HCMV-gB nano antibody as well as preparation method and application thereof

Abstract

The invention provides an HCMV-gB nano antibody as well as a preparation method and application thereof. Specifically, the nano antibody comprises a frame region FR and a complementarity determining region CDR, the frame region FR comprises FR1-4 of which the amino acid sequences are respectively shown as SEQ ID NO1, 3, 5 and 7, and the complementarity determining region CDR comprises CRD1-3 of which the amino acid sequences are respectively shown as SEQ ID NO.2, 4 and 6; the invention also provides a nucleic acid molecule for coding the nano antibody, an expression vector containing the nucleic acid molecule, a host cell containing the expression vector, a medicine or a preparation containing the nucleic acid molecule and the expression vector, and application of the nucleic acid molecule and the expression vector to preparation of a medicine or a reagent for treating or detecting HCMV infection. The product disclosed by the invention not only has the advantages of small molecular weight, simple structure, easiness in high-efficiency expression and the like, but also shows very excellent HCMV-gB binding specificity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a nanobody targeting the HCMV-gB epitope, its preparation method and application. Background Technology

[0002] Human cytomegalovirus (HCMV) is a herpesvirus that is highly prevalent worldwide. This virus has a latent infection characteristic; once it invades the human body, it can remain dormant in various cells, including hematopoietic stem cells and endothelial cells, for a long period. When the body's immune function declines, the latent virus can be reactivated and replicate in large quantities, causing severe clinical symptoms. Studies have shown that the pathogenicity of HCMV is significantly enhanced in immunocompromised individuals.

[0003] Current clinical methods for detecting and treating HCMV infection have limitations. For example, while existing detection methods can quantify viral load, they lack specific biomarkers for latent infection, leading to frequent missed diagnoses. Some methods are cumbersome and time-consuming, failing to meet the need for rapid diagnosis. In terms of treatment, antiviral drugs easily induce drug resistance and often involve serious adverse reactions; passive immunization agents face challenges regarding their availability and efficacy. Furthermore, no drug can effectively eliminate latent virus to prevent its reactivation.

[0004] Human cytomegalovirus (HCMV) glycoprotein B (gB) is the most abundant glycoprotein on the viral envelope and plays a crucial role in the viral life cycle. Its N-terminal domain is highly specific, binding to receptors such as heparan sulfate and platelet-derived growth factor receptor α (PDGFRα) on the host cell surface; this process is the initial step in viral adsorption to the host. When the virus approaches the host cell, the fusion domain at the middle position of gB undergoes a significant conformational change under specific acidic environmental stimulation, causing the viral envelope to gradually approach and eventually fuse with the host cell membrane, creating the necessary conditions for the viral nucleocapsid to successfully enter the host cell. During viral assembly and maturation, the C-terminal intracellular domain of gB plays an indispensable role, participating in the assembly of viral particles in the Golgi apparatus and promoting the formation of infectious mature viral particles. HCMV-gB-mediated invasion works in two ways: firstly, by occupying host receptors with high affinity through its extracellular domain, thereby blocking natural ligand signal transduction; and secondly, by recruiting negatively regulated phosphatases through its intracellular domain, inhibiting host type I interferon pathway signal transduction. Although the copy number of gB on the surface of viral particles is only 1 / 10 to 1 / 20 of that of the gH / gL complex, its affinity for the receptor is more than 50 times that of gH / gL, making it the most critical invading molecule in the early stages of HCMV infection. Therefore, HCMV-gB is widely recognized as an ideal target for developing diagnostic and therapeutic strategies for HCMV infection.

[0005] Nanobody technology is an innovative achievement in antibody engineering, combining molecular biology techniques with the concepts of nanoparticle science, based on traditional antibodies. This has led to the development of functional antibody fragments with the smallest molecular weight. In 1993, Hamers et al. first reported the natural existence of a class of heavy-chain antibodies in camels that lack both the light chain and the heavy chain constant region 1 (CH1). Cloning their variable region yielded single-domain antibodies consisting only of a single heavy-chain variable region, initially named VHH (heavy-chain antibody variable domain), later collectively referred to as "nanobody" (Nb).

[0006] As the smallest antibody fragment with complete antigen-binding capability, nanobodies have an elliptical crystal structure, approximately 2.5 nm in diameter and 4 nm in length, and possess a series of unique advantages: their structure is simpler than that of traditional antibodies, they are easier to conjugate with other molecules, and they can be efficiently expressed by microorganisms. Furthermore, nanobodies exhibit excellent environmental tolerance, high conformational stability, and small molecular weight, making them not only easy to synthesize and cost-effective but also enhancing the targeting and efficacy of clinical treatments. These characteristics make them show great application potential in areas such as precision disease diagnosis and immunotherapy.

[0007] Given the unique advantages of nanobodies, the development of specific nanobodies against HCMV-gB for the diagnosis and treatment of HCMV infection has broad application prospects. Summary of the Invention

[0008] To address the aforementioned problems in the detection or treatment of HCMV infection in existing technologies, the present invention provides, in a first aspect, an HCMV-gB nanobody, wherein the nanobody comprises a framework region (FR) and a complementarity-determining region (CDR), wherein: The frame region FR includes FR1, FR2, FR3 and FR4, and the complementarity determination region CDR includes CRD1, CRD2 and CRD3; The nanobody comprises FR1, CRD1, FR2, CRD2, FR3, CRD3 and FR4 from N-terminus to C-terminus; The amino acid sequence of FR1 is shown in SEQ ID NO: 1, the amino acid sequence of FR2 is shown in SEQ ID NO: 3, the amino acid sequence of FR3 is shown in SEQ ID NO: 5, and the amino acid sequence of FR4 is shown in SEQ ID NO: 7. The amino acid sequence of CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 6.

[0009] Preferably, the HCMV-gB nanobody has the amino acid sequence shown in SEQ ID NO: 9.

[0010] More preferably, the amino acid sequence of the HCMV-gB nanobody is shown in SEQ ID NO: 9.

[0011] In a second aspect, the present invention provides a nucleic acid molecule that encodes the HCMV-gB nanobody described in the first aspect of the present invention.

[0012] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 8.

[0013] More preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 8.

[0014] In a third aspect, the present invention provides an expression vector containing a nucleotide sequence as shown in SEQ ID NO: 8.

[0015] In a fourth aspect, the present invention provides a host cell containing the expression vector described in the third aspect of the present invention.

[0016] In a fifth aspect, the present invention provides a medicament for treating HCMV infection, the medicament comprising the HCMV-gB nanobody described in the first aspect of the present invention, the expression vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.

[0017] In a sixth aspect, the present invention provides a reagent for detecting HCMV infection, the reagent comprising the HCMV-gB nanobody described in the first aspect of the present invention, the expression vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.

[0018] The present invention provides, in its seventh aspect, the use of the HCMV-gB nanobody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the host cell described in the fourth aspect of the present invention in the preparation of a drug for treating HCMV.

[0019] The present invention provides, in its eighth aspect, the use of the HCMV-gB nanobody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the host cell described in the fourth aspect of the present invention in the preparation of reagents for detecting HCMV-gB molecules.

[0020] Compared with the prior art, the present invention has the following technical advantages: This invention first synthesizes HCMV-gB protein and uses this protein to immunize camels, constructing a specific immune library against HCMV-gB molecules. Then, this invention conjugates HCMV-gB molecules onto an ELISA plate, and uses this antigen in this form to screen an immune nanobody gene library (camel heavy chain antibody phage display gene library) using phage display technology. This yields genes for nanobodies with excellent HCMV-gB specificity. These genes are then transferred into *E. coli*, thereby establishing a nanobody strain that can be efficiently expressed in *E. coli*. The nanobodies and related products of this invention can be used for the detection or treatment of HCMV infection. Attached Figure Description

[0021] Figure 1 This is an SDS-PAGE electrophoresis image of the HCMV-gB nanobody after purification by nickel column resin gel affinity chromatography. Analysis using gel imaging software shows that the purity of the HCMV-gB nanobody after this purification process can reach over 90%.

[0022] Figure 2 The binding ability of HCMV-gB nanobodies to rhHCMV-gB antigen was detected using an ELISA method. The results showed that HCMV-gB nanobodies of different concentrations could specifically bind to the coated rhHCMV-gB antigen, but showed no binding reaction with Irrelevant human Ag, indicating that the rhHCMV-gB nanobodies have good specificity.

[0023] Figure 3 The ELISA method was used to detect the specific binding of HCMV-gB nanobodies to rhHCMV-gB antigen. The results showed that the binding ability of HCMV-gB nanobodies to the corresponding antigen decreased after rhHCMV-gB protein blockade.

[0024] Figure 4 The binding ability of HCMV-gB nanobodies to relevant target cells was detected using flow cytometry. The results showed that HCMV-gB nanobodies could specifically bind to HFF fibroblasts expressing rhHCMV-gB molecules. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments.

[0026] Example 1: Construction of a nanobody library targeting HCMV-gB 1 mg of rhHCMV-gB protein (recombinant human HCMV-gB protein) (purchased from Bipsys Biotechnology Co., Ltd.) was dissolved in sterile deionized water to prepare a 1 mL / mg solution. This solution was then thoroughly mixed with an equal volume of Freund's adjuvant and used to immunize dromedary camels once a week for seven consecutive weeks to induce immune cells to produce specific nanobodies against the HCMV-gB antigen. After seven immunizations, lymphocytes were isolated from camel peripheral blood, and total RNA was extracted and reverse transcribed into cDNA. The VHH gene fragment was amplified by PCR using the cDNA as a template. Subsequently, the pComb3 phage display vector and the VHH fragment were digested with restriction endonucleases PstI and NotI, respectively. The digested products were ligated and transformed into TG1 competent cells to construct an HCMV-gB nanobody library. The final library size was determined to be 9.6 × 10⁻⁶. 9 CFU.

[0027] Example 2: Screening of phage libraries containing nanobodies targeting HCMV-gB rhHCMV-gB protein (purchased from Bipsys Biotechnology Co., Ltd.) was dissolved in 100 mM sodium bicarbonate solution to a concentration of 200 μg / mL, and the solution was added to an ELISA plate and incubated overnight at 4°C. The next day, blocking buffer was added, and the plate was blocked at room temperature for 2 hours, followed by washing with PBST buffer. Then, the phage prepared in Example 1 was added, and the plate was incubated at room temperature for 1 hour. The plate was then washed with PBST to remove non-specifically bound phage. Next, phage specifically bound to rhHCMV-gB protein was eluted with elution buffer, and these phages were used to infect TG1 competent cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 hour to expand the phage culture for the next round of screening. This process was repeated multiple times until phage clones specifically bound to rhHCMV-gB protein were enriched.

[0028] Example 3: Screening for HCMV-gB-specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA). Two hundred single colonies were randomly selected from the TG1 phage-enriched cells from the second round of enrichment and inoculated into 96-well plates (each well containing 200 μL of TB medium with ampicillin). The plates were cultured with shaking until the logarithmic growth phase, and then induced overnight with 1 mM IPTG. The next day, the bacterial cells were collected by centrifugation, and a crude antibody supernatant was prepared using the bacterial cells via osmotic shock assay. The supernatant was transferred to an ELISA plate coated with antigen HCMV-gB (dissolved in 100 mM NaHCO3, pH 9.6) that had been incubated overnight at 4°C, with the NaHCO3-coated wells serving as negative controls. The plates were incubated at room temperature for 1 hour. Unbound antibodies were washed away with PBST, and mouse anti-HAtag antibody was added. The plates were incubated at room temperature for 1 hour. Unbound antibodies were washed away again with PBST, and then anti-mouse IgG HRP was added. The plates were incubated at room temperature for 1 hour. Unbound antibodies were washed away with PBST, and then TMB chromogenic buffer was added. The absorbance was read at 450 nm using a microplate reader. Clones with an OD450 ≥ 3 times that of NaHCO3-coated wells were considered positive. A total of 145 positive clones were obtained in this round. Fifty of these positive clones were amplified, plasmids were extracted, and sequenced. DNAMAN was used for sequence analysis; clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different CDR1, CDR2, and CDR3 sequences were considered different clones.

[0029] Six HCMV-gB nanobody genes with different CDR3 values ​​were selected from the obtained sequences and transformed into E. coli expression strains. After large-scale culture, nanobody expression was induced by IPTG. After culture, the crude antibody supernatant was obtained by osmotic shock chromatography, and the crude HCMV-gB nanobody extract was purified by nickel column affinity chromatography. The expression of the target protein was observed by SDS-PAGE, and the binding was screened again by the above-mentioned ELISA method. Combining the SDS-PAGE and ELISA results, the optimal antibody was selected. The amino acid sequence of the VHH chain of this antibody is shown in SEQ ID NO: 9.

[0030] Example 4: Expression and purification of HCMV-gB nanobodies in host bacterium Escherichia coli The selected *E. coli* strain expressing the HCMV-gB nanobody was plated on LA+glucose (containing ampicillin and glucose) plates and incubated overnight at 37°C. Single colonies were inoculated into 5 mL of LB broth containing ampicillin and incubated overnight at 37°C with shaking. 1 mL of the overnight inoculum was then inoculated into 330 mL of TB broth and incubated with shaking until the OD value reached 0.6–1. IPTG was then added, and the culture was incubated overnight with shaking. The cultured *E. coli* were collected by centrifugation. The crude HCMV-gB nanobody extract was obtained using an osmotic shock assay. The crude HCMV-gB nanobody extract was purified by nickel column affinity chromatography. The purified product was identified by SDS-PAGE and analyzed using gel imaging software. Results are shown in [link to relevant documentation]. Figure 1 .from Figure 1 It can be seen that the purity of HCMV-gB nanobody can reach over 90% after purification.

[0031] Example 5: Identification of the binding ability of HCMV-gB nanobody to rhHCMV-gB protein (1) Dilute rhHCMV-gB protein (purchased from Bipsys Biotechnology Co., Ltd.) to 2 μg / mL with 100 mM NaHCO3 (pH 9.6), add 100 μL / well to a 96-well high-adsorption microplate, and coat overnight at 4°C. Discard the coating solution, add 200 μL / well of PBST buffer containing 1% BSA, and block at room temperature for 2 h. Discard the blocking solution and wash 3 times with PBST. Serially dilute the HCMV-gB nanobody purified in Example 4 from 10 μg / mL to 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.63 μg / mL, 0.31 μg / mL, and 0.16 μg / mL to the wells, 100 μL / well, and incubate at room temperature for 1 h. Wash away unbound HCMV-gB nanobody with PBST. Add 100 μL of HRP-labeled anti-VHH to each well and incubate at room temperature for 1 h. Wash away unbound antibody with PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 20 min. Then, stop the reaction by adding 50 μL of 2M H2SO4 to each well. Read the absorbance at 450 nm (OD450) using a microplate reader. Results are shown below. Figure 2 As shown. From Figure 2 It can be seen that HCMV-gB nanobodies of different concentrations can specifically bind to the coated rhHCMV-gB antigen, but do not bind to Irrelevant humanAg, indicating that HCMV-gB nanobodies have good specificity.

[0032] (2) Dilute rhHCMV-gB protein to 2 μg / mL with 100 mM NaHCO3 (pH 9.6), add 100 μL / well to a 96-well high-adsorption microplate, and coat overnight at 4°C. Discard the coating solution, add 200 μL / well of PBST buffer containing 1% BSA, and block at room temperature for 2 h. Discard the blocking solution and wash 3 times with PBST. Add 100 μL / well of the prepared HCMV-gB nanobody according to the group (the experiment is divided into two groups, one group takes 5 μg / mL of purified HCMV-gB nanobody and adds an equal amount of rhHCMV-gB protein, the other group takes 5 μg / mL of purified HCMV-gB nanobody and adds an equal amount of PBS, and incubates at room temperature for 30 minutes), and incubates at room temperature for 1 h. Wash away unbound HCMV-gB nanobody with PBST. Add 100 μL / well of HRP-labeled anti-VHH, and incubate at room temperature for 1 h. Wash away unbound antibody with PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 20 min. Then, stop the reaction by adding 50 μL of 2M H2SO4 to each well. Read the absorbance (OD450) at 450 nm using a microplate reader. See the results below. Figure 3 .from Figure 3 It can be seen that the binding ability of HCMV-gB nanobodies to the corresponding antigens decreased after the rhHCMV-gB protein was blocked.

[0033] Example 6: Identification of the binding ability of HCMV-gB nanobody to cells expressing rhHCMV-gB molecules Senescent fibroblasts were transfected with a lentivirus overexpressing rhHCMV-gB to induce rhHCMV-gB expression. Cells in logarithmic growth phase were collected, and the concentration was adjusted to 1 × 10⁻⁶. 6 Cells / mL. The appropriate antibodies were added according to the following experimental groups: blank group, 2µL PBS; experimental group, 2µg (1mg / mL) HCMV-gB nanobody. After adding the corresponding antibodies, the cells were vortexed and mixed, then incubated at 4℃ in the dark for 30 min. Cells were washed twice with PBS and resuspended. 3µg Anti-His-tagPE flow cytometry antibody was added to each tube, and the cells were incubated at 4℃ in the dark for 30 min. The cells were washed twice with PBS. Flow cytometry was used to detect the binding of HCMV-gB nanobody to senescent fibroblasts expressing rhHCMV-gB molecules, and the data were analyzed using FlowJo software. The results are shown in [link to results]. Figure 4 .from Figure 4 It can be seen that the HCMV-gB nanobody can specifically bind to HFF fibroblasts expressing rhHCMV-gB molecules.

[0034] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An HCMV-gB nanobody, wherein the HCMV-gB nanobody comprises a framework region (FR) and a complementarity-determining region (CDR), characterized in that: The frame region FR includes FR1, FR2, FR3 and FR4, and the complementarity determination region CDR includes CRD1, CRD2 and CRD3; The HCMV-gB nanobody comprises FR1, CRD1, FR2, CRD2, FR3, CRD3 and FR4 from the N-terminus to the C-terminus. The amino acid sequence of FR1 is shown in SEQ ID NO: 1, the amino acid sequence of FR2 is shown in SEQ ID NO: 3, the amino acid sequence of FR3 is shown in SEQ ID NO: 5, and the amino acid sequence of FR4 is shown in SEQ ID NO:

7. The amino acid sequence of CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

6.

2. The HCMV-gB nanobody according to claim 1, characterized in that: The HCMV-gB nanobody has the amino acid sequence shown in SEQ ID NO:

9.

3. The HCMV-gB nanobody according to claim 2, characterized in that: The amino acid sequence of the HCMV-gB nanobody is shown in SEQ ID NO:

9.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the HCMV-gB nanobody as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that: The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

8.

6. The nucleic acid molecule according to claim 5, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

8.

7. An expression carrier, characterized in that, The expression vector contains a nucleotide sequence as shown in SEQ ID NO:

8.

8. A host cell, characterized in that, The host cell expresses the HCMV-gB nanobody as described in any one of claims 1 to 3.

9. A drug or reagent for treating HCMV infection, characterized in that: The drug comprises a therapeutically effective amount of the HCMV-gB nanobody as described in any one of claims 1 to 3, the expression vector as described in claim 7, or the host cell as described in claim 8; The reagent comprises the detection of an effective amount of the HCMV-gB nanobody as described in any one of claims 1 to 3, the expression vector as described in claim 7, or the host cell as described in claim 8.

10. The use of the HCMV-gB nanobody according to any one of claims 1 to 3, the expression vector according to claim 7, or the host cell according to claim 8 in the preparation of a medicament for treating HCMV or in the preparation of a reagent for detecting HCMV-gB molecules.

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