CN115925888B - Alpaca-derived nano-antibody specifically combined with human papilloma virus HPV and application thereof - Google Patents

Abstract

The invention belongs to the technical fields of molecular virology and immunology, and provides alpaca-derived nanobodies specifically combined with human papillomavirus HPV and application thereof, wherein the alpaca-derived nanobodies have a heavy chain variable region VHH, and the VHH comprises the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO. 1, CDR2 with the amino acid sequence shown in SEQ ID NO. 2 and CDR3 with the amino acid sequence shown in SEQ ID NO. 3, wherein the VHH comprises 4 framework regions of FR1-4, and the FR1, FR2, FR3 and FR4 are staggered with the CDR1, CDR2 and CDR3 in sequence. The nanobody provided by the invention can effectively inhibit HPV pseudovirus infection. The HPV nanobody has clinical application value in preventing, treating and/or detecting HPV infection.

Description

Alpaca-derived nano-antibody specifically combined with human papilloma virus HPV and application thereof

Technical Field

The invention belongs to the technical fields of molecular virology and immunology, and particularly relates to an alpaca-derived nano-antibody specifically combined with human papilloma virus HPV and application thereof.

Background

Human papillomaviruses (Human papillomavirus, HPV) are a very common group of viruses worldwide, an epitheliophilic virus with a high degree of host specificity that can induce wart formation in humans and even benign or malignant tumors, almost all cervical cancer cases being caused by human papillomavirus infection. The new cases of cervical cancer worldwide are 52.8 ten thousand per year. Three HPV vaccines (divalent, tetravalent and nine-valent) are marketed in China and can be used for preventing high-risk HPV, but the current inoculation rate is still low, part of individuals still have no antiviral antibody even if the vaccine is inoculated, and the vaccine can not treat the obtained human papillomavirus infection or cervical cancer and other diseases related to human papillomaviruses.

In alpaca there is an antibody naturally lacking the light chain, i.e. a heavy chain antibody, the variable region of which consists of only heavy chains, the variable region protein being less than 10 nanometers in diameter and therefore also known as nanobody. The nano antibody has the advantages of small molecular weight, strong penetrability, easy expression, easy genetic modification, easy combination of a plurality of epitopes and the like.

Nanobodies are the smallest antibody molecules at present, originally found in camel blood by belgium scientists, and are a class of interest in engineering antibody products. The nano antibody has the main advantages that the volume is 1/10 of that of the common antibody, the penetration of the nano antibody in animal tissues is strong because of small volume, the nano antibody can reach the inside of high-density tumors through brain tissues of human bodies, and certain tumors or brain diseases can be treated through the nano antibody; secondly, the antigen specificity is good; thirdly, the gene modification is easy, and the artificial modification is convenient to obtain antibodies against different pathogens; fourthly, the stability is high, the time that the nano antibody is not naturally decomposed in the human body is longer than that of the common antibody, the efficacy time is longer, and the nano antibody can even pass through the human stomach to keep the effectiveness.

Specific nanobodies are obtained by screening phage libraries of nanobodies. Nanobody phage libraries are classified into immune and non-immune libraries. The immune repertoire is prepared by immunizing animals such as alpaca, camel, etc. with proteins. The non-immune library is prepared by randomly editing the variable region by keeping a certain constant region according to the structures of the constant region and the variable region of the nano antibody. When the library capacity of the nano antibody library reaches 10 ⁷ In this way, specific nanobodies against antigens can be obtained. The use of the non-immune repertoire saves time and avoids damage to animals caused by animal immunization and animal blood collection.

At present, no specific therapeutic drug directly aiming at HPV exists clinically, and the in-vivo virus is difficult to thoroughly clear and easy to relapse mainly through physical therapy, interferon and other auxiliary therapies. Therefore, it is required to develop antibodies specific to HPV viruses as therapeutic drugs for diseases associated with HPV infection such as cervical cancer.

Disclosure of Invention

The invention aims to provide alpaca-derived nanobodies specifically combined with human papillomavirus HPV and application thereof, in particular to alpaca-derived nanobodies combined with HPV or antigen-binding fragments thereof, polynucleotides encoding the alpaca-derived nanobodies, nucleic acid constructs containing the polynucleotides, expression vectors containing the nucleic acid constructs, preparation methods of the alpaca-derived nanobodies and the antigen-binding fragments, transformed cells and pharmaceutical compositions containing the nucleic acid constructs.

The alpaca-derived nano antibody or antigen binding fragment thereof is a nano antibody with high neutralization activity, has strong binding capacity with HPV protein, can effectively inhibit HPV infection, has the advantages of small molecular weight, small immunogenicity, better solubility and stability and longer CDR region, and provides a potential treatment strategy for HPV infection.

In order to achieve the above purpose, the present invention provides the following technical solutions:

alpaca-derived nanobodies or antigen binding fragments thereof that specifically bind to human papillomavirus HPV, having a heavy chain variable region VHH comprising the CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO. 1, CDR2 with the amino acid sequence shown in SEQ ID NO. 2 and CDR3 with the amino acid sequence shown in SEQ ID NO. 3, wherein the VHH comprises 4 framework regions of FR1-4, and the FR1, FR2, FR3 and FR4 are staggered with the CDR1, CDR2 and CDR3 in sequence.

The amino acid sequence of the FR1 framework region is shown as SEQ ID NO. 4, the amino acid sequence of the FR2 framework region is shown as SEQ ID NO. 5, the amino acid sequence of the FR3 framework region is shown as SEQ ID NO. 6, and the amino acid sequence of the FR4 framework region is shown as SEQ ID NO. 7.

The amino acid sequence of the heavy chain variable region is shown in the following SEQ ID NO. 8, wherein: positions 1-19 are the FR1 framework region, positions 29-44 are the FR2 framework region, positions 52-74 are the FR3 framework region, positions 100-110 are the FR4 framework region, CDR1 is inserted between the FR1 framework region and the FR2 framework region, CDR2 is inserted between the FR2 framework region and the FR3 framework region, and CDR3 is inserted between the FR3 framework region and the FR4 framework region.

A polynucleotide encoding alpaca-derived nanobody or antigen binding fragment thereof, to which human papillomavirus HPV specifically binds, as described above, the sequence of the polynucleotide being shown in SEQ ID No. 9.

A nucleic acid construct comprising said polynucleotide.

An expression vector comprising said nucleic acid construct.

A transformed cell comprising the polynucleotide, nucleic acid construct or expression vector.

The nucleic acid construct further comprises at least one expression control element, such as a histidine tag, stop codon, etc., operably linked to the polynucleotide.

A pharmaceutical composition comprising said alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to human papillomavirus HPV and a pharmaceutically acceptable carrier.

Use of alpaca-derived nanobodies or antigen binding fragments thereof that specifically bind to human papillomavirus HPV in the preparation of a kit or medicament for preventing, treating and/or diagnosing HPV infection.

The invention aims at human papilloma virus HPV to develop nano-antibody medicine, and acquires peripheral blood after antigen immunization of alpaca, constructs an antibody library, and screens by phage display technology to obtain high-affinity nano-antibody specifically combined with HPV. The alpaca-derived nano-antibody or antigen binding fragment thereof is a nano-antibody with high neutralization activity, has strong binding capacity with HPV protein, can effectively inhibit HPV infection, has the advantages of small molecular weight, small immunogenicity, better solubility and stability and longer CDR region, and provides a potential treatment strategy for HPV infection.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 is a schematic diagram showing the result of PAGE identification of HPV16-L1SDS ≡ # x2011 according to example 1 of the present invention; in the figure: line 1: marker, line 2-4: HPV16-L1;

FIG. 2 is a schematic representation of the VHH fragment amplification results of example 2 of the present invention; in the figure: line 1-12: VHH nucleic acid fragment obtained from lymphocyte amplification, line13: a Marker;

FIG. 3 is a graph showing the library capacity of the nano-library of HPV16-VHH detected by the plate in example 3 of the present invention;

FIG. 4 is a graph showing the library abundance of the flat panel test HPV16-VHH nanolibrary of example 3 of the invention;

FIG. 5 is a schematic representation of the result of prokaryotic expression of HPV16-VHH according to example 4 of the present invention; in the figure: line 1: marker, line 2: e.coli expression supernatant at 16 ℃, line 3: e.coli expression pellet at 16 ℃, line 4: e.coli expression supernatant at 20 ℃, line 5: e.coli expression pellet at 20 ℃, line 6: coli expression supernatant, line 7: coli expression pellet at 25 ℃, line 8: coli expression supernatant at 30 ℃, line 9: coli expression pellet at 30 ℃, line 10: coli expression supernatant, line 11: e.coli expression pellet at 37 ℃;

FIG. 6 is a schematic representation of purification results after HPV16-VHH renaturation according to example 4 of the present invention;

FIG. 7 is a graph showing the kinetics of HPV16-VHH binding to HPV 16L 1 in example 5 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The described embodiments are intended to be illustrative of some, but not all, of the embodiments of the present invention and, based on the embodiments herein, all other embodiments that may be made by those skilled in the art without the benefit of the present disclosure are intended to be within the scope of the present invention. In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention. The present invention will be described in detail below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize that equivalents of the specific embodiments described, as well as those known by routine experimentation, are intended to be encompassed within the present application.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1: expression and purification of target proteins

The coding sequence of 6 histidine tags (hexa & # x2011; his ≡ # x2011; tag) and a translation termination codon TGA are connected to the 3' end of an HPV16-L1 protein coding sequence (L1, genBank accession No. AACO9292, 1879-2385, shown as SEQ ID NO: 10), and the coding sequence and the translation termination codon TGA are constructed into a pCAGGS vector through restriction enzyme sites EcoRI and XhoI, and are transfected into 293F cells for carrying out the expression of HPV16-L1 ≡ # x2011 and His proteins. The cell culture solution containing the target protein is purified by nickel ion affinity chromatography and a gel filtration layer (SuperdexTM 75Increase Hiload column (GE Healthcare)), and then the purer target protein can be obtained. HPV16-L1& # x2011, the SDS ≡ # x2011 of his protein, and the result of the identification of about 56KD by PAGE are shown in FIG. 1.

Example 2: alpaca immunization and antibody library construction

The 6 histidine tagged HPV 16L 1 protein prepared in example 1 was diluted 100. Mu.g with PBS to a final volume of 1 mL, emulsified with 1 ml complete Freund's adjuvant for 5 min, subjected to subcutaneous multipoint injection immunization for alpaca followed by immunization every two weeks, and on day 12 after the fourth immunization, 50-60 mL blood was collected, and jugular vein collected anticoagulated venous blood was used for peripheral blood lymphocyte (PBMC) isolation. The isolated peripheral blood mononuclear cells were subjected to extraction of total RNA according to the procedure of the specification. cDNA was synthesized using the total RNA extracted as a template and using a Superscript II First-Strand Synthesis System for RT-PCR kit with random primer oligo-dT primers.

A. A first round of PCR experiments was performed using specific primers with cDNA as template. The specific method comprises the following steps:

the primers for the first round PCR reaction are shown in Table 1.

Table 1: first round PCR reaction primers

The PCR amplification was continued using the synthesized first strand cDNA as a template, and the reverse transcription product was divided into 30 reactions, each of which was 50. 50 ul.

The continuous PCR reaction system is shown in Table 2, and the conditions for continuous PCR amplification reaction are shown in Table 3.

Table 2: continuing the PCR reaction System

Table 3: conditions for continuing the PCR amplification reaction

The nucleic acid fragment 700 bp was recovered and purified by the gel cutting and recovery DNA fragment gel recovery kit, and 1ul of gel recovery product was connected to pMD19-T simple vector, and the reaction system is shown in Table 4.

Table 4: reaction system

The transient vortex reaction system is reacted overnight at 4 ℃, the heat shock connection product and DH5 alpha competent cells are evenly mixed, incubated on ice for 25 min, heat shock is carried out at 42 ℃ for 90 s, incubated on ice for 4 min, 400 μl of LB culture medium is added for 37 ℃ and 200 rpm for 40 min,50 μl of converted bacterial liquid is evenly coated on the surface of the LB solid culture medium containing AMP, and the culture is carried out upside down at 37 ℃ for overnight. 20 monoclonal colonies were picked the next day and inoculated into 5 ml AMP-containing LB liquid medium overnight for cultivation, and their base sequences were determined. The Vector NTI software analyzes the sequencing results and designs the second round of PCR primers VHH2-F and VHH2-R for library construction.

B. The VHH fragment was amplified using the recovered fragments of 700 bp from the first round of PCR as templates and the primers VHH2-F and VHH2-R designed by sequencing, as shown in FIG. 2, and the PCR could amplify the VHH fragment of the heavy chain antibody.

50. Mu.l of the reaction system is subjected to 24 PCR reactions, and the specific method is as follows: the second round PCR primers are shown in Table 5 and the second round PCR amplification reaction conditions are shown in Table 6.

Table 5: second round PCR primers

Table 6: second round PCR amplification reaction conditions

Example 3: antibody library inventory and abundance detection

(1) The method for measuring library capacity comprises the following steps: the bacteria liquid after electric transformation is diluted in gradient, the dilution is from 10 ^-1 ～10 ^-8 The method comprises the steps of carrying out a first treatment on the surface of the 100 μl of each dilution was plated on solid plates and incubated overnight at 25 ℃; the colonies of the gradient plates were statistically calculated for storage the following day and the results are shown in FIG. 3. 10 ^-6 163 monoclonal colonies were present on the dilution plate, 800×163×10 ⁶ =13.04×10 ¹⁰ And each.

(2) The method for measuring the abundance of the library comprises the following steps: performing gradient dilution on the primary library bacterial liquid, wherein the dilution is 10 ^-4 ～10 ^-10 The method comprises the steps of carrying out a first treatment on the surface of the Each dilution was plated on a solid plate with 100. Mu.l of each broth and incubated overnight at 25 ℃; the colonies of the gradient plates were counted the next day and library abundance was calculated. The results are shown in FIG. 4, where the abundance of the library is 291X 10 ⁷ =2.91×10 ¹⁰ And each ml.

(3) The method for measuring the insertion rate and insertion diversity of the library VHH fragments comprises the following steps: after the library capacity is determined, 34 monoclonal colonies are randomly picked from a solid culture plate for determining the library capacity, and are cultured overnight at 37 ℃ in a shaking way, and bacterial liquid parts are collected for PCR identification. And (3) analyzing the PCR identification result and the sequencing result, wherein the lengths of the PCR sample fragments of the 34 bacterial liquids are consistent with the lengths of the VHH fragments, and the insertion rate of the VHH fragments of the library is calculated to be 100%.

Example 4: screening, identification and expression of nanobodies: the nucleotide sequence of alpaca-derived nano antibody shown in SEQ ID NO. 9 is followed by a coding sequence of 6 histidine tags and a translation stop codon TGA, and the encoding sequence and the translation stop codon TGA are constructed into pET23a plasmid through restriction enzyme sites EcoRI and XhoI to construct an expression vector.

1 mu L of pET23a plasmid constructed to contain the target gene is taken and added into 50 mu L of competent cells of escherichia coli BL21 (DE 3), the mixture is placed on ice for 30 minutes and then placed into a water bath at 42 ℃ for heat shock for 60 seconds, and 450 mu L of LB culture solution is added into the bacterial solution after 5 minutes on ice. Mixing, shaking culture at 200 rpm in shaking table at 37deg.C for 1 hr. Then 200. Mu.L of the bacterial liquid was aspirated and spread on LB+Amp solid plates, and the plates were placed upside down in a 37℃incubator for overnight culture. The method comprises the steps of selecting a monoclonal from a plate cultivated overnight, inoculating the monoclonal to 5 mL of LB+Amp culture solution, shaking and cultivating for 8 hours at 200 rpm of a shaking table at 37 ℃, transferring all bacterial solutions to 4L of LB+Amp culture solution, adding 1mM IPTG for induction until the logarithmic phase, cultivating at 16 ℃ overnight, centrifugally collecting bacterial precipitate the next day, adding a proper amount of 1 XPBS for resuspension, and carrying out ultrasonic crushing under the conditions of 112.5 w, crushing for 3 seconds and intermittent 5 seconds. After centrifugation at 12000 rpm at 4℃for 20 min, the supernatant after disruption and the pellet were collected, respectively, and as shown in FIG. 5, the supernatant after disruption was free of nanobodies as detected by SDS-PAGE, demonstrating that the nanobody construct could not be expressed in a soluble form in E.coli BL21 (DE 3).

The inclusion bodies in the pellet were collected and dissolved with 10mmol/L Tris-HCl (pH 7.0, containing 8mol/L urea, 1mmol/L DTT), and the insoluble matter was removed by centrifugation, after which a renaturation buffer (100 mmol/L Tris-HCl pH 8.0, 400mmol/L arginine, 5mmol/L reduced glutathione, 0.5 mmol/L oxidized glutathione, 0.1mmol/L PMSF) was prepared for dilution renaturation. The renaturation product was analyzed by nickel ion affinity chromatography and gel filtration chromatography (Superdex 75Increase Hiload column (GE Healthcare)), and as shown in FIG. 6, the objective peak was determined by SDS-PAGE to obtain purer nanobody.

The antibody: a VHH having a heavy chain variable region, said VHH comprising CDRs as follows: CDR1 (GRTISSYAM) with the amino acid sequence shown in SEQ ID NO. 1, CDR2 (FSSRSGAI) with the amino acid sequence shown in SEQ ID NO. 2, CDR3 (CAGYSIGSYL) with the amino acid sequence shown in SEQ ID NO. 3, said VHH comprising 4 framework regions of FR1-4, said FR1, FR2, FR3 and FR4 being staggered in sequence with CDR1, CDR2 and CDR3.

The amino acid sequence of the FR1 framework region is shown as SEQ ID NO. 4 (ESGGGLVPGGSLRLSCAAS), the amino acid sequence of the FR2 framework region is shown as SEQ ID NO. 5 (GWFRQAPGKE REFVA), the amino acid sequence of the FR3 framework region is shown as SEQ ID NO. 6 (KYYADSVKGR FTISRGNAKN TVYLQMNSLK PEDTAVYY), and the amino acid sequence of the FR4 framework region is shown as SEQ ID NO. 7 (HTTSYTYWGQ G).

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8 (ESGGGLVPGG SLRLSCAASG RTISSYAMGW FRQAPGKERE FVAFSSRSGA IKYYADSVKG RFTISRGNAK NTVYLQMNSL KPEDTAVYYC AGYSIGSYLH TTSYTYWGQG TEVIVSS);

wherein: positions 1-19 are the FR1 framework region, positions 29-44 are the FR2 framework region, positions 52-74 are the FR3 framework region, positions 100-110 are the FR4 framework region, CDR1 is inserted between the FR1 framework region and the FR2 framework region, CDR2 is inserted between the FR2 framework region and the FR3 framework region, and CDR3 is inserted between the FR3 framework region and the FR4 framework region.

The sequence of the polynucleotide for encoding the alpaca-derived nano-antibody specifically combined with human papillomavirus HPV is shown as SEQ ID NO. 9.

Example 5: antigen and nanobody affinity experiments

And (3) selecting an NTA chip, fixing HPV 16L 1 protein on the chip through the NTA chip, wherein the fixed amount is about 100 RU, diluting HPV16-VHH protein by a PBST buffer solution in a multiple ratio, and loading the HPV16-VHH protein from low concentration to high concentration one by one. The kinetics of antibody binding to HPV 16L 1 protein is shown in figure 7.

The equilibrium dissociation constant (KD) between HPV16-VHH and HPV 16L 1 is less than 0.1 nM. The HPV16-VHH nanobody was demonstrated to bind with higher affinity to HPV 16L 1.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (9)

1. Alpaca-derived nanobodies or antigen binding fragments thereof, which specifically bind to human papillomavirus HPV, are characterized in that: it has a heavy chain variable region VHH comprising the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO. 1, CDR2 with the amino acid sequence shown in SEQ ID NO. 2 and CDR3 with the amino acid sequence shown in SEQ ID NO. 3, wherein the VHH comprises 4 framework regions of FR1-4, and the FR1, FR2, FR3 and FR4 are staggered with the CDR1, CDR2 and CDR3 in sequence.

2. Alpaca-derived nanobodies or antigen binding fragments thereof that specifically bind to human papillomavirus HPV according to claim 1, characterized in that: the amino acid sequence of the FR1 framework region is shown as SEQ ID NO. 4, the amino acid sequence of the FR2 framework region is shown as SEQ ID NO. 5, the amino acid sequence of the FR3 framework region is shown as SEQ ID NO. 6, and the amino acid sequence of the FR4 framework region is shown as SEQ ID NO. 7.

3. Alpaca-derived nanobodies or antigen binding fragments thereof that specifically bind to human papillomavirus HPV according to claim 1, characterized in that: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8, wherein: positions 1-19 are the FR1 framework region, positions 29-44 are the FR2 framework region, positions 52-74 are the FR3 framework region, positions 100-110 are the FR4 framework region, CDR1 is inserted between the FR1 framework region and the FR2 framework region, CDR2 is inserted between the FR2 framework region and the FR3 framework region, and CDR3 is inserted between the FR3 framework region and the FR4 framework region.

4. A polynucleotide, characterized in that: encoding alpaca-derived nanobodies or antigen binding fragments thereof which specifically bind to human papillomavirus HPV according to any one of claims 1-3, wherein the sequence of the polynucleotide is shown in SEQ ID No. 9.

5. A nucleic acid construct comprising the polynucleotide of claim 4.

6. An expression vector comprising the nucleic acid construct of claim 5.

7. A transformed cell comprising the polynucleotide of claim 4, the nucleic acid construct of claim 5, or the expression vector of claim 6.

8. A pharmaceutical composition comprising a alpaca-derived nanobody or antigen-binding fragment thereof which specifically binds to human papillomavirus HPV according to any one of claims 1-3 and a pharmaceutically acceptable carrier and/or excipient.

9. Use of alpaca-derived nanobodies or antigen binding fragments thereof which specifically bind to human papillomavirus HPV as claimed in any one of claims 1-3 in the preparation of a kit or medicament for the treatment and/or diagnosis of HPV infection.

Images