HIV-1 nano antibody as well as preparation method and application thereof

Abstract

The invention provides an HIV-1 (human immunodeficiency virus-1) nano antibody as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The antibody is expressed by fusing a His tag at the C terminal of a candidate amino acid sequence. The preparation method of the antibody comprises the following steps: immunizing alpaca for multiple times by using a purified antigen cov-NHR VQ capable of simulating an HIV-1gp41NHR tripolymer; peripheral blood of the immunized alpaca is obtained; an intermediate product to be screened is obtained; screening the intermediate product to be screened to obtain a candidate amino acid sequence of the HIV-1 nano antibody; the VHH gene of the candidate amino acid sequence of the antibody is cloned into a prokaryotic expression vector, and nano antibody protein is expressed in the form of VHH-his to obtain the antibody. The antibody can be applied to a modified antibody in a CDR region and preparation of a drug HIV-1 detection reagent for preventing and / or treating HIV-1. The antibody has high specificity and high affinity in a targeted gp41NHR trimer conserved region.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an HIV-1 nano antibody, a preparation method and application thereof. Background Art

[0002] AIDS, a highly lethal infectious disease, is one of the most vexing medical problems today. Currently, more than 37 million people are infected with the human immunodeficiency virus (HIV), and approximately 1.8 million new infections each year continue to fuel the HIV epidemic. As of September 2022, 29.8 million people worldwide are receiving antiretroviral therapy (https: / / www.unaids.org / en / resources / fact-sheet). Initially, the advent of antiretroviral therapy (ART) brought a ray of hope to HIV patients, effectively suppressing HIV replication in treated individuals and significantly reducing the morbidity and mortality associated with AIDS-related diseases. However, because HIV establishes a latent viral reservoir in the host CD4+ T cells, interruption of ART can lead to rapid viral rebound and continued disease progression, while lifelong treatment often leads to the accumulation of toxicity and drug resistance. Therefore, there is a need to study drugs to prevent and / or treat AIDS, as well as to study test reagents to detect HIV virus. Summary of the invention

[0003] In view of this, the present invention provides an HIV-1 nanobody, a preparation method and application thereof, constructs a protein capable of simulating the gp41 NHR trimer as an antigen, immunizes alpacas, and obtains highly specific and high-affinity nanobodies (Nbs) targeting the conserved region of the gp41 NHR trimer by screening the nanobody library, thereby being more suitable for practical use.

[0004] In order to achieve the above first purpose, the technical solution of HIV-1 nanobody provided by the present invention is as follows:

[0005] The HIV-1 nanobody provided by the present invention is characterized in that a His tag is fused to the C-terminus of the candidate amino acid sequence for expression, wherein the candidate amino acid sequence is selected from any one of the following sequences:

[0006] SEQ ID NO: 1Nb-113 EVQVVESGGGLVQPGDSLRLSCVASGRTLSNLN VGWFRQAPGKEREFVAAITWILGTPYYADSAKGRFTISRDNAKNAIYLQMNS LKPEDTAVYRCAAPHGNRYASPEQYVYWGQGTQVTVSS;

[0007] SEQ ID NO:2Nb-114 EVQVVESGGGLVQTGGSLRLSCTVSDTHIFSTY VLAWFRQAPGKEREFVGWTDRLTFYADSVEGRFTISRDGAQNTVHLQMDSL RPEDTAVYYCAAGPISDTSPYIEARSYEYWGQGTQVTVSS;

[0008] SEQ ID NO:3Nb-117 QVQLVESGGGLVQPGGSLRLSCASSGFSLTYYTI TWFRQASGKERESVSCIESDGTTQDPTSVKGRFTISRDNAKNTVYLQMNDLK PEDTAIYTCATSIAICAWDDPIYDRWGQGTQVTVSS;

[0009] SEQ ID NO:4Nb-142 QVQLVESGGGLVQAGGSLTLSCVVSGITFDGRA AGWYRQVPGKQRELVARIDTGGSTNYADSAKGRFTISRDIAENTMYLQMNSL KPEDTAVYYCNVESWFTIAGSYWGQGTQVTVSS;

[0010] SEQ ID NO:5Nb-145 EVQVVESGGGLVQPGGSLRLSCTASGFILDFYA MGWFRQAPGKEREGISCISSSDDSTYYADSVKGRFIISRDNAKSTVYLQMNNL KPEDTAIYYCATDWDVVCGSSWSSPRHGSDFGAWGQGTQVTVSS;

[0011] SEQ ID NO:6Nb-152 EVQLVESGGGLVQPGGSLRLSCASSGFSLTYYT VAWFRQAPGRERERVSCIESNDGRSRYANSVKGRFTISKDNAKNTVYLQMND LKPDDTAVYVCATTLSICDWDDPTYDRWGQGTQVTVSS;

[0012] SEQ ID NO:7Nb-162 EVQVVESGGGLAQAGGSLRLSCATSGRTFNTYP VGWFRQAPGKEREFVAAINWFGERTSYADSVKGRFTISRNNAKNTVYLEMN NLKPEDTAVYRCAARADGTQGIGGANPGSYEYWGQGTQVTVSS;

[0013] SEQ ID NO: 8Nb-172 EVQVVESGGGQVQPGGSLRLSCAASGSTFDNF GAWYRQAPGKQRELVAQILLGGTTNYADSVKGRFTISRDNDKNTMYLQMNS LKPEDTAVYYCHTFYRAQFYWGQGTQVTVSS;

[0014] SEQ ID NO: 9Nb-184 QVQLVESGGGLVQAGGILRLSCIGSGGTLSGWR MGWYRQAPGQEREFVAASSSHIGNTYYADSVKGRFTISRDNAKNTVYLQMN SLQPEDTAIYYCNSHPTAVFDWGKGTLVTVSS;

[0015] SEQ ID NO: 10Nb-187 QVQLVESGGGLVQAGGSLRLSCAASGLPFTNY AMGWFRQAPGKEREFVTAISRNGGTTNYTDSVKGRFTISRDNARNTVYLQM NSLKPEDTAVYVCAAGWTIFSVSRLPTEYAFWGQGTQVTVSS;

[0016] SEQ ID NO: 11Nb-190 EVQLVESGGGLVQPGGSLRISCVASGRTFAYYI MGWFRQAPGKEREFVANIRWSDGETYYSDSVKDRFTISRDNTKNTVYLQMN SLKPEDTAVYHCAASQALGIMPWYTRTDNYKYWGQGIQVTVSS.

[0017] In order to achieve the above second purpose, the technical scheme of the method for preparing HIV-1 nanobody provided by the present invention is as follows:

[0018] The method for preparing HIV-1 nanobody provided by the present invention is characterized by comprising the following steps:

[0019] Purified cov-NHR VQ is used to immunize alpacas multiple times to obtain immunized alpacas, so that HIV-1 neutralizing antibodies are produced in the immunized alpacas, wherein the amino acid sequence of the purified antigen cov-NHR VQ that can simulate HIV-1 gp41 NHR trimer is ARQELSGIVQK QNNLLRQIEAQQHLLQLTVSKIKQLQARILAVERYLKDQQLGKGNQPQQDKLYREVALIRAQLQKIESETLQLLHQQAEIERELNNQEQEIGSLKQRGLIDGPLLSGIDQQQNNLKRAIEAQKHLLQLTVWGIKQLQARILTVERYLKDQQLGGGGSHHHHHH;

[0020] Obtaining peripheral blood from the immunized alpaca;

[0021] Processing the peripheral blood of the immunized alpaca to obtain an intermediate product to be screened;

[0022] Screening the intermediate product to be screened to obtain a candidate amino acid sequence of HIV-1 nanobody;

[0023] The VHH gene of the candidate amino acid sequence of the HIV-1 nanobody is cloned into a prokaryotic expression vector, and the nanobody protein is expressed in the form of VHH-his to obtain the HIV-1 nanobody, wherein in the HIV-1 nanobody, the C-terminus of the candidate amino acid sequence is fused with a His tag for expression.

[0024] The method for preparing the HIV-1 nanobody provided by the present invention can also be further implemented by adopting the following technical measures.

[0025] Preferably, in the step of using purified cov-NHR VQ to immunize alpacas multiple times to obtain immunized alpacas, so that HIV-1 neutralizing antibodies are produced in the immunized alpacas, the method for obtaining the purified cov-NHR VQ includes the following steps:

[0026] According to the amino acid sequence, obtaining a nucleotide sequence;

[0027] The nucleotide sequence was cloned into the pComb3x vector to obtain the pComb3x-covNHR-VQ plasmid;

[0028] The pComb3x-covNHR-VQ plasmid was transformed into the HB2151 strain, and a single clone colony was selected and activated in LB at 37° C. overnight and purified to obtain the purified cov-NHR VQ, wherein the purity of the purified cov-NHR VQ was >90%.

[0029] Preferably, the purified cov-NHR VQ is used to immunize alpacas multiple times to obtain immunized alpacas, so that neutralizing antibodies to HIV-1 are produced in the immunized alpacas, and the alpacas are immunized multiple times, comprising the following steps:

[0030] The first immunization was performed by mixing 0.5 mg of the immunogen covNHR-VQ with equal volumes of Freund's complete adjuvant and emulsifying them.

[0031] Subsequently, booster immunizations were performed in the third, sixth, and ninth weeks using an equal volume of 0.25 mg covNHR-VQ mixed with Freund's incomplete adjuvant and emulsified, for a total of four immunizations to obtain immunized alpacas.

[0032] Preferably, the method further comprises the following steps after the step of immunizing the alpaca with purified cov-NHR VQ for multiple times to obtain the immunized alpaca, so that the immunized alpaca produces HIV-1 neutralizing antibodies:

[0033] The step of detecting the antigen-specific antibodies in the alpaca serum and the neutralization titer of the alpaca antiserum to the HIV-1 laboratory adapted strain for the alpaca after the fourth immunization to obtain the immune response result of the alpaca.

[0034] Preferably, obtaining the peripheral blood of the immunized alpaca specifically comprises the following steps:

[0035] Obtaining peripheral blood from alpacas that have undergone the third immunization and the fourth immunization;

[0036] Separating the alpaca peripheral blood mononuclear cells according to the instructions for use of the lymphocyte separation solution;

[0037] The peripheral blood of the immunized alpaca was obtained by lysing and freezing the blood using the Trizol method.

[0038] Preferably, the peripheral blood of the immunized alpaca is processed to obtain the intermediate product, which specifically comprises the following steps:

[0039] The total RNA of alpaca peripheral blood mononuclear cells was extracted by Trizol method;

[0040] Reverse transcription is performed on the total RNA to obtain cDNA;

[0041] Using nested PCR to amplify the nanobody gene fragments from cDNA, a nanobody phage library targeting covNHR-VQ was constructed;

[0042] The nanobody phage library against the covNHR-VQ is incubated and eluted to obtain an intermediate product to be screened.

[0043] Preferably, screening the intermediate product to be screened to obtain a candidate amino acid sequence of HIV-1 nanobody specifically comprises the following steps:

[0044] Randomly selecting among the intermediate products to be screened to obtain randomly selected clones;

[0045] Performing monoclonal phage supernatant ELISA identification on the randomly selected clones to obtain ELISA identification results of each randomly selected clone;

[0046] According to the ELISA identification results, analysis software is used to sequence the clones positive for the covNHR-VQ antigen to obtain sequencing results;

[0047] According to the sequencing results, similar sequences are removed to obtain the candidate amino acid sequence of the HIV-1 nanobody.

[0048] Preferably, the VHH gene of the candidate amino acid sequence of the HIV-1 nanobody is cloned into a prokaryotic expression vector, and the nanobody protein is expressed in the form of VHH-his. After obtaining the HIV-1 nanobody, the method further includes a step of confirming that the HIV-1 nanobody is successfully expressed.

[0049] In order to achieve the third purpose above, the technical solution for the application of HIV-1 nanoantibodies provided by the present invention is as follows:

[0050] The applications of the HIV-1 nanoantibodies provided by the present invention include one or more of the following applications:

[0051] Application of modified antibodies containing the HIV-1 nanobody CDR region;

[0052] Use of the HIV-1 nanobody in the preparation of a drug for preventing and / or treating HIV-1;

[0053] Application of the HIV-1 nano antibody in the preparation of HIV-1 detection reagent.

[0054] The present invention obtains an NHR-specific nanoantibody library by immunizing alpacas, and obtains nanoantibodies with high affinity to gp41 NHR by phage display technology, which have the advantages of small molecular weight and can overcome the steric hindrance and kinetic obstacles caused by the transient exposure of NHR trimers.

[0055] The nano antibody obtained by the present invention can be expressed through a prokaryotic expression system, and can effectively bind to the gp41NHR trimer protein or NHR-derived polypeptide in vitro, thereby inhibiting the formation of 6-HB.

[0056] The nanoantibodies Nb-162 and Nb-172 developed by the present invention have a strong binding force with the NHR trimer protein, can well inhibit the entry of multiple subtypes of HIV-1 pseudoviruses in vitro, and can effectively neutralize HIV-1 laboratory adapted strains, clinical isolates of different subtypes and tropisms, and T-20 resistant strains.

[0057] The small molecular weight nano-antibodies obtained by the present invention can not only be used as entry inhibitors to inhibit viral infection, but can also be used in combination with HIV-1 inactivators to promote their inactivation of free viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0059] Figure 1 The SDS-PAGE provided in the present invention-embodiment verifies the integrity and purity of the covNHR-VQ protein;

[0060] Figure 2 Alpaca immunization strategy provided by the present invention-embodiment;

[0061] Figure 3 Antigen-specific antibody titer in alpaca serum provided by the present invention-embodiment;

[0062] Figure 4 The neutralization titer of the alpaca antiserum provided in the embodiment of the present invention against HIV-1Bal;

[0063] Figure 5 A schematic diagram of obtaining nanobodies by phage display technology provided in the present invention-embodiment;

[0064] Figure 6 The SDS-PAGE provided in the present invention-embodiment identifies the molecular weight and purity of the nanobody;

[0065] Figure 7 The binding ability of the nanobodies provided in the embodiments of the present invention to the N63 trimer protein;

[0066] Figure 8The inhibitory activity of the Nanobodies provided in the present invention-Examples against HIV-1Bal;

[0067] Fig. 9 The inhibitory effect of Nb-162 & Nb-172 provided in the embodiment of the present invention on HIV-1 clinical isolates;

[0068] Fig.10 In vitro inactivation effect of Nb-172 combined with mD1.22 on HIV-1 clinical isolates provided in the present invention

[0069] Fig.11 The affinity of Nb172 and N63 trimer proteins provided in the embodiments of the present invention;

[0070] Fig.12 The binding ability of Nb172 provided in the embodiment of the present invention to the gp41 NHR-derived polypeptide;

[0071] Fig.13 The Nb-172 provided in the present invention-embodiment competes with the CHR-derived polypeptide for binding to the N63 trimer protein;

[0072] Fig.14 A schematic diagram of the interaction between HIV-1gp41 NHR and CHR provided in the embodiments of the present invention;

[0073] Fig.15 Nb-172 provided in the present embodiment inhibits the formation of 6-HB by binding to NHR. DETAILED DESCRIPTION

[0074] In view of this, the present invention provides an HIV-1 nanobody, a preparation method and application thereof, constructs a protein capable of simulating the gp41 NHR trimer as an antigen, immunizes alpacas, and obtains highly specific and high-affinity nanobodies (Nbs) targeting the conserved region of the gp41 NHR trimer by screening the nanobody library, thereby being more suitable for practical use.

[0075] In order to further explain the technical means and effects taken by the present invention to achieve the predetermined invention purpose, the following is a detailed description of an HIV-1 nanobody, its preparation method and application, its specific implementation method, structure, characteristics and effects proposed by the present invention in combination with the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0076] The term "and / or" in this article is merely a description of the association relationship of associated objects, indicating that three relationships may exist, for example, A and / or B. Specifically, it is understood that: A and B may be included at the same time, A may exist alone, or B may exist alone, and any of the above three situations may be met.

[0077] The first aspect of the present invention is to obtain a series of nano antibodies by immunizing alpacas. The present invention obtains an alpaca HIV-1NHR-specific nano antibody library by immunizing alpacas four times using cov-NHRVQ, and then screens out nano antibodies with high affinity to gp41 NHR protein through phage display, and constructs its sequence into an expression vector for prokaryotic expression. On the other hand, the present invention reveals that the obtained nano antibodies can be used as entry inhibitors of HIV-1. The present invention performs activity detection on the obtained nano antibodies and finds that Nb-162 and Nb-172 can well inhibit the entry of HIV-1 pseudoviruses, and can effectively neutralize HIV-1 laboratory adapted strains, clinical isolates and T-20 resistant strains. Further, the present invention explores the mechanism of action of nano antibodies and finds that the target of Nb-172 is the conserved region of gp41 NHR, which can inhibit the formation of 6-HB by binding to NHR, thereby inhibiting HIV-1 infection. When the nano antibody Nb-172 obtained in the present invention is used in combination with the HIV-1 inactivator mD1.22 targeting gp120, the inhibitory activity of mD1.22 on HIV-1 entry into cells and the inactivation effect on free HIV-1 can be improved, which indicates that the nano antibody targeting gp41 can be used in combination with the gp120 targeting to treat HIV-1 infection. The nano antibody targeting gp41 obtained in the present invention has a broad-spectrum inhibitory effect on HIV-1, providing guidance for the development of new strategies for HIV-1 treatment.

[0078] Example 1: Purification of cov-NHR VQ and immunization of alpacas

[0079] Step 1: The amino acid sequence of covNHR-VQ (ARQELSGIVQKQNNLLRQIEAQQHL LQLTVSKIKQLQARILAVERYLKDQQLGKGNQPQQDKLYREVALIRAQLQKIE SETLQLLHQQAEIERELNNQEQEIGSLKQRGLIDGPLLSGIDQQQNNLKRAIEA QKHLLQLTVWGIKQLQARILTVERYLKDQQLGGGGSHHHHHH) was obtained from the literature (Jurassic Park, M, Morel B, et al. Structural and Thermodynamic Analysi s of HIV-1Fusion Inhibition Using Small gp41 Mimetic Proteins. J Mol Biol, 2019), and the nucleotide sequence was obtained based on the amino acid sequence and cloned into the pComb3x vector.

[0080] Step 2: The obtained pComb3x-covNHR-VQ plasmid was transformed into the HB2151 strain, and a single clone was selected and activated in LB at 37°C overnight. The culture was transferred to 1L LB medium at a ratio of 1:100 for culture. When the OD value reached 0.6, IPTG was added to a final concentration of 0.1mmol / L, and the culture was shaken at 30°C for 20h. The above culture system was centrifuged at 6,000rpm for 5min to collect the bacteria, and the collected bacteria were resuspended in PBS, and polymyxin was added to a final concentration of 0.5mM at 37°C for 2h, and then centrifuged at 8,000rpm for 30min. The filtered supernatant was combined with Ni Sepharose (GE) for 2h. The impurities were washed with Tris buffer containing 40mM imidazole, and the target protein was eluted with a buffer containing 250mM imidazole. The protein was concentrated by ultrafiltration using an ultrafiltration tube, and the buffer was replaced with PBS. The protein purity was determined to be >90% by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 ).

[0081] Step 3: Apac immunizes alpacas according to the following strategy ( Figure 2 ): 0.5 mg of the immunogen covNHR-VQ was mixed and emulsified with Freund's complete adjuvant (purchased from Beijing Solebow Technology Co., Ltd.) in equal volumes for the first immunization, followed by booster immunizations in the third, sixth, and ninth weeks using 0.25 mg of covNHR-VQ mixed and emulsified with Freund's incomplete adjuvant (purchased from Beijing Solebow Technology Co., Ltd.) in equal volumes. Blood was collected one week after each booster immunization to detect the antibody titer in the alpaca serum.

[0082] Example 2 Alpaca immune response detection

[0083] After the fourth immunization, in order to measure the immune response of the alpaca, the antigen-specific antibodies in the alpaca serum and the neutralizing titer of the alpaca antiserum against the HIV-1 laboratory adapted strain were tested. The specific steps are as follows:

[0084] Step 1: Coat 2 μg / ml covNHR-VQ in an ELISA plate at 4°C overnight; block with PBST (phosphate buffer containing 0.05% Tween-20) containing 5% skim milk powder, dilute the alpaca serum inactivated at 56°C for 30 minutes in a 2-fold ratio, add to the ELISA plate, and incubate at 37°C for 1 hour; wash five times with PBST, add Anti-Alpaca IgG (H+L), Goat antibody (HRP) (purchased from Chengdu Critical Point Biotechnology Co., Ltd.), incubated at 37°C for 1 h; after washing with PBST 5 times, the substrate TMB / H 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 .

[0085] Antigen-specific antibody titers in alpaca serum Figure 3 As shown, when the serum dilution factor is 1.28×10 5 When the covNHR-VQ specific antibody was detected, Figure 3 ).

[0086] Step 2: The inactivated alpaca serum was diluted 3-fold in RPMI 1640 medium (Dalian Meilun Biotechnology Co., Ltd.) in a 96-well plate, and an equal volume of HV-1Bal virus was added. After incubation at 37°C for 30 min, CEMx174 5.25M7 cells were added to the mixture, and 1×10 4 / well. After culturing at 37°C for 12 hours, the medium was replaced with RPMI 1640 medium containing 10% FBS. After 4 days, the cell supernatant was collected, treated with 5% TritonX-100, and P24 was detected by ELISA to measure the virus content in the cell supernatant.

[0087] Step 3: Coat the ELISA plate with 5 μg / ml HIV-1 antibody at 4°C overnight; after blocking with PBST containing 5% skim milk powder, add the cell supernatant to the ELISA plate and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add mouse 183 polyclonal antibody and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 minutes; after washing with PBST for 5 times, add substrate TMB / H 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450The inhibition rate was calculated by the following formula: Inhibition rate (%) = (OD 450病毒孔 -OD 450血清孔 ) / (OD 450病毒孔 -OD 450细胞孔 )×100.

[0088] The neutralization titer of alpaca antiserum against HIV-1Bal is as follows Figure 4 As shown, when alpaca serum was incubated with HIV-1 at 37°C, the serum's neutralizing activity against HIV-1 could not be detected. This may be because the antibody naturally present in the alpaca is a heavy chain antibody that naturally lacks light chains, has an Fc structure, and has a molecular weight of about 90kD, which is not easy to bind to gp41 in a fusion state. Golding et al. proved that an incubation temperature of 31.5°C can slow down the transition of gp41 from the pre-hairpin intermediate state to the post-hairpin fusion state, thereby allowing more gp41 in the fusion intermediate state to accumulate (Golding H, Zaitseva M, de Rosny E, et al. Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates. J Virol, 2002). When we incubated serum with HIV-1 at 31.5°C, alpaca serum had the ability to neutralize HIV-1. This indicates that HIV-1 neutralizing antibodies are produced in alpacas, so we will further screen, express and purify the variable region (VHH) of the alpaca heavy chain antibody.

[0089] Example 3 Screening of Nanobodies Targeting NHR by Phage Display Technology

[0090] Step 1: Collect 50 mL of alpaca peripheral blood after three and four immunizations, separate alpaca peripheral blood mononuclear cells according to the instructions of lymphocyte separation solution (Tianjin Haoyang Huake Biotechnology Co., Ltd.), and lyse and freeze with Trizol (Thermo Fisher Scientific). The total RNA of alpaca peripheral blood mononuclear cells was extracted by Trizol method, and PrimeScript TM The extracted total RNA was reverse transcribed using II1st Strand cDNA Synthesis Kit (Takara, catalog number: 6210A) to synthesize cDNA. Nested PCR was used to amplify the nanobody gene fragment:

[0091] First round of PCR:

[0092] Upstream primer: 5'-CTTGGTGGTCCTGGCTGC-3'

[0093] Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3'

[0094] Second round of PCR:

[0095] The second round of PCR uses the recovered product of the first round of PCR as a template.

[0096] Upstream primer:

[0097] 5'-CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC-3' downstream primer-1:

[0098] 5'-CATGCCATGACTCGCGGCCGGCCTGGCCGCTGGGGTCTTCGCTGTGGTGCG-3'

[0099] Downstream Primer-2:

[0100] 5'-CATGCCATGACTCGCGGCCGGCCTGGCCTGGTTGTGGTTTTGGTGTCTTGGG-3'

[0101] The second round of PCR amplification of the VHH nucleic acid fragment product was recovered and digested with the restriction endonuclease SfiI (NEB, catalog number: R0123L) at 50°C overnight, after which the digested VHH nucleic acid fragment was recovered. The digested VHH nucleic acid fragment was inserted into the phage display vector pComb3xss (Chengdu Critical Point Biotechnology Co., Ltd.) and connected using T4 DNA Ligase (NEB, catalog number: M0202L). The ligation product was electroporated into Escherichia coli TG1 competent cells to construct a nanoantibody phage library against covNHR-VQ. After electroporation, 100 μL of the product was plated by gradient dilution and plated at 10 -4 There are 102 clones on the plate for the dilution gradient determination of the library capacity, so the library capacity is 1.02×10 9 At the same time, 48 clones were randomly selected from the library capacity test plate for bacterial liquid PCR identification, and the results showed that the library insertion rate was 100%.

[0102] Step 2: Dilute the target molecule covNHR-VQ antigen with carbonate buffer at pH 9.6 to a final concentration of 5 μg / mL, add 100 μL / well to the enzyme-labeled wells, coat 8 wells, and coat overnight at 4°C; discard the coating solution, wash 3 times with PBS, and add 300 μL of 3% OVA-PBS blocking solution, blocked at 37°C for 1h; washed 3 times with PBS, added 100μL of nanoantibody phage library against covNHR-VQ, incubated at 37°C for 1h; aspirated unbound phages, washed 6 times with PBST, and washed 2 times with PBS; added 100μL of Gly-HCl elution solution, incubated at 37°C for 8min, and eluted specifically bound phages; transferred the eluate to a 1.5mL sterile centrifuge tube and quickly neutralized with 10μL Tris-HCl neutralization buffer; took 10μL for gradient dilution, determined the titer, calculated the panning recovery rate, and the remaining eluate was mixed for amplification and purification for the next round of affinity panning.

[0103] Step 3: After three rounds of screening, 192 clones were randomly selected from the second and third rounds of titer determination plates for monoclonal phage supernatant ELISA identification. The target molecule covNHR-VQ antigen was diluted with a carbonate buffer with a pH value of 9.6 to a final concentration of 2μg / mL, and 100μL / well was added to the enzyme-labeled wells, and coated overnight at 4℃; the coating solution was discarded, washed 3 times with PBST, 300μL 5% skim milk was added to each well, and blocked at 37℃ for 1h; washed once with PBST, 50μL phage culture supernatant and 50μL 5% skim milk were added to each well, and incubated at 37℃ for 1h; washed 5 times with PBST, Anti-M13 Bacteriophage was added, Rabbit antibody (HRP) (Chengdu Critical Point Biotechnology Co., Ltd.), 100 μL / well, 37°C for 1h; PBST wash plate 6 times. TMB colorimetric solution was added for color development, 100 μL / well, 37°C, 7min, and the reaction was terminated by adding stop solution, 50 μL / well, and the optical density was measured at 450nm. The clones positive for covNHR-VQ antigen in the ELISA results were sequenced, and the sequencing was completed by the Chengdu Branch of Beijing Qingke Biotechnology Co., Ltd. The sequencing results were compared and sorted, and after removing similar sequences, 11 specific clones with different sequences were screened and obtained. Their sequence CDRs were analyzed by KABAT, Chothia and IMGT software, respectively, and the corresponding sequence information is shown in Table 1.

[0104] Table 1. VHH amino acid sequence information

[0105]

[0106]

[0107] Example 4 Expression and identification of nanobodies

[0108] The obtained VHH gene was cloned into a prokaryotic expression vector to express the nanobody protein in the form of VHH-his. The constructed recombinant plasmid was transformed into the Bacillus B strain, and the monoclonal colonies were selected and activated in MT medium at 37°C. After 4 hours, samples were taken for PCR positive clone identification. According to the identification results, positive clones were selected and transferred to 200mL of MTN medium at a ratio of 1:200 for culture, and cultured at 30°C shaker at 220rpm for 72 hours. The above-obtained culture system was centrifuged at 10,000g for 10 minutes to collect the supernatant, and the collected supernatant was affinity chromatographed with Ni Sepharose (Biolink). The impurities were washed with Tris buffer containing 20mM imidazole, and the target protein was eluted with a buffer containing 250mM imidazole. The protein was concentrated by ultrafiltration using an ultrafiltration tube, and the buffer was replaced with 0.1M NaHCO3 (pH 8.3). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) test confirmed that the nanobody was successfully expressed with a molecular weight of approximately 15 kD ( Figure 6 ).

[0109] Example 5 Binding ability of nanobodies to gp41 NHR protein

[0110] The N63 trimer protein was used to detect the binding ability of the nanobody to the gp41 NHR protein, where the N63 sequence is STMGAASMTTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ, and the trimer is formed with the help of the fibritin-foldon motif of T4 phage.

[0111] Step 1: The nucleotide sequence of N63-Foldon-Fc (N63Fd) was synthesized by BGI and cloned into the pSectag vector through the two restriction sites of SfiI and XhoI. After being sequenced correctly by BGI, it was transfected into Expi293F cells for expression. 40 μg of plasmid was incubated with 120 μL EZ-trans transfection reagent for 15 min and then transferred into 50 ml Expi293F (2×10 6After 5 days, the cell culture medium was collected and centrifuged at 3,000 rpm for 30 min. The supernatant was filtered with a 0.45 μm filter membrane and combined with Protein AResin FF (GenScript Biotech Co., Ltd.) overnight. The protein was washed with PBS and eluted with 0.1 M glycine (pH 2.5-3) and neutralized with 1 M Tris-HCl (pH 8.5). The protein purity and integrity were detected by SDS-PAGE, and the protein was concentrated by ultrafiltration and the buffer was replaced with PBS.

[0112] Step 2: 2 μg / ml N63 trimer protein was coated on the ELISA plate at 4°C overnight; after blocking with PBST (phosphate buffer containing 0.05% Tween-20) containing 5% skim milk powder, the nanobody was diluted 4 times and added to the ELISA plate, and incubated at 37°C for 1 hour; after washing with PBST for 5 times, HRP-labeled 6×His monoclonal antibody (Proteintech Biotechnology Company) was added and incubated at 37°C for 30 minutes; after washing with PBST for 5 times, the substrate TMB / H was added. 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 .

[0113] The binding ability of nanobodies to gp41 NHR protein is as follows Figure 7 As shown in the figure, the binding of most nanobodies to the N63 trimer protein is concentration-dependent, but some individual antibodies such as Nb-162, Nb-172 and Nb-187 show strong binding specificity to the N63 trimer protein, and no decrease in binding ability is observed within the detected concentration range ( Figure 7 ).

[0114] Example 6 Inhibitory activity of nanobodies against HIV-1 laboratory adapted strain Bal

[0115] Antibodies targeting gp41 NHR can block the formation of 6-HB structure between NHR and CHR, thereby inhibiting HIV virus from entering target cells. Therefore, we further verified the inhibitory activity of the screened nanoantibodies against HIV-1Bal. The specific steps are as follows:

[0116] Step 1: The nanobody was diluted 4-fold in RPMI 1640 medium (Dalian Meilun Biotechnology Co., Ltd.) in a 96-well plate, and an equal volume of HIV-1Bal virus was added. After incubation at 37°C for 30 min, CEMx174 5.25M7 cells were added to the mixture, and 1×10 4 / well. After culturing at 37°C for 12 hours, the medium was replaced with RPMI 1640 medium containing 10% FBS. After 4 days, the cell supernatant was collected, treated with 5% TritonX-100, and P24 was detected by ELISA to measure the virus content in the cell supernatant.

[0117] Step 2: Coat the ELISA plate with 5 μg / ml HIV-1 antibody at 4°C overnight; after blocking with PBST containing 5% skim milk powder, add the cell supernatant to the ELISA plate and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add mouse 183 polyclonal antibody and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 minutes; after washing with PBST for 5 times, add substrate TMB / H 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 The inhibition rate was calculated by the following formula: Inhibition rate (%) = (OD 450病毒孔 -OD 450血清孔 ) / (OD 450病毒孔 -OD 450细胞孔 )×100.

[0118] The inhibitory activity of nanoantibodies against HIV-1Bal Figure 8 As shown in Figure 2, Nb-162 and Nb-172 had the best inhibitory effects on Bal virus, with IC 50 1.19 and 0.33 μM respectively ( Figure 8 ).

[0119] Example 7 Inhibitory Effects of Nb-162 and 172 on HIV-1 Pseudovirus

[0120] Step 1. Pseudovirus packaging: Use EZ-trans transfection reagent to co-transfect 293T cells (ATCC) with PcDNA3.1-env plasmids carrying different HIV-1 capsid protein genes and HIV backbone plasmid pNL4-3.Luc.RE- (obtained from NIH AIDS reagent program) at a ratio of 1:2. Collect the cell supernatant after 48 hours to obtain the pseudovirus.

[0121] Step 2. Use TZM-b1 cells (ATCC) to plate 96-well plates, with 1×10 4 The cells were cultured at 37°C with 5% CO2 overnight.

[0122] Step 3. Use DMEM culture medium (Dalian Meilun Biotechnology Co., Ltd.) to dilute Nb-162, Nb-172 or D5 scFv 3-fold or 4-fold in a 96-well plate, and then add equal volumes of pseudovirus. After incubation at 37°C for 30 minutes, add the mixed solution to TZM-b1 cells. After 12 hours, replace the cells with fresh DMEM culture medium containing 10% FBS (Yisheng Biotechnology).

[0123] Step 4. 48 hours after the medium was replaced, lysis solution (Promega) was added to the cells and lysed for 30 minutes, and the relative content of pseudovirus in the cells was measured by luciferase detection system (Promega). The RLU value was detected by microplate reader, and the IC 50 .

[0124] The inhibitory activities of Nb-162 and Nb-172 against different subtypes of HIV-1 pseudoviruses are shown in Table 1. Among them, Nb-172 has the best inhibitory effect on pseudoviruses, IC 50 It was 0.149~1.594μM (Table 2).

[0125] Table 2. Inhibitory effect of Nb-162 & 172 on HIV-1 pseudovirus

[0126]

[0127] Example 8 Inhibitory Effect of Nb-162 & 172 on HIV-1 Clinical Isolates

[0128] We further validated the inhibitory activity of Nb-162&172 nanoantibodies against HIV-1 clinical isolates.

[0129] Step 1. Nb-162 / Nb-172 / D5 scFv was diluted 3 or 4 times in RPMI 1640 medium (Mei Lun) in a 96-well plate, and then an equal volume of HIV-1 clinical isolate was added. After incubation at 37°C for 30 min, CEMx174 5.25M7 (with CCR5 and CXCR4 dual receptors) or MT-2 (expressing only CCR5 receptor) cells were added to the mixture, and 1×10 4 / well. After culturing at 37°C for 12 hours, the medium was replaced with RPMI 1640 medium containing 10% FBS. After 4 days, the cell supernatant was collected, treated with 5% TritonX-100, and P24 was detected by ELISA to measure the virus content in the cell supernatant.

[0130] Step 2. Coat the ELISA plate with 5 μg / ml HIV-1 antibody at 4°C overnight; after blocking with PBST containing 5% skim milk powder, add the collected cell supernatant to the ELISA plate and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add mouse 183 polyclonal antibody and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 minutes; after washing with PBST for 5 times, add substrate TMB / H 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 , and calculated the inhibition rate.

[0131] The inhibitory activities of Nb-162 and Nb-172 against clinical isolates of HIV-1 are shown in Fig.12 As shown, with D5 scFv as the positive control, it can be seen that Nb-172 also exhibits excellent inhibitory effects on HIV-1 clinical isolates, with IC values ​​of 92UG029, 92UG024, and 93 / BR / 020 being 0. 50 0.21, 0.21 and 0.34 μM, respectively, while the inhibitory activity of Nb-162 against these three clinical isolates was slightly lower than that of Nb-172, IC 50 0.73~1.32( Fig. 9 ), therefore, we used Nb-172 for further research later.

[0132] Example 9 Inhibitory Effect of Nb-162 & 172 on HIV-1 T20 Drug-Resistant Strain

[0133] Next, we verified the inhibitory activity of Nb-162&172 nanoantibodies against HIV-1T20 drug-resistant strains. The specific steps are as follows:

[0134] Step 1. Nb-162 / Nb-172 / T20 was diluted 4-fold in RPMI 1640 medium (Mei Lun) in a 96-well plate, and then an equal volume of HIV-1 T20 resistant strain was added. After incubation at 37°C for 30 min, CEMx174 5.25M7 cells were added to the mixture, and 1×10 4 / well. After culturing at 37°C for 12 hours, the medium was replaced with RPMI 1640 medium containing 10% FBS. After 4 days, the cell supernatant was collected, treated with 5% TritonX-100, and P24 was detected by ELISA to measure the virus content in the cell supernatant.

[0135] Step 2. Coat the ELISA plate with 5 μg / ml HIV-1 antibody at 4°C overnight; after blocking with PBST containing 5% skim milk powder, add the collected cell supernatant to the ELISA plate and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add mouse 183 polyclonal antibody and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 minutes; after washing with PBST for 5 times, add substrate TMB / H 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 , and calculated the inhibition rate.

[0136] The inhibitory activities of Nb-162 and Nb-172 against HIV-1T20 resistant strains are shown in Table 2. Among them, Nb-172 has the best inhibitory effect on HIV-1T20 resistant strains, IC 50 It is 0.102~0.535μM (Table 3).

[0137] Table 3. Inhibitory effect of Nb-162 & Nb-172 on HIV-1 T20 resistant strains

[0138]

[0139] Example 10 Inhibitory effect of Nb-172 combined with mD1.22 on HIV-1 clinical isolates

[0140] Since the pocket region of the HIV-1gp41 NHR trimer is only transiently exposed during membrane fusion, although nanobodies targeting NHR can inhibit HIV-1 from entering cells, they have no inhibitory effect on free virus particles in the blood or free virus particles newly released after latent cells are activated. CD4 mimics can bind to HIV-1gp120, induce exposure of HIV-1CXCR4 / CCR5 binding sites and gp41 NHR, actively attack virus particles before they attach and enter host cells, and have a direct inactivation effect on free HIV-1. mD1.22 is a soluble CD4 subunit D1 analog obtained by phage display technology. It has high solubility and stability in a neutral environment and can inhibit a variety of HIV-1 strains at low nM levels (Chen W, Feng Y, Prabakaran P, et al. Exceptionally potent and broadly cross-reactive, bispecific multivalent HIV-1 inhibitors based on single human CD4 and antibody domains. J Virol, 2014). Therefore, we first explored whether the combination with Nb-172 could improve the in vitro inhibition of HIV-1 by mD1.22. The specific operation is as follows:

[0141] Step 1. Nb-172 and mD1.22 were mixed according to IC 50 For example, the IC50 of mD1.22 and Nb-172 against 92UG024 were about 9 nM and 208 nM, respectively. Therefore, the two were mixed at a ratio of 1:23 and the inhibitory activity against 92UG024 was tested again. The final concentration of the mixture was 250 nM or 500 nM. The mixture was diluted 4 times in RPMI 1640 medium in a 96-well plate, and an equal volume of HIV-1 clinical isolates was added. After incubation at 37°C for 30 min, CEMx1745.25M7 (93 / BR / 020) or MT-2 (92UG029 and 92UG024) cells were added to the mixture, and 1×10 4 / well. After culturing at 37°C for 12 hours, the medium was replaced with RPMI 1640 medium containing 10% FBS. After 4 days, the cell supernatant was collected, treated with 5% TritonX-100, and P24 was detected by ELISA to measure the virus content in the cell supernatant.

[0142] Step 2. Coat the ELISA plate with 5 μg / ml HIV-1 antibody at 4°C overnight; after blocking with PBST containing 5% skim milk powder, add the collected cell supernatant to the ELISA plate and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add mouse 183 polyclonal antibody and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 minutes; after washing with PBST for 5 times, add substrate TMB / H 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 , and calculated the inhibition rate.

[0143] The inhibitory effect of Nb-172 combined with mD1.22 on HIV-1 clinical isolates is shown in Table 3. The results show that the combination of mD1.22 and Nb-172 has a strong synergistic inhibitory effect on HIV-1 infection, and the combination index (CI value) is 0.24, 0.34 and 0.45, respectively.

[0144] Table 3. The inhibitory effect of mD1.22 and Nb-172 combined on HIV-1 clinical isolate infection

[0145]

[0146] Example 11 In vitro inactivation effect of Nb-172 combined with mD1.22 on HIV-1 clinical isolates

[0147] Next, we verified whether the combination with Nb-172 would enhance the in vitro inactivation effect of mD1.22 on HIV-1 clinical isolates, as follows:

[0148] Step 1. Mix mD1.22 and Nb-172 in a 1.5 ml EP tube at a ratio of 1:1 (final concentration of 125 nM or 250 nM), vortex to mix, dilute 4-fold using RPMI 1640 medium, add an equal volume of 500 TCID50 virus, pipette to mix, and incubate at 4°C for 1 h.

[0149] Step 2. Add 30% PEG6000 to a final concentration of 3%, vortex to mix, and continue incubation at 4°C for 1 h.

[0150] Step 3. Centrifuge at 4°C 13,000 rpm for 30 min in a refrigerated centrifuge and carefully remove the supernatant. The virus precipitates at the bottom of the EP tube. Wash the virus particles with sterile PBS containing 10 mg / ml BSA and 3% PEG6000, centrifuge at 4°C 13,000 rpm for 30 min, and repeat 3 times.

[0151] Step 4. Aspirate the supernatant, resuspend the virus in serum-free RPMI 1640 medium, pipette repeatedly to mix, and add to a 96-well cell culture plate. Add the corresponding target cells according to the virus tropism, CEMx174 5.25M7 (93 / BR / 020) or MT-2 (92UG029 and 92UG024) cells, count them, and add them to the plate, 1×10 per well. 4 The cells were placed in a 37°C 5% CO2 incubator and cultured. After 12 hours, the medium was replaced with RPMI 1640 medium containing 10% FBS.

[0152] Step 5. When CPE is observed under a microscope, 50 μL of supernatant is aspirated, and an equal volume of 5% TritonX-100 is added for treatment. P24 is then detected by ELISA to measure the viral content in the cell supernatant.

[0153] Step 6. Elisa detection of P24: Coat the ELISA plate with 5 μg / ml HIV-1 antibody at 4°C overnight; after blocking with PBST containing 5% skim milk powder, add the collected cell supernatant to the ELISA plate and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add mouse 183 polyclonal antibody and incubate at 37°C for 1 hour; after washing with PBST for 5 times, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 minutes; after washing with PBST for 5 times, add substrate TMB / H 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 , and calculated the virus survival rate.

[0154] The in vitro inactivation effect of Nb-172 and mD1.22 on clinical isolates of HIV-1 Fig.13 As shown in Figure 2, when the two were used in combination, the inactivation effect of mD1.22 on three HIV-1 strains, 92UG029, 92UG024 and 93 / BR / 020, was increased by about 3 times ( Fig.10 ).

[0155] Example 12 Detection of the affinity of Nb-172 and NHR protein using biofilm interferometry

[0156] Next, we further detected the real-time binding kinetics between Nb-172 and N63 trimer protein using biofilm interferometry (BLI). The specific steps are as follows:

[0157] Step 1: Soak the anti-human IgG Fc biosensor (probe) in pure water for 10 minutes and then place it in the first column of a 96-well blackboard. 2 O, 2 μg / ml N63Fd in the second column, 0.02% PBST in the third column, and serially diluted Nb-172 / D5 scFv in the fourth column, with a total volume of 200 μL in each well.

[0158] Step 2: Use the biomolecular interaction instrument (Fortebio Octet RED96) and run the program according to baseline 60s (first column), loading 300s (second column), Baseline-2 300s (third column), Association 300s (fourth column), Disassociation 300s (third column). Equilibrium dissociation constant (K D ) was calculated using Octet QK software according to a 1:1 binding model.

[0159] D5 scFv has been confirmed to be a single-chain antibody targeting the gp41 NHR pocket region (Eckert DM, Shi Y, Kim S, et al. Characterization of the steric defense of the HIV-1gp41 N-trimer region. Protein Sci, 2008). BLI results showed that Nb-172 showed a similar affinity to D5 scFv for the N63 trimer protein, with a K D 24.5 and 24.8 nM respectively. Fig.11 shown.

[0160] Example 13 Binding ability of nanobody Nb-172 to gp41 NHR-derived polypeptide

[0161] Next, to further explore the target of Nb-172, we used ELISA to verify the binding ability of the nanobody to the gp41 NHR-derived peptide. IQN17 is a trimeric peptide containing the gp41 NHR hydrophobic pocket region, and its sequence is RMKQIEDKIEEIESKQKKIENEIARIKKLLQLT VWGIKQLQARIL. Biotin-labeled IQN17 was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd.

[0162] Step 1: 2 μg / ml streptavidin was coated on the ELISA plate at 4°C overnight; after blocking with PBST (phosphate buffer containing 0.05% Tween-20) containing 5% skim milk powder, 5 μg / ml biotin-labeled IQN17 was added and incubated at 37°C for 1 hour; after washing with PBST 5 times, the nanobody was diluted 5-fold and added to the ELISA plate, and incubated at 37°C for 1 hour; after washing with PBST 5 times, HRP-labeled 6×His monoclonal antibody (Proteintech Biotechnology Company) was added and incubated at 37°C for 30 minutes; after washing with PBST 5 times, the substrate TMB / H was added. 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 .

[0163] like Fig.12 As shown, the affinity of Nb-172 to IQN17 is weaker than that of D5 scFv, which indicates that Nb-172 acts on the hydrophobic pocket of gp41NHR, and its target site overlaps with but does not coincide with that of D5 scFv.

[0164] Example 14: Nb-172 and CHR-derived polypeptides compete for binding to N63 trimeric protein

[0165] In order to further narrow the target range of Nb-172, we used ELISA experiments to verify the ability of Nb-172 to competitively bind to NHR trimer protein with gp41CHR derived peptides. The C34 peptide sequence is WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL and was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd.

[0166] Step 1: 2 μg / ml N63 trimer protein was coated in the ELISA plate at 4°C overnight; after blocking with PBST (phosphate buffer containing 0.05% Tween-20) containing 5% skim milk powder, the gradient diluted C34 / T20 polypeptide was mixed with the Nb-172 antibody dilution with a final concentration of 1 μM and added to the plate, and incubated at 37°C for 30 min; after washing with PBST for 5 times, HRP-labeled 6×His monoclonal antibody (Proteintech Biotechnology Co., Ltd.) was added and incubated at 37°C for 30 min; after washing with PBST for 5 times, the substrate TMB / H was added. 2 O 2 After 10 min of color development, H 2 SO 4 Terminate the reaction. Detect OD using an ELISA reader 450 .

[0167] like Fig.13 As shown, C34 inhibited the binding of Nb-172 to N63 trimer protein in a concentration-dependent manner, while T20 showed a weak inhibitory effect on the binding of Nb-172, which indicates that the target sites of Nb-172 and C34 polypeptides overlap, but almost do not overlap with the binding site of T20.

[0168] Sequence information of CHR-binding peptides and spatial binding site analysis of the interaction between gp41 NHR and CHR ( Fig.14 ), we can roughly infer that the target of Nb-172 is located in the hydrophobic pocket region (highlighted) and the amino acid residues at its front end.

[0169] Example 15 Nb-172 blocks the formation of 6-HB

[0170] Given that Nb-172 can bind to the hydrophobic pocket of NHR, we infer that it has the ability to inhibit the formation of 6-HB by NHR and CHR, so we used Native-page to verify whether Nb-172 can inhibit the formation of 6-HB by C34 and N36 peptides. The sequence of N36 peptide is SGIVQQQNNLLRAIEAQ QHLLQLTVWGIKQLQARIL, which was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. The specific operation is as follows:

[0171] Step 1: Nb-172 was diluted in a gradient manner and mixed with an equal volume of 40 μM N36 peptide. After incubation at 37°C for 1 hour, an equal volume of C34 peptide was added to make the final concentration 5 μM. After standing at room temperature for 30 minutes, 5× loading-buffer was added and sample was applied. The electrophoresis conditions were 120 V and the duration was 2 hours.

[0172] Depend on Fig.15 It can be seen that as the concentration of Nb-172 in the mixture increases, the band of the complex formed by N36 and C34 becomes lighter, and the remaining amount of free C34 that has not formed a complex becomes more, indicating that Nb-172 can inhibit the formation of 6-HB between C34 and N36 by binding to NHR.

[0173] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0174] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

[0175]

[0176]

Claims

1. A HIV-1 nanobody, characterized in that: A His tag is fused to the C-terminus of the candidate amino acid sequence for expression, wherein the candidate amino acid sequence is selected from any one of the following sequences: SEQ ID NO: 1Nb-113 EVQVVESGGGLVQPGDSLRLSCVASGRTLSNLN VGWFRQAPGKEREFVAAITWILGTPYYADSAKGRFTISRDNAKNAIYLQMNS LKPEDTAVYRCAAPHGNRYASPEQYVYWGQGTQVTVSS; SEQ ID NO: 2Nb-114 EVQVVESGGGLVQTGGSLRLSCTVSDTHIFSTY VLAWFRQAPGKEREFVGWTDRLTFYADSVEGRFTISRDGAQNTVHLQMDSL RPEDTAVYYCAAGPISDTSPYIEARSYEYWGQGTQVTVSS; SEQ ID NO: 3Nb-117 QVQLVESGGGLVQPGGSLRLSCASSGFSLTYYTI TWFRQASGKERESVSCIESDGTTQDPTSVKGRFTISRDNAKNTVYLQMNDLK PEDTAIYTCATSIAICAWDDPIYDRWGQGTQVTVSS; SEQ ID NO: 4Nb-142 QVQLVESGGGLVQAGGSLTLSCVVSGITFDGRA AGWYRQVPGKQRELVARIDTGGSTNYADSAKGRFTISRDIAENTMYLQMNSL KPEDTAVYYCNVESWFTIAGSYWGQGTQVTVSS; SEQ ID NO: 5Nb-145 EVQVVESGGGLVQPGGSLRLSCTASGFILDFYA MGWFRQAPGKEREGISCISSDDSTYYADSVKGRFIISRDNAKSTVYLQMNNL KPEDTAIYYCATDWDVVCGSSWSSPRHGSDFGAWGQGTQVTVSS; SEQ ID NO: 6Nb-152 EVQLVESGGGLVQPGGSLRLSCASSGFSLTYYT VAWFRQAPGRERERVSCIESNDGRSRYANSVKGRFTISKDNAKNTVYLQMND LKPDDTAVYVCATTLSICDWDDPTYDRWGQGTQVTVSS; SEQ ID NO: 7 Nb-162 EVQVVESGGGLAQAGGSLRLSCATSGRTFNTYP VGWFRQAPGKEREFVAAINWFGERTSYADSVKGRFTISRNNAKNTVYLEMN NLKPEDTAVYRCAARADGTQGIGGANPGSYEYWGQGTQVTVSS; SEQ ID NO: 8 Nb-172 EVQVVESGGGQVQPGGSLRLSCAASGSTFDNF GAWYRQAPGKQRELVAQILLGGTTNYADSVKGRFTISRDNDKNTMYLQMNS LKPEDTAVYYCHTFYRAQFYWGQGTQVTVSS; SEQ ID NO: 9 Nb-184 QVQLVESGGGLVQAGGILRLSCIGSGGTLSGWR MGWYRQAPGQEREFVAASSSHIGNTYYADSVKGRFTISRDNAKNTVYLQMN SLQPEDTAIYYCNSHPTAVFDWGKGTLVTVSS; SEQ ID NO: 10 Nb-187 QVQLVESGGGLVQAGGSLRLSCAASGLPFTNY AMGWFRQAPGKEREFVTAISRNGGTTNYTDSVKGRFTISRDNARNTVYLQM NSLKPEDTAVYVCAAGWTIFSVSRLPTEYAFWGQGTQVTVSS; SEQ ID NO: 11 Nb-190 EVQLVESGGGLVQPGGSLRISCVASGRTFAYYI MGWFRQAPGKEREFVANIRWSDGETYYSDSVKDRFTISRDNTKNTVYLQMN SLKPEDTAVYHCAASQALGIMPWYTRTDNYKYWGQGIQVTVSS.

2. The method for preparing the HIV-1 nanobody according to claim 1, characterized in that: Comprising the following steps: Purified cov-NHR VQ is used to immunize alpacas multiple times to obtain immunized alpacas, so that HIV-1 neutralizing antibodies are produced in the immunized alpacas, wherein the amino acid sequence of the purified antigen cov-NHR VQ that can simulate HIV-1 gp41 NHR trimer is ARQELSGIVQK QNNLLRQIEAQQHLLQLTVSKIKQLQARILAVERYLKDQQLGKGNQPQQDKLYREVALIRAQLQKIESETLQLLHQQAEIERELNNQEQEIGSLKQRGLIDGPLLSGIDQQQNNLKRAIEAQKHLLQLTVWGIKQLQARILTVERYLKDQQLGGGGSHHHHHH; Obtaining peripheral blood from the immunized alpaca; Processing the peripheral blood of the immunized alpaca to obtain an intermediate product to be screened; Screening the intermediate product to be screened to obtain a candidate amino acid sequence of HIV-1 nanobody; The VHH gene of the candidate amino acid sequence of the HIV-1 nanobody is cloned into a prokaryotic expression vector, and the nanobody protein is expressed in the form of VHH-his to obtain the HIV-1 nanobody, wherein in the HIV-1 nanobody, the C-terminus of the candidate amino acid sequence is fused with a His tag for expression.

3. The method for preparing HIV-1 nanobody according to claim 2, characterized in that: In the process of using purified cov-NHRVQ to immunize alpacas multiple times to obtain immunized alpacas, so that HIV-1 neutralizing antibodies are produced in the immunized alpacas, the method for obtaining the purified cov-NHR VQ includes the following steps: According to the amino acid sequence, obtaining a nucleotide sequence; The nucleotide sequence was cloned into the pComb3x vector to obtain the pComb3x-covNHR-VQ plasmid; The pComb3x-covNHR-VQ plasmid was transformed into the HB2151 strain, and a single clone colony was selected and activated in LB at 37° C. overnight and purified to obtain the purified cov-NHR VQ, wherein the purity of the purified cov-NHR VQ was >90%.

4. The method for preparing HIV-1 nanobody according to claim 2, characterized in that: The purified cov-NHRVQ is used to immunize alpacas multiple times to obtain immunized alpacas, so that HIV-1 neutralizing antibodies are produced in the immunized alpacas, and the alpacas are immunized multiple times, including the following steps: The first immunization was performed by mixing 0.5 mg of the immunogen covNHR-VQ with equal volumes of Freund's complete adjuvant and emulsifying them. Subsequently, booster immunizations were performed in the third, sixth, and ninth weeks using an equal volume of 0.25 mg covNHR-VQ mixed with Freund's incomplete adjuvant and emulsified, for a total of four immunizations to obtain immunized alpacas.

5. The method for preparing HIV-1 nanobody according to claim 2, characterized in that: The method of using purified cov-NHRVQ to immunize alpacas multiple times to obtain immunized alpacas, and producing HIV-1 neutralizing antibodies in the immunized alpacas, further comprises the following steps: The step of detecting the antigen-specific antibodies in the alpaca serum and the neutralization titer of the alpaca antiserum to the HIV-1 laboratory adapted strain for the alpaca after the fourth immunization to obtain the immune response result of the alpaca.

6. The method for preparing HIV-1 nanobody according to claim 2, characterized in that: Obtaining the peripheral blood of the immunized alpaca specifically comprises the following steps: Obtaining peripheral blood from alpacas that have undergone the third immunization and the fourth immunization; Separating the alpaca peripheral blood mononuclear cells according to the instructions for use of the lymphocyte separation solution; The peripheral blood of the immunized alpaca was obtained by lysing and freezing the blood using the Trizol method.

7. The method for preparing HIV-1 nanobody according to claim 2, characterized in that: The process of processing the peripheral blood of the immunized alpaca to obtain the intermediate product specifically comprises the following steps: The total RNA of alpaca peripheral blood mononuclear cells was extracted by Trizol method; Reverse transcription is performed on the total RNA to obtain cDNA; Using nested PCR to amplify the nanobody gene fragments from cDNA, a nanobody phage library targeting covNHR-VQ was constructed; The nanobody phage library against the covNHR-VQ is incubated and eluted to obtain an intermediate product to be screened.

8. The method for preparing HIV-1 nanobody according to claim 2, characterized in that: Screening the intermediate product to be screened to obtain a candidate amino acid sequence of HIV-1 nanobody specifically comprises the following steps: Randomly selecting among the intermediate products to be screened to obtain randomly selected clones; Performing monoclonal phage supernatant ELISA identification on the randomly selected clones to obtain ELISA identification results of each randomly selected clone; According to the ELISA identification results, analysis software is used to sequence the clones positive for the covNHR-VQ antigen to obtain sequencing results; According to the sequencing results, similar sequences are removed to obtain the candidate amino acid sequence of the HIV-1 nanobody.

9. The method for preparing HIV-1 nanobody according to claim 2, characterized in that: The VHH gene of the candidate amino acid sequence of the HIV-1 nanobody is cloned into a prokaryotic expression vector, and the nanobody protein is expressed in the form of VHH-his. After obtaining the HIV-1 nanobody, the method further includes the step of confirming that the HIV-1 nanobody is successfully expressed.

10. The use of the HIV-1 nanobody according to claim 1, characterized in that: The application includes one or more of the following applications: Application of modified antibodies containing the HIV-1 nanobody CDR region; Use of the HIV-1 nanobody in the preparation of a drug for preventing and / or treating HIV-1; Application of the HIV-1 nano antibody in the preparation of HIV-1 detection reagent.

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