NL2029160B1 - Anti-ev-d68 monoclonal antibody and preparation method and use thereof - Google Patents
Anti-ev-d68 monoclonal antibody and preparation method and use thereof Download PDFInfo
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Abstract
Described is an anti-EV-D68 monoclonal antibody and a preparation method and use thereof. A single B cell sorting technology is firstly used to sort a specific single memory B cell of macacamu/az‘z‘a infected with an EV-D68, paired antibody light and heavy chain genes are obtained, and an EV-D68 specific monoclonal antibody with a neutralizing activity is obtained through further screening. The monoclonal antibody comprises heavy chains and light chains, and has nucleotide coding sequences and amino acid coding sequences as shown in the sequence listing. The present disclosure provides an alternative drug for preventing and treating an infection by EV-D68 and also provides a new tool and a new idea for identifying the EV-D68.
Description
ANTI-EV-D68 MONOCLONAL ANTIBODY AND PREPARATION METHOD AND USE
TECHNICAL FIELD The present disclosure belongs to the technical field of a preparation of an anti-infective monoclonal antibody and specifically relates to a monoclonal antibody capable of effectively inhibiting an EV-D88 infection. At the same time, the present disclosure also relates to a preparation method of the monoclonal antibody and use of the antibody in identifying an EV- D68 virus, preparing an anti-infective drug, and the like.
BACKGROUND Enterovirus D88 (EV-D68) belongs to the family picornaviridae, the genus enterovirus and was first isolated in the United States in 1962. In 2014, EV-D68 had a large-scale outbreak and a wide spread in the United States and Canada. Subsequently, respiratory diseases caused by the EV-D68 are also prevalent in Europe, Thailand, China, New Zealand, etc. The EV-D68 not only can cause serious respiratory diseases such as pneumonia, but also central nervous system symptoms such as acute flaccid paralysis (AFP). Since effective therapeutic drugs and preventive vaccines are still in an exploration stage, a research on sorting and identification of a monoclonal antibody based on an infected animal model is expected to provide a new breakthrough for preventing and treating EV-D68.
In recent years, a monoclonal antibody technology has been developed. An anti-infective monoclonal antibody has the following advantages of rapid and immediate protection, specificity against pathogens and relatively low clinical side effects. In addition, an antigen epitope recognized by the monoclonal antibody can be used as a target for designing a novel genetic engineering vaccine. By means of these advantages, the anti-infective monoclonal antibody has become an ideal therapeutic agent for many diseases, is widely used in cancers, autoimmune disorders and infectious diseases, and has become an important supplement to preventive vaccines. An earliest monoclonal antibody is a murine-derived monoclonal antibody obtained by a hybridoma technology based on B cell fusion. Although the technology can obtain monoclonal antibodies aiming at a large portion of antigens, such monoclonal antibodies are susceptible to eliciting an immune response against a heterologous protein in human body. To alleviate this limitation, the murine-derived monoclonal antibody is humanized, but such humanized antibody still do not mimic a precise conformation of a natural antibody in human body and specificity aiming at a target antigen. To completely address an interference of the murine-derived system, a fully humanized hybridoma technology begins to be used by some scientists. Other display technologies of a monoclonal antibody are emerging, including phage display, yeast display, mammalian cell display and the like. However, these technologies still have disadvantages: a produced antibody light and heavy chain gene pairing is unnatural and random, and a produced antibody has different activity from a natural antibody. In view of the above disadvantages, based on direct amplification of light and heavy chain genes of an antibody encoded in a human single B cell, a monoclonal antibody isolation strategy has emerged. Such single B cell separation technology can more efficiently separate to obtain a functional monoclonal antibody naturally occurring in vivo.
SUMMARY The present disclosure is to provide an anti-EV-D68 monoclonal antibody aiming at the deficiencies of the prior art and provide a candidate drug for preventing and treating an EV-D68 infection. The present disclosure also aims to provide a method for preparing the monoclonal antibody. The present disclosure also aims to provide use of the monoclonal antibody in preparing a drug for treating or preventing an EV-D88 infection. The purpose of the present disclosure is achieved through the following technical solutions. An anti-EV-D68 monoclonal antibody includes heavy chains and light chains, wherein CDR2-FR3-CDR3-FR4 of a heavy chain variable region of the monoclonal antibody has a nucleotide coding sequence as shown in SEQ ID NO.1 of the sequence listing; CDR2-FR3- CDR3-FR4 of a light chain variable region has a nucleotide coding sequence as shown in SEQ ID NO.2 of the sequence listing; CDR2-FR3-CDR3-FR4 of the heavy chain variable region of the monoclonal antibody has an amino acid coding sequence as shown in SEQ ID NO.3 of the sequence listing; and CDR2-FR3-CDR3-FR4 of the light chain variable region has an amino acid coding sequence as shown in SEQID NO.4 of the sequence listing. A method for preparing the anti-EV-D68 monoclonal antibody includes the following steps: (1) infecting Macaca mulatta with an EV-D68 intranasally as a donor to sort an EV-D68 specific single B cell; (2) using an EV-D68VP1 protein prepared in a previous stage as a sorting antigen to sort anEV-D88 specific single memory B cell; (3) establishing a nested PCR system for single B cell antibody light and heavy chain genes and amplifying the antibody light and heavy chain genes from the single memory B cell; (4) ligating the antibody light and heavy chain genes to an expression vector and conducting an expression in 293T cells, 293A cells and CHO cells; and (5) purifying an expressed antibody. Use of the anti-EV-D68 monoclonal antibody in preparing a drug for treating or preventing an EV-D68 infection. Use of the anti-EV-D88 monoclonal antibody in identifying an EV-D68. Use of the anti-EV- D68 monoclonal antibody in inhibiting an EV-D68 infection.
The monoclonal antibody of the present disclosure is isolated and obtained from Macaca mulatta. A single B cell separation technology is used. An anti-EV-D68VP1-protein monoclonal antibody A6-1 can be prepared according to a conventional preparation method known in the art and the monoclonal antibody A6-1 can also be prepared by referring to the relevant literature (Christopher S, Ganesh P, Lyadh D, et al. 2012, J Immunol Methods, 388, 85-93).
In the present disclosure, EV-D88 is used to infect Macaca mulatta intranasally. The Macaca mulatta produces a memory B cell specific to an EV-D6&8 antigen and an EV-D68 VP1 specific sorting antigen is used to sort out a memory B cell of an EV-D68-specific membrane B- cell receptor (BCR).
In order to prepare an EV-D68 VP1 sorting antigen, an EV-D68 VP1 protein is firstly expressed, and the gene cloning, restriction digestion and ligation of target gene fragments, cloning and expression vector construction, transformation of competent cells, and expression and purification of proteins are performed according to a molecular cloning technology well known to those skilled in the art (Sambrook et al. Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, 2001} or referring to the previously declared patent (patent application number: 201710579444.9) of our laboratory.
The EV-D68 VP1 protein can be fluorescently labelled according to a preferred embodiment of the present disclosure. A fluorescent labelling method refers to instructions of a kit for protein labelling.
The fluorescently labelled EV-D68 VP1-FITC is mixed with anti-monkey CD20" and anti- monkey CD27" flow cytometry antibodies in a certain ratio, and a single memory B cell is sorted from EV-D68 infected Macaca mulatta. Peripheral blood mononuclear cells (PBMCs) are firstly isolated from Macaca mulatta. A monkey lymphocyte separation solution can be used for separation according to a conventional PBMC sorting method in the art. A mixture of the EV- D868 VP1-FITC antibody, anti-monkey CD20" antibody and anti-monkey CD27* antibody is used to stain and label the PBMCs, and single B cell sorting is conducted by a flow cell sorter.
A nested PCR method is used to amplify antibody light and heavy chain genes of an obtained EV-D68 specific memory B cell by sorting. Design of primers for the nested PCR is optimized according to a literature (Meng W, Li L, Xiong W, et al. 2016, mAbs, 7(4):707-718): upstream primers of a first round are mainly for a leader peptide sequence and downstream primers mainly cover a constant region of an antibody; and upstream primers of a second round mainly target framework region 1 (FR1) of a variable region and downstream primers mainly target a J region of the variable region. According to a preferred embodiment of the present disclosure, nested PCR amplification of the antibody light and heavy chain genes is performed.
The obtained antibody light and heavy chain gene fragments by amplification are ligated to a cloning vector and an expression vector. According to a molecular cloning technology well known to those skilled in the art (Sambrook et al. Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, 2001), gene cloning, restriction digestion and ligation of target gene fragments, cloning and expression vector construction, and transformation of competent cells are conducted. The selected cloning vector in the present disclosure is a Topo vector. According to a conventional molecular cloning operation, when the vector is used, a positive cloning rate of the gene fragments reaches 90% or more.
The identified expression vector containing correct antibody light and heavy chain gene sequence is transfected into 293T, 293A and CHO cell lines for antibody expression. In 293T and 293A expression systems, a serum-free medium and a liposome transfection reagent are used. In a CHO cell expression system of a suspension expression manner, a serum-free medium and a FreeStyleMax reagent as a transfection reagent are used. The rest operation methods are conducted in accordance with conventional operation methods of the 293T, 293A and CHO expression systems in the art. Protein A is used for purifying an antibody by affinity chromatography and the purified antibody is collected.
Nucleotide sequence analysis results show that light and heavy chain genes of the purified monoclonal antibody AS6-1 of the present disclosure have a nucleotide coding sequence as shown in SEQ ID NO:1 to SEQ ID NO:2 in the sequence listing; and according to the nucleotide coding sequence of the light and heavy chain genes of the monoclonal antibodyA8-1, the monoclonal antibodyAB-1 has an amino acid sequence as shown in SEQ ID NO:3 to SEQ ID NO:4 in the sequence listing. All amino acid sequences are expressed in standard one-letter abbreviations for amino acids.
The monoclonal antibody A6-1 of the present disclosure can effectively recognize an EV- D68 VP1 protein, such that the monoclonal antibody can be used to identify an EV-D68 in the future.
In order to identify biological functions of an antibody A6-1, an EV-D68 neutralizing test is conducted in the present disclosure. The neutralizing antibody test is important to test a neutralizing efficacy of an antibody in vitro. The test shows that the A6-1 can effectively inhibit adsorption and infection of EV-D68 to cells. In order to identify a mechanism by which the antibody A6-1 exerts a neutralizing effect, a virus adsorption inhibiting experiment is conducted in the present disclosure. The experiment can reflect an inhibitory effect of an antibody on virus adsorption to host cells. The result shows that the A6-1 can effectively inhibit an adsorption of the EV-D68 on cells and has a dose-effect relationship. An anti-EV-D88 infective effect of the AB-1 in suckling mice is further verified. The result shows that the A6-1 can effectively inhibit an infection of the suckling mice caused by EV-D68 nasal cavity challenge. Therefore, the above experiments show that the antibody A6-1 can be used in treating an anti-EV-D68 infection.
Compared with the prior art, the present disclosure has the following beneficial technical effects: A single B cell sorting technology is firstly used to sort a specific single memory B cell of Macaca mulatta infected with an EV-D68, paired antibody light and heavy chain genes are obtained, and an EV-D68 specific monoclonal antibody with a neutralizing activity is obtained through screening. The finding of the monoclonal antibody not only provides an alternative drug for preventing and treating an infection by EV-D68, a new tool for identifying the EV-D68, but also provides a technical reference for research and development of an antiviral monoclonal antibody drug. 5
BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 shows viral load levels in blood, nasal swab, and faecal samples at different time points after Macaca mulatta is infected with an EV-D68.
FIG. 2 shows flow sorting of an EV-D88 VP1 specific single memory B cell. The EV-D68 VP1 specific single memory B cell (CD20+CD27+EV-D68-VP1APC+) is sorted into a 96-well plate. SSC means side scattered light and FSC means forward scattered light.
FIG. 3 shows targeting of a single memory B cell antibody light heavy chain gene sequence of Macaca mulatta infected with an EV-D68. (A) Distribution of different subgroups of IGHV, IGHK and IGHL antibody gene sequences in a specific single B cell, and (B) Analysis of amino acid lengths in a CDR3 region of different antibody gene sequences.
FIG.4 shows silver staining analysis of a purified EV-D68 and western-blot analysis of an antibody-specific recognition of an EV-D&8 particle.
FIG.5 shows affinity analysis of an EV-D68 VP1 specific paired monoclonal antibody and activity analysis of binding of 31 paired EV-D68specific monoclonal antibodies.
FIG.6 shows viral load of an EV-D68 adsorbed on surfaces of Hela and Vero cells detected by qRT-PCR. * represents p<0.05 and has a statistical difference.
FIG.7 shows effectiveness analysis of neutralization of A6-1 at different concentrations on viruses at a pre-attachment stage.
FIG.8 shows a verification of an in-vivo protection effect of A6-1 on suckling mice. HE pathological section staining analysis on lung tissues and brain tissues of a treatment group and a control group of the suckling mice, wherein a magnification of a microscope is 200 times. Congestion (black arrows} and filtration of inflammatory cells (white arrows).
DETAILED DESCRIPTION The present disclosure will be further described in detail below with reference to the drawings and embodiments, but the drawings and embodiments are not intended to limit technical solutions of the present disclosure. All changes and equivalent substitution made based on the present disclosure shall fall within a protection scope of the present disclosure.
Example 1 Detailed description of preparation process of anti-EV-D68 monoclonal antibody Infection of Macaca mulatta with EV-D68
1) Animal experiments conformed to a 3R principle, namely replacement, reduction and refinement and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Medical Biology, Chinese Academy of Medical Sciences. Two healthy 6-month- old Macaca mulatta weighing 1.2kg+0.3kg were intranasally injected with an EV-D68 with a titre of 104.5CCID50 via the upper respiratory tract. Before the experiments, an EV-D68 antibody test of the two Macaca mulatta was negative.
2)At1, 3,5, 7, 9, 11 and 14 days after the Macaca mulatta was intranasally infected, viral load of faeces, nasal swab, and blood samples was detected; and detection of neutralizing antibody levels was conducted at 0, 7, 14, 21 and 28 days; 3) Result analysis: the viral load level in the blood began to increase on the 3rd day, reached a peak on the 9th day after the infection, after which it began to decline, which was consistent with the aforementioned change time points of blood cells. The viral load in the nasal swab and faeces samples peaked on the third to fifth days. The viral load of the faeces samples of the two Macaca mulatta were 28,900 copies/100mg and 26,000 copies/100mg, respectively.
Viremia and high viral load in the nasal swab and faeces samples indicated that the Macaca mulatta was infected with the EV-D68 (FIG. 1). After the EV-D68 infection, the levels of the neutralizing antibody produced by two Macaca mulatta were relatively low, not exceeding 1:16. However, the result still showed that the Macaca mulatta had an antibody immune response to the EV-D68. Based on the results of the above blood routine examination, viral load and neutralizing antibody levels, it can be seen that the EV-D68-infected Macaca mulatta produced a specific immune response against the EV-D68.
Marking of EV-D68 VP1 sorting protein According to instructions of a Lightning-Link allophycocyanin (APC) conjugation kit (a protein labelling kit, Innova Biosciences), a VP1 protein was marked as a VP1-APC protein capable of being used for flow sorting and the labelling was conducted according to the following steps: 1) 1 ul of an LL-modifier reagent was added to every 10 HI of a purified VP1 recombinant protein and the materials were mixed gently; 2) a lid of Lightning-Link was opened, the VP1 protein labelled with the LL-modifier reagent was added, the materials were mixed gently with a micropipette, and the liquid was pipetted 1-2 times; 3) the above suspension was placed at room temperature (20-25°C) and incubation was conducted in the dark for 3 hours; or incubation was conducted overnight at 4°C in a dark room; and 4) after the incubation was completed, 1 pl of an LL-quencher was added to every 10 pl of the suspension and labelling was conducted for 30 min before use.
Sorting of EV-D68 specific single memory B cell In this section, flow sorting of an EV-D88 specific single B cell was conducted.
1) Peripheral blood mononuclear cells (PBMCs) were isolated from the Macaca mulatta infected with the EV-D68, the obtained PBMCs by the isolation were subjected to flow cytometry antibody staining and the PBMCs were resuspended with 100pl of a phosphate buffer saline (PBS) solution; 2) CD20+-PE, CD27+-FITC and EV-D68VP1+-APC flow cytometry antibodies were added into the obtained suspension, and incubation was conducted in the dark at 4°C for 30 min; 3) washing twice with PBS was conducted and resuspending with 300 ul of PBS was conducted; 4) machine sorting: CD20+-PE and CD27+-FITC double-positive cells were gated, this group of the positive cells was gated for EV-D68VP1*-APC, and the cells in a gate were sorted into a 96-well plate; 5) the 96-well plate containing B cells after the sorting was immediately stored at -80°C; and 6) the results showed that in two Macaca mulatta intranasally infected with the EV-D68, the memory B cells specific for an EV-D68VP1 antigen have a percentage of 3.56% and 2.02%, respectively, which was obviously increased, compared with 0.58% and 0.97% before the infection (FIG. 2). RT-PCR of single B cell and nested PCR amplification of antibody light and heavy chain genes A RT-PCR reaction solution was supplemented into the 96-well plate, reaction was conducted according to a reaction ratio and reaction conditions of a PrimeScript TM II 1st Strand kit (TAKARA), cDNA was obtained as a nested PCR template and a nested PCR reaction was conducted.
A first round of a nested PCR reaction system for a heavy chain gene of the antibody was as follows: PCR reaction conditions: pre-denaturation at 98°C for 1min; PCR reaction at 98°C for 10s, 55°C for 5s and 72°C for 5s, a total of 50 cycles; and final extension at 72°C for 5min; A first round of a nested PCR reaction system for a light chain gene kappa of the antibody was as follows: PCR reaction conditions: pre-denaturation at 98°C for 2min; 98°C for 10s, 60°C for 5s and 72°C for bs, a total of 45 cycles; and final extension at 72°C for 5min; A first round of a nested PCR reaction system of a light chain gene Lambda of the antibody was as follows: PCR reaction conditions: pre-denaturation at 98°C for 2min; PCR reaction at 98°C for 10s, 54°C for 5s and 72°C for 5s, a total of 45 cycles; and final extension at 72°C for 5min; A second round of the nested PCR reaction system of the heavy chain gene of the antibody was as follows:
PCR reaction conditions: pre-denaturation at 98°C for 1min; 98°C for 10s, 60°C for 5s and 72°C for 5s, a total of 50 cycles; and 72°C for 5min; A second round of the nested PCR reaction system of the light chain gene kappa of the antibody was as follows: PCR reaction conditions: pre-denaturation at 98°C for 2min; 98°C for 10s, 60°C for 5s and 72°C for 5s, a total of 45 cycles; and final extension at 72°C for 5min; A second round of the nested PCR reaction system of the light chain gene Lambda of the antibody was as follows: PCR reaction conditions: pre-denaturation at 98°C for 2 min; 98°C for 10s, 60°C for 5s and 72°C for bs, a total of 40 cycles; and final extension at 72°C for 5 min; and
1.2% agarose gel electrophoresis was used to detect the second round of a PCR product. Gel of the PCR product with a positive fragment at 300-400 bp was recovered and the concentration of the recovered DNA fragment was measured by nanodrop.
Ligation of purified PCR fragments of light and heavy chain genes with TOP vector The target fragments were ligated to a cloning vector Topo:
1. Operation in an air bath (20-30°C) according to the following system (10 pl) After the above reagents were conducted, a pipette was used to mix evenly or a tube bottom was flicked gently to mix evenly, all liquid on the tube bottom was collected by instantaneous centrifugation at a low speed and ligation was conducted at room temperature for 5 min.
2. Transformation of ligation product into competent cell DH5a 1) Competent cells were placed on an ice bath; 2) 5 ul of a ligation reaction solution was added to the competent cells, gentle flicking and even mixing were conducted, and reaction was conducted on an ice bath for 30 min; 3) a centrifuge tube was placed in a 42°C water bath for 90 s, quickly transferred to an ice bath, and cooled for 2-3 min; the centrifuge tube should not be shaken during this process; 4) 900 ul of an antibiotic-free LB medium was added to the centrifuge tube and after even mixing, a shaking culture was conducted at 37°C for 45 min (150 r/min); and 5) the contents in the centrifuge tube were mixed evenly and 100 ul of the obtained suspension was taken and evenly spread on an LB solid agar plate (containing 100 mg/ml of ampicillin) by using a sterile spreading rod; the plate was placed upside at 37°C for 15 min until the liquid was completely absorbed by the LB culture plate; and the plate was placed upside down and cultivated for 16-18 h until a single colony was formed.
3. Shaking The single colony grown in the above ampicillin-resistant LB culture plate was picked, the single colony was added to an ampicillin-resistant liquid LB medium, a shaking culture was conducted at 37°C for 18-18 h (200 r/min) and bacteria liquid was collected.
4. Small-amount extraction of plasmid
1) Column balance: 500 pl of a balance solution BL was added to an adsorption column CP3, centrifugation was conducted at 12,000 rpm for 1 min, waste liquid in a collection tube was discarded, and the adsorption column was put back into the collection tube; 2) 3 ml of the bacterial liquid was taken to be cultured overnight, the cultured bacteria were added into a centrifuge tube, centrifugation was conducted at 12,000 rpm for 1min by using a conventional desk centrifuge and the supernatant was pipetted out as much as possible; 3) 250 HI of a solution P1 (whether RNase A was added should be checked first) was added to the centrifuge tube with a bacterial precipitate and the bacterial precipitate was completely suspended by using a pipette and a vortex oscillator; 4) 250 pl of a solution P2 was added to the centrifuge tube and the centrifuge tube was gently turned up and down 6-8 times to fully lyse the bacteria; 5) 350 pl of a solution P3 was added to the centrifuge tube and the centrifuge tube was gently turned up and down 6-8 times immediately to fully and evenly mix the liquid, at this time, a white flocculent precipitate appeared and centrifugation was conducted at 12,000rpm for 10min; 6) the collected supernatant in the previous step was transferred to an adsorption column CP3 (the adsorption column was put into the collection tube) by using a pipette and the precipitate should not be pipetted out as much as possible. Centrifugation was conducted at 12,000 rpm/min for 60s, the waste liquid in the collection tube was discarded, and the adsorption column CP3 was put into the collection tube; 7) 600 pl of a rinsing solution PW was added to the adsorption column CP3 (whether absolute ethanol was added should be checked first), centrifugation was conducted at 12,000 rpm/min for 30-60s, the waste liquid in the collection tube was discarded, and the adsorption column CP3 was put into the collection tube; 8) the operation in step 7) was repeated; 9) the adsorption column CP3 was put in the collection tube and centrifugation was conducted at 12,000 rpm/min for 2min to remove the residual rinsing solution in the adsorption column; and 10) the adsorption column CP3 was placed in a clean centrifuge tube, about 30pl of an elution buffer was added dropwise to a centre of an adsorption membrane, the adsorption column CP3 was placed at room temperature for 2 min, centrifugation was conducted at 12,000rpm/min for 2 min, and a plasmid solution was collected in the centrifuge tube.
5. Identification of positive clones 1) Electrophoresis identification: the extracted plasmids were subjected to an electrophoresis and comparison with the negative control was conducted to screen out the plasmids that may be positive;
2) enzyme digestion identification: double digestion with EcoR I/Nhe | or EcoR |/Pvull was conducted and the molecular weight of a digested product was compared with that of a DNA Marker by electrophoresis; and 3) sequencing confirmation of recombinant plasmids: the clones identified positive by electrophoresis and enzyme digestion were sent to Takara Biotechnology (Dalian) for sequencing, and expression vector plasmids with correct sequencing were stored.
6. Targeting of antibody light and heavy chain gene sequences and result analysis. The result showed that in a heavy chain variable region (VH) of all antibodies, /GHV3 was the most representative subgroup (61.5%), followed by /GHV1 (23.1%), IGHV4 (11.8%) and IGHV7 (3.8%) (FIG.3). In V-Kappa, /GKV1 and /GKV3 were the most representative subgroups with a ratio of 37.5%, /GKV2 had a ratio of 8.3% and /GKV4had a ratio of 16.7% (FIG.3). For a Lambda variable region gene germline, there were only /GLV1 and /GLV3 subgroups, which accounted for 14.3% and 85.7% respectively (FIG.3). Length of a heavy chain variable region CDRH3 varied between 11-22 amino acids and length of all clones of CDR3 was greater than 11 amino acids. Length of CDRL3 amino acids of a light chain Kappa was less than or equal to 9 and length of CDRL3 amino acids of all cloned light chain Lambda was between 10-14(FIG.3). Therefore, after Macaca mulatta was infected with the EV-D68, the Macaca mulatta was stimulated by foreign antigens and antibody genes were rearranged, which was different from a germline gene source before the infection.
Ligation and identification of antibody light and heavy chain gene fragments with expression vector
1. A pTOPO plasmid with correct antibody light and heavy chain gene fragments and an antibody eukaryotic expression vector were respectively subjected to double enzyme digestion and a double enzyme digestion system (EcoR I/Nhe |) for the pTOPO plasmid ligated with the antibody light chain gene and the eukaryotic expression vector was prepared according to the following table: Reaction conditions: reaction was conducted at 37°C for 3 h and gel of a digested product was recovered.
2. A double enzyme digestion system (EcoR I/Pvull) for the pTOPO plasmid ligated with the antibody heavy chain gene and the eukaryotic expression vector was prepared according to the following table: Reaction conditions: reaction was conducted at 37°C for 3 h and gel of a digested product was recovered.
3. Gel recovery of antibody target gene fragments and expression vector after digestion 1) Gel blocks were crushed; 2) the gel blocks were weighed and the volume of the gel blocks was calculated according to a conversion ratio of 1mg to 1p;
3) the gel blocks were dissolved; 1.0%-1.5% gel and a dissolving solution GM were added to the gel blocks at a volume ratio of 1:4; 4) the gel blocks were dissolved at room temperature and intermittent shaking and mixing were conducted to fully dissolve the gel blocks; 5) a Spin column in a kit was arranged in the collection tube, a gel solution in step (4) was transferred to the Spin column, centrifugation was conducted at 12,000 rpm for 1 min and a filtrate was discarded; 6) 700 ul of a washing solution was added to the Spin column, centrifugation was conducted at 12,000 rpm at room temperature for 30 s, and a filtrate was discarded; 7) the operation in step 6) was repeated; 8) the Spin column was placed in a new 1.5-ml centrifuge tube, 30pl of sterile water or an elution buffer was added to a centre of a Spin Column membrane, and standing at room temperature was conducted for 1 min; and 9) centrifugation was conducted at 12,000 rpm for 1 min at room temperature to elute DNA.
4. Ligation of expression vector with antibody light and heavy chain gene fragments A ligation reaction system was as follows: The above-mentioned reaction solution was mixed evenly, ligation reaction was conducted at 16°C for 16 h, and after the reaction was completed, heat inactivation was conducted at 65°C for 10 min.
5. Transformation of ligation product into DH5a competent cell 1) Competent cells were placed on an ice bath; 2) 5 ul of a ligation reaction solution was added to the competent cells, gentle flicking and even mixing were conducted, and reaction was conducted on an ice bath for 30 min; 3) a centrifuge tube was placed in a 42°C water bath for 90 s, quickly transferred to an ice bath, and cooled for 2-3 min; the centrifuge tube should not be shaken during this process; 4) 900 ul of an antibiotic-free LB medium was added to the centrifuge tube and after even mixing, a shaking culture was conducted at 37°C for 45 min (150 r/min); and 5) the contents in the centrifuge tube were mixed evenly and 100 ul of the obtained suspension was taken and evenly spread on an LB solid agar plate (containing 100 mg/ml of ampicillin) by using a sterile spreading rod; the plate was placed upside at 37°C for 15 min until the liquid was completely absorbed by the LB culture plate; and the plate was placed upside down and cultivated for 16-18 h until a single colony was formed.
6. Shaking The single colony grown in the above ampicillin-resistant LB culture plate was picked, the single colony was added to an ampicillin-resistant liquid LB medium, a shaking culture was conducted at 37°C for 16-18 h(200 r/min) and bacteria liquid was collected.
7. Small-amount extraction of plasmid
1) Column balance: 500 pl of a balance solution BL was added to an adsorption column CP3, centrifugation was conducted at 12,000 rpm for 1min, waste liquid in a collection tube was discarded, and the adsorption column was put back into the collection tube; 2) 3 ml of the bacterial liquid was taken to be cultured overnight, the cultured bacteria were added into a centrifuge tube, centrifugation was conducted at 12,000rpm for 1min by using a conventional desk centrifuge and the supernatant was pipetted out as much as possible; 3) 250 HI of a solution P1 (whether RNase A was added should be checked first) was added to the centrifuge tube with a bacterial precipitate and the bacterial precipitate was completely suspended by using a pipette and a vortex oscillator; 4) 250 pl of a solution P2 was added to the centrifuge tube and the centrifuge tube was gently turned up and down 6-8 times to fully lyse the bacteria; 5) 350 pl of a solution P3 was added to the centrifuge tube and the centrifuge tube was gently turned up and down 6-8 times immediately to fully and evenly mix the liquid, at this time, a white flocculent precipitate appeared and centrifugation was conducted at 12,000rpm for 10min; 6) the collected supernatant in the previous step was transferred to an adsorption column CP3 (the adsorption column was put into the collection tube) by using a pipette and the precipitate should not be pipetted out as much as possible. Centrifugation was conducted at 12,000rpm/min for 60s, the waste liquid in the collection tube was discarded, and the adsorption column CP3 was put into the collection tube; 7) 600 pl of a rinsing solution PW was added to the adsorption column CP3 (whether absolute ethanol was added should be checked first), centrifugation was conducted at 12,000 rpm/min for 30-60s, the waste liquid in the collection tube was discarded, and the adsorption column CP3 was put into the collection tube; 8) the operation in step 7) was repeated; 9) the adsorption column CP3 was put in the collection tube and centrifugation was conducted at 12,000rpm/min for 2min to remove the residual rinsing solution in the adsorption column; and 10) the adsorption column CP3 was placed in a clean centrifuge tube, about 30pl of an elution buffer was added dropwise to a centre of an adsorption membrane, the adsorption column CP3 was placed at room temperature for 2 min, centrifugation was conducted at 12,000 rpm/min for 2 min, and a plasmid solution was collected in the centrifuge tube.
8. Identification of positive clones 1) Electrophoresis identification: the extracted plasmids were subjected to an electrophoresis and comparison with the negative control was conducted to screen out the plasmids that may be positive;
2) enzyme digestion identification: double digestion with EcoR I/Nhe | or EcoR |/Pvull was conducted and the molecular weight of a digested product was compared with that of a DNA Marker by electrophoresis; and 3) sequencing confirmation of recombinant plasmids: the clones identified positive by electrophoresis and enzyme digestion were sent to Takara Biotechnology (Dalian) for sequencing, and expression vector plasmids with correct sequencing were stored.
Expression conditions of antibodies of different cell lines
1. Antibody expression in 293A cell system The successfully ligated antibody light chain and heavy chain gene expression vectors were transfected into 293A cells transiently: 1) a 12-well cell culture plate was used for a transfection experiment, the number of cells inoculated in each well was 1.5x105 cells/well; when confluence of 293T cells reached 70-90%, transfection was conducted; 2) a medium was changed to a serum-free medium before transfection; 3) a lipofectamine 2000 reagent suspension was prepared and added to 75 pl of Opti-MEM medium 4) the light and heavy chain plasmids to be transfected (according to a mass ratio of 1:1) were added into 75 pl of Opti-MEM medium; 5) the solutions of 3) and 4) were mixed evenly in equal volumes gently, standing was conducted at room temperature for 5 min until a DNA-lipid complex was formed; 6) the DNA-lipid complex was gently added to the 293T cells; 7) after 4-6 h of transfection, antibiotics were added and incubation was conducted at 37°C for 1-3 days; and 8) antibodies were harvested from the transfected cells: the supernatant (including cells) was harvested and centrifugation was conducted at 5,000 rpm for 10 min; the harvested supernatant at was stored at -20°C; 1ml of pre-cooled PBS was added into a centrifuge tube, an obtained precipitate after the centrifugation was washed, centrifugation was conducted at 5,000 rpm for 5 min, washing was conducted twice, the PBS was discarded, 100 ul of RIPA was added into each tube, ice bath was conducted for 30 min, centrifugation was conducted at 5,000 rpm for 5 min, and the supernatant was harvested; and the original supernatant and the harvested supernatant after the lysis and precipitation were subjected to SDS-PAGE and western-blot detection, respectively;
2. Antibody expression in 293T cell system The experiment steps of antibody expression in 293T cell system were consistent with those of the antibody expression in 293A cell expression.
3. Large-amount expression of antibodies by using CHO cell 1) Escherichia coli glycerol stock with a target gene was shaken, large-scale extraction was conducted by using an endotoxin-free plasmid large-scale extraction kit, plasmids after the large-scale extraction were filtered through a 0.22-um filter; the plasmid concentration was best guaranteed to be 1 mg/ml and when the plasmid concentration was low, the plasmids needed to be concentrated; 2) The heavy and light chain expression vector plasmids, an Opti-MEM solution and a FreeStyle TM CHO cell culture medium were taken out from a refrigerator and returned to a room temperature. Glutamine (at a final concentration of 8mM) was added to the FreeStyle TM CHO cell culture medium according to a volume ratio of 1/100, and double antibodies were supplemented, 3) Before transfection, a density of CHO suspension cells was ensured to be 1.2x10°-
1.5x10° cells/ml; and in order to ensure high transfection efficiency, a proportion of live cells should be greater than 95%; 4) before a FreeStyle TM MAX transfection reagent was used, the solution was slightly mixed and should not be shaken and mixed evenly; 5) 37.5 ug of plasmid DNA was added to the Opti-MEM solution to enable a total volume of0.6ml and mixed well; and 37.5pl of the FreeStyle TM MAX transfection reagent was added to the Opti-MEM solution to enable a total volume of 0.6m; 6) the immediately diluted FreeStyle TM MAX transfection reagent was added to the diluted DNA solution to enable a total volume of 1.2 ml and the materials were mixed gently; 7) the DNA-FreeStyle TM MAX mixed solution was placed at room temperature for 10 min to form a DNA-FreeStyle TM MAX complex; and
1.2 ml of the DNA-FreeStyle TM MAX complex was added to a 125-ml suspension cell culture flask; and the transfected cells were cultured in a CO: incubator at 37°C, shaking culture was conducted for 6-7 days and the transfected cells were collected.
Purification of antibody 1) Protein A agarose beads and all solutions were equilibrated to a room temperature; 2) a column was filled with 2ml of resin mortar; 3) a packed column was equilibrated with 5ml of a binding buffer; 4) a collected antibody expression supernatant passed through an affinity chromatography column to maintain a proper salt ion concentration and a pH value; 5) the operation in step 4) was repeated;; 6) 15 ml of the binding buffer was used to clean the protein A beads; 7) the affinity chromatography column was washed with 5ml of an elution buffer; after the elution, Tris-HCL (1 mmol) with a pH of 8.0 was immediately used; and for neutralization, 100 pl of a Tris-HCL solution was added into per ml of an eluate; 8) the protein eluate was concentrated by using a 10KD ultrafiltration tube for concentration; and
9) a BCA method was used to conduct protein quantification on purified antibodies; and the quantitative result showed that the purified antibodies had a concentration of 0.1 mg/ml-0.5 mg/ml.
Affinity screening of monoclonal antibody 1) Antigen coating; a VP1 recombinant purified protein was added to a 96-well plate with
0.2 ug per well and coating was conducted overnight at 4°C; 2) blocking: a blocking solution containing 5% of BSA was added to the 96-well plate and blocking was conducted at room temperature for 2 h; 3) monoclonal antibody adding: a purified monoclonal antibody diluted with PBST (containing 0.05% BSA) was added at 0.1-0.5 pg/well at room temperature for 2 h; 4) detection antibody adding: washing was conducted three times with 0.1% PBST for 10 min each time; an HRP-labelled rabbit anti-human IgGH&L detection antibody was diluted at a ratio of 1:5,000 and added into the 96-well plate at 100 pl/well at a room temperature for 1 h; 5) colour development: washing was conducted three times with 0.1% PBST for 10 min each time; a TMB colour development solution was added to the 96-well plate at 100 pl/well and reaction was conducted at 37°C for 10 min; 6) colour development stopping: 50 ul/well of a colour development stop solution was added to the 96-well plate and on-machine detection was conducted within 10 min after the stop solution was added; 7) determination: a blank well was adjusted to zero, an absorbance value (an OD value) of each well was measured at a wavelength of 450 nm wavelength and it was determined to be positive when OD positive serum/OD negative serum was greater than 2.5; and 8) analysis of detection results: among all the detection antibodies, A6-1, E2-2, F4-3 and D7-4 could bind strongly to a purified EV-D68VP1 antigen and had obvious affinity activity (FIG.
5).
Analysis of neutralization activity of monoclonal antibody 1) Preparation of EV-D68 attack solution A virus stock solution was diluted to 2000 CCIDs9/ml with a virus diluent, that is, 100 CCIDs5/0.05ml of the virus solution was added to each well; a virus backdrop control was set at the same time: the virus diluent was used for dilution in a manner of 10 times from 100 CCIDso to 3 gradients of 10 CCIDs9/0.05ml, 1 CCIDs5/0.05ml and 1 CCIDs0/0.05m, the virus solutions at the 3 gradients and the virus solution at 100 CCIDs9/0.05ml were added to a virus backdrop plate, each concentration gradient of the virus solution was added in 4 multiple wells, 50 HI of the virus solution corresponding to the concentration gradient was added to each well and 50 HI of the virus diluent was additionally added to make up to 100 pl (equivalent to the amount of serum).
3) Dilution of antibody
The purified antibody was diluted, 50 ul/well of the virus diluent was added to the 96-well cell culture plate, 50 pl of the purified antibody sample was taken out to be added into a first role of the 96-well cell culture plate, the liquid was pipetted and mixed with a multichannel pipette, the liquid was pipetted according to 50 pl/well to a second row, the liquid was pipetted and mixed with a multichannel pipette, the liquid in the second row was pipetted according to 50ul/well to a third row, and so on, until the last row; and the liquid in the last row was pipetted and mixed, and 50pl of the liquid was pipetted to be discarded. 4) Adding of EV-D68 attack solution The diluted EV-D868 attack solution was added to the above cell culture plate at 50ul/well and pipetted and mixed evenly. 5) Neutralization of virus and antibody The above 96-well culture plate was put in a 5% CO: incubator at 37°C and neutralization was conducted for 2 h; 6) Adding of cell suspension Hela cells in a logarithmic growth phase were used and subjected to trypsinization, a complete cell culture medium was used, pipetting and even mixing were conducted, the cells were diluted to 2x10° cells/ml, and the cell suspension was added into the 96-well cell culture plate at 100 pl/well. 7) Detection by MTS reagent Standing was conducted at room temperature for 90 min or water bath was conducted at 37°C for 10 min until a cell titre 96 aqueous one solution reagent was completely dissolved; in the 96-well cell culture plate, 20 pl of the cell titre 96 aqueous one solution reagent was added to 100 pl of a medium in each well; the 96-well cell culture plate was placed in the environment of 37°C and 5%CO: and incubation was conducted for 4 h; and the absorbance value of each well was read under a wavelength of 490 nm by a microplate reader. 8) Processing and analysis of results Graph pad 5 was used to calculate a half effective dose. The result showed that A6-1 could effectively neutralize the EV-D68 with an inhibition rate of 100%. TheA6-1 had the half maximal inhibitory concentration (IC50) of 0.6ug/ml for a KM strain and1.57 pg/ml for a Fermon strain, and thus had better neutralizing activity than F4-3, E2-2 and D7-4 antibodies. The F4-3, E2-2 and D7-4 neutralizing antibodies had similar IC50 values to the two different EV-D68 strains, ranging from 2-6 pg/ml, and had the maximum effective inhibition percentages of not reaching 100%. Therefore, among the four antibodies, the antibodyA6-1 had a higher neutralizing activity.
Example 2 Identification of EV-D68 by monoclonal antibody A6-1 obtained in Example 1 Western-blot verification of antibody specificity
1) Sample preparation: 80 pl of purified whole EV-D68 was added to 20 pl of 5xloading buffer, even mixing was conducted and cooking was conducted at 100°C for 5 min; 2) SDS-PAGE gel protein electrophoresis of the whole EV-D68 was conducted; 3) after the electrophoresis, gel was taken out, the concentrated gel was cut off, the separated gel was soaked in a transfer buffer, a PVDF membrane with the same size as the gel and 2 pieces of filter paper were prepared and soaked in the transfer buffer; on a positive electrode plate, the filter paper, the PVDF membrane, the gel and the filter paper were stacked neatly in order, bubbles were removed during the stacking process, a negative plate was covered and membrane transfer was conducted at 25V and 1.3A for 20 min; 4) blocking: after the membrane transfer was completed, the PVDF membrane was taken out and put in a glass dish, 40 ml of a freshly prepared blocking solution was added, and shaking was conducted at 4°C overnight; 5) primary antibody incubation: the blocking solution was discarded and washing was conducted twice with PBST for 5 min each time; a primary antibody was added at a concentration of 1:200 and incubation was conducted at room temperature for 2 h; 6) secondary antibody incubation: washing was conducted 4-6 times with PBST for 15min each time, a goat anti-rabbit secondary antibody was added at a concentration of 1:10,000 and incubation was conducted at room temperature for 1 h; 7) ECL chemiluminescence colour development: washing was conducted 4-6 times with PBST for 10min each time; a prepared ECL reagent evenly covered a PVDF membrane for luminescence development, tableting was conducted under dark room for exposure at exposure time of 10s, and development and fixing were conducted; and 8) the result showed that the monoclonal antibody A6-1 obtained in Example 1 could recognize an EV-D68VP1 virus protein(FIG.4).
Example 3 Inhibition of EV-D68 infection by monoclonal antibody A6-1 obtained in Example 1
1. Virus contact inhibition experiment 1) Hela and Vero cells in a logarithmic growth phase were used and subjected to trypsinization, a complete cell culture medium was used, pipetting and even mixing were conducted, the cells were diluted to 4x10° cells/ml, and the cell suspension was added into a 12-well cell culture plate at 500 pl/well until the cells grew into a single layer for later use; 2) viruses were neutralized with monoclonal antibodies of different dilutions, an obtained neutralized virus-antibody mixture was added to a 96-well cell culture plate where the cells grew into a single layer, reaction was conducted at room temperature for 1 h, and the plate was washed 3 times with PBS to remove excess viruses;
3) the cells in the above-mentioned 12-well plate were harvested, the total RNA of the cells was extracted, and qRT-PCR quantitative detection of the copy number of a virus gene was conducted; and 4) result analysis: from the result of the qRT-PCR, it could be seen that an inhibitory effect of A6-1 on the viruses was dose-dependent. When theantibodyA6-1 had a concentration of 2.5 pg/ml, only 20% of the original number of virus particles could be adsorbed to surfaces of the Hela cells; a similar antibody dose-dependent inhibitory effect was also observed on the Vero cells; and from the above result, it was preliminarily speculated that the antibody A6-1 could inhibit the adsorption of the viruses to the cells (FIG.6).
2. Neutralization effect of pre-attachment detected by MTS 1) Hela cells in a logarithmic growth phase were used and subjected to trypsinization, a complete cell culture medium was used, pipetting and even mixing were conducted, the cells were diluted to 2x10° cells/ml, and the cell suspension was added into a96-well cell culture plate at 100ul/well until the cells grew into a single layer for later use; 2) steps of preparation of EV-D68 attack solution, dilution of monoclonal antibody, adding of EV-D68 attack solution, neutralization of virus and antibody, etc. are the same as the steps in the section of "analysis of neutralization activity of monoclonal antibody"; 3) viruses were neutralized with monoclonal antibodies of different dilutions, an obtained neutralized virus-antibody mixture was added to a 96-well cell culture plate where the cells grew into a single layer, reaction was conducted at room temperature for 1 h, the plate was washed 3 times with PBS, a cell culture medium containing 2% serum was supplemented, and the cell culture plate was placed at 37°C and 5%CO: to be continuously cultured for 72 h; 4) detection by MTS: standing was conducted at room temperature for 90 min or water bath was conducted at 37°C for 10 min until a cell titre 96 aqueous one solution reagent was completely dissolved; in the 96-well cell culture plate, 20 pl of the cell titre 96 aqueous one solution reagent was added to 100 pl of a medium in each well; the 96-well cell culture plate was placed in the environment of 37°C and 5%CO: and incubation was conducted for 4 h; and the absorbance value of each well was read under a wavelength of 490 nm by a microplate reader; and 5) analysis of results: the results showed that before the viruses contacted the cells, the antibody A6-1 was used for treatment and the antibody interfered with the adsorption of the viruses to the cells, thereby inhibiting infection with an IC50 value of 1.39 pg/ml; therefore, the monoclonal antibody A6-1 could effectively inhibit the infection of the cells by the EV-D68 (FIG. 7).
3. Evaluation of effect of monoclonal antibody A6-1 against EV-D68 infection in animals 1) Grouping of suckling mice and experimental procedures: suckling mice were divided into an experimental group and a control group with 20 suckling mice in each group, injected with different antibodies and treated with virus attack (Table 2). An injection dose of the antibody was 30 pg/mouse and the attack dose was 10+5CCIDse/mouse.
Table 2 Experiment and grouping of suckling mice 2) At the time points of 0, 3, 7 and 10 days, 3 mice were randomly selected for dissection at each time point, and samples of lung tissue and brain tissue were collected. A tissue suspension was prepared for determination of viral infective titre and detection of absolute quantitative PCR of the copy number of a viral gene; and preparation of pathological frozen sections and HE staining were conducted on 0, 3 and 7 days.
3) Analysis of results: the results showed that the monoclonal antibody A6-1 could inhibit the respiratory tract infection of the EV-D88 in suckling mice and alleviate pathological symptoms of the infected suckling mice, and had a better preventive effect (FIG.8).
SEQUENCE LISTING <110> INSTITUTE OF MEDICAL BIOLOGY CHINESE ACADEMY OF MEDICAL
SCIENCES <120> Anti-EV-D68 monoclonal antibody and preparation method and use thereof <130> MAb-EV-D68 NL <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 296 <212> DNA <213> artificial <220> <223> CDR2-FR3-CDR3-FR4 of a heavy chain variable region of anti-EV-D68 VP1 monoclonal antibody A6-1 <400> 1 atgacttggg tccgccagcc tccagggaag ggcctcgaat gggttggtcg tattacgggt 60 ccaggtgaag gttggtcagt ggactatgct gcacccgtgg aaggcagatt taccatctcg 120 agactcaatt caataaattt cttatatttg gagatgaaca atttaagaat ggaagactca 180 ggcctttact tctgtgcccg cacgggaaaa tattatgatt tttggagtgg ctatccgccg 240 ggagaagaat acttccaaga ctggggccgg ggcaccctgg tcaccgtctc ctcagc 296 <210> 2 <211> 275 <212> DNA <213> artificial <220> <223> CDR2-FR3-CDR3-FR4 of a light chain variable region of anti-EV-D68 VP1 monoclonal antibody A6-1 <400> 2 gcaagttggt accaaaagaa gccaggacag gcccctatac ttctcttcta tggtaaaaat 60 aatcgtcctt caggggtccc agaccgattc tctggctceg cctcaggaaa cagagcttcc 120 ttgaccatct ctggggctca ggcggaagac gacgcggaat attattgtag ttctcgggac 180 aagagtggca gccgtctgtc ggtcttecggc ggggggacca aactgaccgt cctcagtcag 240 cccaaggctg ccccctcggt cactctgttc ccacc 275 <210> 3 <211> 98 <212> PRT <213> artificial <220>
<223> CDR2-FR3-CDR3-FR4 of a heavy chain variable region of anti-EV-D68 VP1 monoclonal antibody A6-1
<400> 3
Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val Gly
1 5 10 15
Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala Pro
Val Glu Gly Arg Phe Thr Ile Ser Arg Leu Asn Ser Ile Asn Phe Leu 40 45 Tyr Leu Glu Met Asn Asn Leu Arg Met Glu Asp Ser Gly Leu Tyr Phe 50 55 60
Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro Pro
65 70 75 80
Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Thr Val 85 90 95
Ser Ser
<210> 4
<211> 91
<212> PRT
<213> artificial
<220>
<223> CDR2-FR3-CDR3-FR4 of a light chain variable region of anti-EV-D68
VP1 monoclonal antibody A6-1
<400> 4
Ala Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Ile Leu Leu Phe
1 5 10 15
Tyr Gly Lys Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly
20 25 30 Ser Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Ser Gly Ala Gln Ala 35 40 45 Glu Asp Asp Ala Glu Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser 50 55 60 Arg Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln 65 70 75 80
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 85 90
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