Preparation and application of anti-HPV6 virus L1 murine monoclonal antibody

Abstract

The application relates to the technical field of biological medicine, in particular to preparation and application of an anti-HPV6 virus L1 murine monoclonal antibody; the anti-HPV6 virus L1 murine monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1; and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2. The murine monoclonal antibody is a unique single-chain antibody, and can be specifically combined with HPV6 virus L1. Western blot experiment results show that the HRP-labeled murine monoclonal antibody recombined in vitro is combined with HPV6 virus L1 to form obvious bands, and the murine monoclonal antibody has the characteristic of targeting HPV6 virus L1.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the preparation and application of anti-HPV6 virus L1 murine monoclonal antibody. BACKGROUND

[0002] Human papillomavirus (HPV) is a small DNA virus without envelope, and there are currently more than 200 types. HPV mainly infects skin and mucosal tissue, and more than 40 types of HPV infection can cause human diseases. According to the relationship between HPV infection and cancer, HPV can be divided into high-risk and low-risk types, and HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 are high-risk types. Persistent infection with high-risk HPV can cause cervical cancer, anal cancer and vaginal cancer and other malignant tumors. Other HPV subtypes are low-risk types, which mainly cause warty hyperplasia of the skin and mucosa, and show lesions such as condyloma acuminatum and flat warts. There is no good treatment method in clinical practice. Epidemiological investigations and clinical studies have shown that the detection rate of HPV 6 and HPV 11 is the highest in genital condyloma acuminatum tissue, and statistics show that the rate of genital warts caused by HPV 6 and HPV 11 infection and recurrent respiratory papillomatosis is more than 90%.

[0003] Currently, experimental and clinical detection methods for HPV6 include PCR, DNA probe hybridization, etc., but these methods are often tedious, time-consuming and costly. Therefore, using monoclonal antibodies to detect and prevent HPV6 L1 virus proteins has high practical value and wide application prospect. The research and application of antibodies (murine) against HPV6 L1 virus proteins can provide a highly specific, sensitive and economical experimental technique. It has important application value in the fields of vaccine research, pathogen detection, virus protein function and antigenicity analysis, etc.

[0004] However, there are still many problems and challenges in the preparation of antibodies against HPV6 L1 virus proteins, such as insufficient specificity of antibodies in the preparation process and insufficient sensitivity to meet the detection and diagnosis requirements. Therefore, in the present application, a new method is used to prepare anti-HPV6 virus L1 murine monoclonal antibody, and the advantages in laboratory and clinical application are discussed, in order to promote the development of anti-HPV6 virus screening and research. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a preparation and application of anti-HPV6 virus L1 murine monoclonal antibody

[0006] The purpose of the present application can be achieved by the following technical solutions:

[0007] An anti-HPV6 virus L1 murine monoclonal antibody, comprising a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2.

[0008] Preferably, the nucleotide sequence of the heavy chain variable region is shown as SEQ ID No. 3; the nucleotide sequence of the light chain variable region is shown as SEQ ID No. 4.

[0009] Preferably, the heavy chain variable region comprises the following three complementarity determining regions CDRs:

[0010] a CDR1 shown as SEQ ID NO. 5,

[0011] a CDR2 shown as SEQ ID NO. 6,

[0012] a CDR3 shown as SEQ ID NO. 7.

[0013] Preferably, the light chain variable region comprises the following three complementarity determining regions CDRs:

[0014] a CDR1' shown as SEQ ID NO. 8,

[0015] a CDR2' shown as SEQ ID NO. 9,

[0016] a CDR3' shown as SEQ ID NO. 10.

[0017] Preferably, a recombinant protein, the recombinant protein has: the sequence of the antibody.

[0018] Preferably, a polynucleotide, preferably the polynucleotide encodes a polypeptide selected from the group consisting of: the antibody; or the recombinant protein.

[0019] Preferably, a vector comprising the polynucleotide.

[0020] Preferably, a pharmaceutical composition comprising the antibody, or the recombinant protein, or the pharmaceutically acceptable carrier.

[0021] Preferably, the anti-HPV6 virus L1 murine monoclonal antibody is used for preparing a kit for detecting HPV6 type capsid protein L1.

[0022] The application provides a mouse-derived monoclonal antibody targeting HPV6 virus L1, which comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1; and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2. The mouse-derived monoclonal antibody is a unique single-chain antibody, and can be specifically combined with HPV6 virus L1. Western blot experiment results show that the HRP-labeled mouse-derived monoclonal antibody recombined in vitro can form obvious bands with HPV6 virus L1, and the mouse-derived monoclonal antibody has the property of targeting HPV6 virus L1. The results show that the mouse-derived monoclonal antibody 6M4 has the strongest binding capacity to HPV6 virus L1 structure. The prepared mouse-derived monoclonal antibody against HPV6 virus L1 has high specificity, can accurately recognize and combine HPV6 L1 virus protein, reduces false positive results caused by non-specific combination, and thus improves the accuracy of detection results. In addition, it is found through experiments that the mouse-derived monoclonal antibody against HPV6 virus L1 can effectively detect samples with low HPV6 concentration, and is helpful for realizing early diagnosis and treatment of HPV6 virus.

[0023] Compared with existing detection and diagnosis methods for HPV6, such as PCR and DNA probe hybridization, the mouse-derived monoclonal antibody against HPV6 L1 is more economical and easy to operate, and makes large-scale screening and research possible. The mouse-derived monoclonal antibody against HPV6 virus L1 has important application value and broad application prospect in the fields of vaccine research, pathogen detection, virus protein function and antigenicity analysis. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 ELISA results of sera of three mice against HPV6 L1;

[0026] Figure 2 Amplification of light and heavy genes of the mouse antibody;

[0027] Figure 3 Specificity determination of 6M4-IgG monoclonal antibody and HPV L1 antigen combination;

[0028] Figure 4 Pseudovirus neutralization effect determination; DETAILED DESCRIPTION

[0029] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work are within the scope of the present application.

[0030] 1. Experimental materials:

[0031] 293F cells were purchased from ATCC and were preserved by the laboratory after subculture; EX Taq enzyme was purchased from TaKaRa company; reverse transcription kit Transcriptor First Strand cDNA Synthesis Kit was purchased from Roche company; plasmid extraction kit and gel recovery kit were purchased from QIAGen company; cell culture medium OPM-293CD05 Medium was purchased from Shanghai Oupu Mai company; endonuclease was purchased from NEB company; library construction vector pComb3XSS was preserved by the laboratory; pseudovirus vector pLenti6.3 and pseudovirus packaging plasmid were purchased from Invitrogen company; mouse IgG double plasmid expression vector was constructed and preserved by the laboratory; HPV6 L1 protein was donated by Jiangsu Ruikuo Biotechnology Company; animal immunization, primer synthesis and sequencing were completed by Chuzhou Tongyong.

[0032] 2. Experimental methods

[0033] 2.1 Animal immunization Three 6-week-old female BALB / c mice were selected and were immunized subcutaneously at a dose of 30 μg per mouse. Complete Freund's adjuvant was used for the first time, and incomplete Freund's adjuvant was used for the subsequent boost immunization. The immunization was performed for 3 times. The mice were euthanized 7 days after the third immunization, and blood and spleen were prepared for use. This part was completed by Chuzhou Tongyong Biotechnology Company.

[0034] 2.2 Mouse ScFv antibody library construction: Mouse spleen was ground through 40 mesh sieve with normal saline, then PBMC was isolated by Ficoll density gradient centrifugation, total cell RNA was extracted and reverse transcribed into cDNA. The variable region of heavy chain and light chain were amplified respectively using series of mouse antibody specific primers, the PCR condition was: 94℃ 3min, 94℃ 30s, 57℃ 30s, 72℃ 45s, 30 cycles. About 350bp fragments were recovered by gel, then light and heavy chains were connected into ScFv fragments by Overlapping PCR. ScFv was digested by Sfi I and connected with phage vector pComb3xss, then TG1 competent cells were electroporated to form antibody library. The library construction method was basically according to the literature (Breitling F, Broders O, Helmsing S, Hust M, Dübel S (2010) Human antibody gene libraries. In: Kontermann R, Dübel S (eds) Antibody Engineering, vol 1, 2nd edn. Springer, Heidelberg, pp 197-206 and PMID: 22481024).

[0035] 2.3 Antibody library screening: HPV6 L1 protein was coated with 0.1M PBS PH=7.4 at 4℃ overnight, and then blocked with MPBST (PBS added with 1% skim milk and 0.05% Tween-20) at room temperature for 2h. Then it was washed with PBST for 3 times. 1012 phage antibody library was added, and incubated at room temperature for 2h. The supernatant was discarded, and washed with PBST for 10 times. Glycine-HCI (pH 2.2) was added for elution, and then 2mol / L Tris was added to neutralize to PH=7.0. The eluted phage infected fresh TG1 (OD600=0.5), and then infected with helper phage VCSM13 for the next round of screening. This was repeated for 3 times. The specific enrichment method and ScFv antibody induction expression were basically according to the literature PMID: 31255726. After 3 rounds of enrichment, 96 single clone colonies were selected, and the bacterial expression supernatant was detected by ELISA for the binding specificity with HPV6 L1 protein after IPTG induction. The positive colonies were sequenced, and the antibody sequence was analyzed by IMGT database. The antibodies with different sequences were selected for whole antibody plasmid construction and expression and purification.

[0036] 2.4 Mouse IgG2a whole antibody expression plasmid construction and antibody purification: the selected mouse antibody light chain was cloned into the pGI expression vector containing the mouse light chain constant region through the Age I / Bsiw I enzyme cutting site, and the heavy chain was cloned into the pGI expression vector containing the mouse heavy chain constant region through the Age I / Sal I enzyme cutting site. The double plasmid was co-transfected into 293F cells by PEI, and the supernatant was collected after 7 days of 37°C 8% CO2 shaking culture. The expression supernatant was purified by Protein A column, desalted by dialysis, and then quantified by BCA protein before being ready for use.

[0037] 2.5 Indirect ELISA assay of mouse whole antibody and HPV6 L1 binding specificity: HPV6 L1 protein was coated on the enzyme-labeled plate, and the recombinant monoclonal antibody was added in a dilution ratio. After 1h of 37°C incubation, it was washed with PBST for 3 times; HRP-labeled anti-mouse IgG was added and incubated at 37°C for 1h, and then washed with PBST for 3 times; TMB was used for color development, and 2M H2SO4 was used to terminate the reaction, and the OD450nm value was read by the enzyme-labeled instrument. Similarly, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58 L1 proteins were coated on the enzyme-labeled plate, and the recombinant mouse monoclonal antibody was added, and then HRP-labeled anti-mouse IgG secondary antibody was added, followed by color development and OD value reading.

[0038] 2.6 HPV6 pseudovirus neutralization test The HPV pseudovirus was packaged by pLenti system, the HPV6 L1 gene was cloned into pLenti-CMV-GFP-BSD vector, and then mixed with pLP1, pLP2 and pLP-VSVG in equal amounts, and then transfected into 293T cells using Lipofectamine TM 2000, and the specific operation was carried out according to the manual. The expression supernatant was collected after 72h, concentrated 10 times by 100kDa ultrafiltration tube, and then frozen at -80°C. The neutralizing antibody detection method was established according to the reported literature (DOI: 10.3969 / j.issn.1005-5673.2012.06.005). 1x104 / well 293T cells were inoculated in a 96-well cell plate and cultured at 37°C 5% CO2 overnight. The antibody to be tested was diluted in culture medium starting from 1mg / ml, mixed with an equal amount of virus with MOI = 0.2, and incubated at room temperature for 1h. 100μL of virus-antibody complex was added to the 96-well cell plate, and the results were observed under a fluorescence microscope after 72h. When the fluorescence intensity of the test well was about half of that of the negative control, the dilution factor of the antibody was the neutralizing titer of the antibody.

[0039] 3、Experimental results

[0040] 3.1 Mouse immunization results: 3 BALB / c mice were immunized with HPV6 L1 protein, and blood was taken from the mice one week after the third immunization. The mice were euthanized. The HPV6 L1 protein was used to coat the enzyme-labeled plate, and the serum samples of the 3 mice were detected. The antibody titers were all 1:640,000, as shown in Figure 1 This result indicates that the mice produced a strong immune response to the protein, which can be used for subsequent library construction.

[0041] 3.2 Mouse ScFv antibody library construction: After the mouse spleen cells were separated and total RNA was extracted, the total RNA was reverse transcribed into cDNA using OligodT primers. The cDNA was used as a template to amplify the antibody light chain and heavy chain genes, respectively. After the light and heavy chain genes were recovered, they were mixed in equal proportions and used as a template to connect the ScFv fragment by Overlapping PCR. As shown in Figure 2

[0042] 3.3 Library screening and antibody acquisition: The library was enriched for 3 rounds using HPV6 L1 protein, and the amount of phage used each time was 5x1011 phage. It was found that the output efficiency of the second round was slightly lower than that of the first round, but the third round showed a 10-fold increase. After the third round of enrichment, 96 single clones were randomly picked and induced, and then ELISA was used to detect the binding specificity of the antigen. Of the 96 single clones, 32 wells had an OD>0.4. The 32 clones were sent for sequencing, and it was found that the 32 sequences were exactly the same, and were named 6M4. The screening results are shown in Table 1.

[0043] Table 1 Phage library screening results

[0044]

[0045] 2.4 Whole antibody plasmid construction and mouse IgG2a whole antibody expression: The 6M4 light and heavy chains were cloned into the pGI-Mouse-Kappa and pGI-Mouse-IgG2a two expression vectors, respectively. After sequencing, two expression plasmids of the light and heavy chains were obtained. The two expression plasmids were large-scale extracted and transfected into 293F cells. The recombinant IgG antibody was obtained by purifying the expression supernatant using a Protein A column, and was named 6M4-IgG.

[0046] 2.5 Indirect ELISA to verify the binding specificity of 6M4-IgG to HPV6 L1 and the cross-reactivity of other types of L1 protein The enzyme-labeled plate was coated with HPV6 L1 protein, and indirect ELISA was used to detect the binding of 6M4-IgG to HPV6 L1 protein. As shown in Figure 3 A, the detection limit of 6M4-IgG for HPV6 L1 protein was less than 7.8 ng / ml, and the blank control did not bind to the coated protein. Figure 3 ​B is the cross-reactivity results of two monoclonal antibodies with nine HPV L1 proteins. Q9-IgG has certain cross-reactivity with HPV18 and HPV45 in addition to binding to HPV6 L1. Q11-IgG is only specific to HPV6 L1 and does not produce a significant reaction with L1 antigens of other types.

[0047] 2.6 Evaluation of the neutralization effect of the antibody pseudovirus: The neutralization activity of two recombinant mouse monoclonal antibodies was evaluated by the HPV6 pseudovirus system. The results are shown in Table 2. Figure 4

[0048] In the description of the present specification, the description of the terms "one embodiment", "example", "specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0049] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the present application is not limited by the above embodiments, and the above embodiments and descriptions in the specification are only illustrative of the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.​

Claims

1. A murine monoclonal antibody against HPV6 virus L1, characterized in that, It includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The application of the anti-HPV6 virus L1 murine monoclonal antibody as described in claim 1 in the preparation of a kit for detecting HPV6 capsid protein L1.

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