and contact with fomites. They spread rapidly in densely populated or enclosed areas. They have broad tissue tropism and can affect multiple tissues and organs. Respiratory infections are the most common, affecting people of all ages. While generally self-limited, they can cause severe pneumonia, acute respiratory distress syndrome, respiratory failure, obliterative bronchitis, encephalitis, and even death in children and immunocompromised individuals. The incidence of severe illness caused by adenovirus infection ranges from 6.5% to 40.6%, with a mortality rate of 0.8% to 12%. HAdV infection accounts for up to 24.5% of severe pneumonia cases. HAdV-7 is the most common adenovirus subtype responsible for severe pneumonia.

[0003] Currently, the diagnosis of HAdV-7 mainly relies on the pathogen's antigens, antibodies and viral copy number. The disease status after human infection mainly relies on routine examination indicators such as blood routine, liver and kidney function, C-reactive protein (CRP), and procalcitonin (PCT). These cannot directly reflect the status of the human body after HAdV-7 infection, and it is also difficult to evaluate the effectiveness of adenovirus infection treatment.

[0004] There is currently no specific treatment for HAdV-7 infection. Severe adenovirus pneumonia is mainly treated with broad-spectrum antiviral drugs and symptomatic treatment. Broad-spectrum antiviral drugs include cidofovir, ganciclovir, and ribavirin, but these broad-spectrum antiviral drugs have obvious limitations. Among them, cidofovir is a cytosine analog that inhibits viral deoxyribonucleic acid synthesis. It was originally an antiviral drug for cytomegalovirus infection and was later proven to have anti-adenovirus effects. It binds to the adenoviral DNA chain through the virus-encoded E2B polymerase, forming an irreversible stable structure that prevents viral DNA synthesis. However, cidofovir is subject to some restrictions due to its low bioavailability and nephrotoxicity, especially in children and immunocompromised people; ganciclovir, as a synthetic analog of deoxyguanosine, was originally used to treat herpes virus infection and was later proven to have inhibitory effects on the replication of multiple serotypes of adenovirus, but ganciclovir has serious adverse reactions such as bone marrow suppression, and ganciclovir needs to be activated by viral thymidine kinase to work, but adenovirus does not encode this kinase; ribavirin is a nucleoside inosine nucleoside analog, and its main indications are respiratory syncytial virus and cutaneous herpes virus infections. Relevant studies have shown that it can be used as a way to treat adenovirus, but it carries the risk of fetal teratogenicity and hemolytic anemia. Symptomatic treatment mainly includes human immunoglobulin (IVIG), plasma exchange and extracorporeal membrane oxygenation (ECOM), but these treatments are expensive and have limited therapeutic effects.

[0005] To this end, the present invention proposes diagnostic and therapeutic targets and applications for treating HAdV-7 infection. Summary of the Invention

[0006] The purpose of the present invention is to address the shortcomings of the prior art and to propose diagnostic and therapeutic targets and applications for treating HAdV-7 infection.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The therapeutic target for treating HAdV-7 infection is the transferrin receptor TFRC, whose protein sequence is:.

[0009] The use of diagnostic and therapeutic targets for treating HAdV-7 infection is characterized by being applied to the diagnosis and treatment of human adenovirus type 7 HAdV-7 infection.

[0010] The beneficial effects of the present invention are:

[0011] 1. The present invention experimentally verified that transferrin receptor TFRC is a marker molecule for human adenovirus type 7 HAdV-7 infection. Knocking down transferrin receptor TFRC can effectively reduce the viral load of human adenovirus type 7 HAdV-7. Therefore, transferrin receptor TFRC can be used as a target for the diagnosis and treatment of human adenovirus type 7 HAdV-7 infection, thereby providing broad prospects for the diagnosis and treatment of human adenovirus type 7 HAdV-7. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The diagnostic and therapeutic targets for the treatment of HAdV-7 infection proposed in the present invention and the expression level of TFRC protein were detected by Western Blot after HAdV-3 / HAdV-7 / HAdV-55 / rAd3 / 7-F infected HPAEpic cells;

[0013] Figure 2 The diagnostic and therapeutic targets for the treatment of HAdV-7 infection proposed in the present invention and the expression level of TFRC protein were detected by Western Blot after HAdV-3 / HAdV-7 / HAdV-55 / rAd3 / 7-F infected A549 cells;

[0014] Figure 3 Laser confocal images of the therapeutic targets for treating HAdV-7 infection proposed in the present invention and taken 24 hours after HAdV-3 / HAdV-7 infected HPAEpic cells;

[0015] Figure 4 The present invention proposes a diagnostic and therapeutic target for treating HAdV-7 infection and its application, which is a graph of TFRC protein expression levels detected by Western Blot after HPAEpic cells were infected with different HAdV-7 virus infection doses;

[0016] Figure 5 Figure 2 is a diagram showing the diagnostic and therapeutic targets for the treatment of HAdV-7 infection proposed in the present invention and the statistical analysis of TFRC protein expression levels, where ns, no statistical difference, *, p < 0.05, ****, p < 0.0001, n = 3;

[0017] Figure 6 The present invention provides a diagnostic and therapeutic target for the treatment of HAdV-7 infection and a graph showing the viral load of cells and supernatant detected by HPAEpic cells infected with HAdV-7 and knocking down TFRC;

[0018] Figure 7 This is a diagram of the diagnostic and therapeutic target for the treatment of HAdV-7 infection proposed in the present invention and the detection of cell and supernatant viral loads in HPAEpic cells infected with HAdV-7 and knocked down NC. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0020] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "connected," "connected," and "disposed" should be understood in a broad sense. For example, they may refer to fixed connection or disposition, detachable connection or disposition, or integral connection or disposition. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0021] Example 1:

[0022] The above-mentioned different types of adenovirus, including HAdV-3, HAdV-7, HAdV-55 and rAd3 / 7-F, a total of 4 different subtypes of respiratory infection adenovirus subtypes, were used to infect alveolar epithelial cells, including normal human alveolar type II epithelial cells (HPAEPic) and tumor alveolar epithelial cells (A549). When obvious staphylococcal lesions appeared in each group, specimens were collected, and cells were lysed using Western Blot lysis buffer to prepare protein samples for protein immunoblotting (Western Blot) experiments. The experimental results showed that TFRC was significantly increased in the HAdV-7 infection group ( Figure 1 and Figure 2 HPAEpic cells were infected with HAdV-3 / HAdV-7 at the same infection dose for 24 hours, and then fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton for 20 minutes, blocked with 5% BSA for 30 minutes, incubated with primary antibodies (TFRC and Hexon) at 4°C overnight, incubated with secondary antibodies at room temperature for 1.5 hours, stained with DAPI for 20 minutes, and sealed with anti-fluorescence quencher to complete specimen preparation. The expression of Hexon and TFRC proteins was detected using laser confocal microscopy. The experimental results showed that the green fluorescence corresponding to Hexon was significantly expressed in cells infected with HAdV-3 or HAdV-7, but the red fluorescence corresponding to TFRC was not significantly expressed in the HAdV-3 group, but was significantly expressed in the HAdV-7 group ( Figure 3 ), suggesting that TFRC is a hallmark molecule of HAdV-7 infection.

[0023] Example 2:

[0024] Studies have shown that the higher the adenovirus viral load, the more severe the patient's disease. In this example, HPAEpic cells were infected with different HAdV-7 viral infection doses to simulate mild, moderate, and severe HAdV-7 infection in humans. Samples were collected 24 hours after infection, and cells were lysed using Western Blot lysis buffer to prepare protein samples for Western Blot analysis. The experimental results showed that the protein expression level of TFRC was positively correlated with the HAdV-7 viral infection dose ( Figure 3 and Figure 4 ). This indicates that TFRC is a diagnostic target for HAdV-7 virus infection and can indicate the severity of the disease caused by HAdV-7 virus infection in humans.

[0025] Example 3:

[0026] In this example, RNA interference technology was used to construct HPAEpic cells with TFRC knockdown and NC control cells. The same HAdV-7 infection dose was used, and samples were collected 48 hours later. Viral DNA was extracted using a viral DNA / RNA purification kit. The viral load of HAdV-7 was detected using Q-PCR technology using Hexon plasmid as a standard curve. It was found that knocking down TFRC could significantly reduce the viral load ( Figure 5 and Figure 6 In addition, in order to explore the relationship between TFRC and Hexon, two drugs were introduced in this example: MG132 was used to inhibit the degradation of TFRC; and cycloheximide (CHX) was used to inhibit the synthesis of TFRC. First, HPAEPic cells were pretreated with MG132 and CHX for 12 hours, respectively, and then infected with the same infection dose of HAdV-7 for 48 hours. Then, the cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton for 20 minutes, blocked with 5% BSA for 30 minutes, incubated with primary antibodies (TFRC and Hexon) at 4°C overnight, incubated with secondary antibodies at room temperature for 1.5 hours, stained with DAPI for 20 minutes, and sealed with anti-fluorescence quencher to complete specimen preparation. The expression of Hexon and TFRC proteins was detected by laser confocal microscopy. The experimental results showed that compared with the +HAdV-7 group, the red fluorescence (TFRC) and green fluorescence (Hexon) intensities of the MG132+HAdV-7 group were significantly increased, while those of the CHX+HAdV-7 group were significantly decreased ( Figure 7 ), indicating that regulating TFRC can effectively affect Hexon protein expression, indicating that TFRC is a therapeutic target for HAdV-7 infection, and drugs designed to target TFRC are expected to become effective drugs for the treatment of HAdV-7 infection

[0027] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A therapeutic target for treating HAdV-7 infection, characterized in that: It is the transferrin receptor TFRC, and its protein sequence is: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF。 2. Use of a therapeutic target for treating HAdV-7 infection, characterized in that: Applied to the diagnosis and treatment of human adenovirus type 7 (HAdV-7) infection.

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