CN116496388A - Chimpanzee 68 adenovirus-based monoclonal antibody, preparation method and application thereof - Google Patents
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Abstract
The invention discloses a method for preparing recombinant monoclonal antibody with stable and high-efficiency expression, which comprises the steps of immunizing mice with chimpanzee 68 type adenovirus Hexon protein as antigen to obtain hybridoma cells secreting anti-AdC 68 Hexon protein monoclonal antibody, sequencing an antibody variable region to obtain corresponding sequences, constructing to an expression vector plasmid, obtaining recombinant monoclonal antibody with stable and high-efficiency expression, using the recombinant antibody for virus titration and verification of chimpanzee 68 type adenovirus, and adopting the obtained recombinant monoclonal antibody to detect specificity, linear range, repeatability, intermediate precision and durability of the chimpanzee 68 type adenovirus.
Description
Technical Field
The invention relates to a chimpanzee 68 adenovirus-based monoclonal antibody, a preparation method and application thereof, and belongs to the field of biotechnology in the field of recombinant proteins.
Background
Replication-defective adenoviruses have been widely used in gene therapy drug carriers and vaccine carriers because of their low toxicity, multi-tissue preference, high expression level of heterologous genes, strong induced immune response, easy preparation and production, and the like. Among them, the conventional adenovirus vector represented by human adenovirus type 5 has the defects of high pre-existing immunity ratio in the population and the like, and the clinical effect is not ideal, so the non-human adenovirus serotype vector gradually becomes a focus of attention. Chimpanzee adenovirus type 68 is a chimpanzee adenovirus vector reported in the first literature for vaccine development, has immunogenicity similar to that of human adenovirus 5, has low pre-existing antibody proportion in human, is not cross-neutralized by human adenovirus antibodies and the like, and has increasingly been applied to vaccine and gene therapy drug development. The commonly used adenovirus quantitative method is ultraviolet spectrophotometry to detect the number of virus particles, fluorescence quantitative PCR method to detect the copy number of virus group genes, plaque method or TCID50 method to detect the virus titer, etc. The ultraviolet spectrophotometry is convenient and quick, but is easy to be influenced by a matrix in a test sample, and is only suitable for quantification of highly purified adenovirus. The quantitative range of fluorescent quantitative PCR is wide, the sensitivity is high, and the sample nucleic acid extraction step can reduce or eliminate the interference of the sample matrix, but the adenovirus quantity with infectivity can not be detected as the ultraviolet spectrophotometry is the same [12]. The plaque method or TCID50 method calculates the number of infectious adenoviruses by inoculating adenoviruses into cell culture and observing cytopathic conditions, but the test period is long (usually 10 days or more), and the test reproducibility is poor.
The Hexon protein is one of the most important structural proteins of adenovirus, and has a core structure which is highly conserved, 7 hypervariable regions are arranged, and species specificity and serum specificity determining sites are also main targets of serotype specific neutralizing antibodies.
In the process of gene therapy and vaccine development, the titer of an adenovirus vector is an important quality control index, and the development of an accurate and specific titer detection method has great significance for the development of gene therapy and vaccine development process and monitoring of production process.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to prepare a recombinant monoclonal antibody for specifically recognizing the chimpanzee adenovirus 68 by taking the chimpanzee adenovirus 68 type Hexon protein as an antigen, which is used for determining the titer of the chimpanzee adenovirus 68 type vector by a cell immunostaining method and provides guidance for research on gene therapy medicaments and vaccine development processes by taking the chimpanzee adenovirus 68 type as a viral vector.
For the above reasons, the present invention provides a monoclonal antibody based on chimpanzee type 68 adenovirus, which can effectively detect the viral titer of chimpanzee type 68 adenovirus, so as to solve the above problems.
To achieve one of the above objects, the present invention provides a chimpanzee 68 adenovirus-based monoclonal antibody comprising a heavy chain and a light chain, the heavy chain and the light chain comprising 3 CDR regions and 4 FR regions, respectively, wherein the amino acid sequence of the CDR1 region of the heavy chain is GLPFNTKA (SEQ ID NO: 1), the amino acid sequence of the CDR2 region of the heavy chain is IRTKSNNYAT (SEQ ID NO: 2), the amino acid sequence of the CDR3 region of the heavy chain is VRDEAS (SEQ ID NO: 3), the amino acid sequence of the FR1 region of the heavy chain is EVQLVETGGGLVQPKGSLKLSCAVS (SEQ ID NO: 4), the amino acid sequence of the FR2 region of the heavy chain is MDWVRQAPGEGLEWVAR (SEQ ID NO: 5), the amino acid sequence of the FR3 region of the heavy chain is YYADSVKDRFTISRDDAQSMLYLQMTNLKTEDTAMYYC (SEQ ID NO: 6), the amino acid sequence of the FR4 region of the heavy chain is WGHGTLVTVSA (SEQ ID NO: 7), the amino acid sequence of the CDR1 region of the light chain is QSLVHSDGNTY (SEQ ID NO: 8), the amino acid sequence of the FR2 region of the heavy chain is 5698 (SEQ ID NO: 5), the amino acid sequence of the FR2 region of the heavy chain is 563 (SEQ ID NO: 9); the amino acid sequence of the FR1 region of the light chain was DIVMTQIPLSLPVSLGDQASISCRSS (SEQ ID NO: 11), the amino acid sequence of the FR2 region of the light chain was LHWYLQKPGQSPKLLIY (SEQ ID NO: 12), the amino acid sequence of the FR3 region of the light chain was NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO: 13) and the amino acid sequence of the FR4 region of the light chain was FGGGTKLEIK (SEQ ID NO: 14).
Preferably, the antibody is an anti-AdC 68 antibody and the sequence of the antibody heavy chain is: EVQLVETGGGLVQPKGSLKLSCAVSGLPFNTKAMDWVRQAPGEGLEWVARIRTKSNNYATYYADSVKDRFTISRDDAQSMLYLQMTNLKTEDTAMYYCVRDEASWGHGTLVTVSA (SEQ ID NO: 15);
the sequence of the light chain is:
DIVMTQIPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFPWTFGGGTKLEIK(SEQ IDNO:16)。
preferably, the chimpanzee type 68 adenovirus has the following Hexon protein sequence:
DGETATEKTYTYGNAPVQGINITKDGIQLGTDTDDQPIYADKTYQPEPQVGDAEWH
DITGTD
EKYGGRALKPDTKMKPCYGSFAKPTNKEGGQANVKTGTGTTKEYDIDMAFFDNR
SAAAAGL APEIVLYTENVDLETPDTHIVYKAGTDDSSSSINLG(SEQ ID NO:17)。
preferably, the antibody is an IgG antibody.
In order to achieve one of the above objects, the present invention adopts the following technical scheme of a method for preparing a chimpanzee 68 adenovirus-based monoclonal antibody:
the method provided by the invention uses chimpanzee 68 type adenovirus hexon protein as antigen to immunize and obtain hybridoma cells, constructs the hybridoma cells to expression vector plasmid, and obtains recombinant monoclonal antibody.
The method further comprises the following steps:
a) Adopting chimpanzee 68 adenovirus Hexon protein sequence, synthesizing target gene according to the codon preference of colibacillus, inserting into expression vector pET-32a, converting colibacillus, and picking positive clone;
b) Antigen expression and purification;
c) Immunizing animals;
d) Cell fusion and subcloning screening monoclonal hybridoma cell strains;
e) The monoclonal hybridoma cell line was expanded and purified.
Preferably, the preparation method further comprises an identification step of detecting an antibody subtype of hybridoma cell supernatant by using a monoclonal antibody subtype kit; the method comprises the following steps: cell supernatants were added to each of 6 wells of 50ng of the enzyme-labeled plate coated with the AdC68-Hexon protein, 100. Mu.L/well. Incubation at 37℃for 1h, washing the plates with PBST three times, adding the diluted enzyme-labeled secondary antibodies against mouse IgM, igA, igG1, igG2a, igG2b and IgG3 to the 6 wells, incubating at 37℃for 1h, washing the plates with PBST three times, and developing TMB.
More preferably, the specific expression of the antigen of step b is: transforming BL21 DE3 competent cells with the constructed plasmid, inoculating a resistant LB plate medium (ampicillin), and culturing overnight at 37 ℃; selecting a monoclonal colony, inoculating to 3ml of resistant LB culture medium (ampicillin), culturing at 37 ℃ at 220rpm until the OD reaches 1.0, transferring 60 μl to an Erlenmeyer flask containing 200ml of resistant LB culture medium, and culturing at 200rpm at 37 ℃ overnight; 800mL of fresh resistant LB liquid medium is added into the Erlenmeyer flask in the next day, and the culture is carried out for 1-2 h until the OD600nm is 0.5-0.6. 200 μl of 1M IPTG was added to the Erlenmeyer flask and the mixture was induced at 37℃for 3.5h.
More preferably, the step b antigen purification is specifically: the cells were collected by centrifugation at 4000rpm at 4℃for 15min, the supernatant was discarded, 30ml of PBST was added to resuspend the cells, PMSF was added to a final concentration of 1mM, and the cells were sonicated at 200W under ice bath conditions for 6min. Centrifuging at 8000rpm for 15min at 4deg.C, collecting supernatant, purifying target protein by nickel column affinity chromatography, and dialyzing the purified target protein to obtain PBS buffer.
More preferably, the specific steps of step c are: BALB/c mice were immunized four times with equal volumes of purified AdC68-Hexon protein and complete and incomplete Freund's adjuvant, 2 weeks apart, with a dose of 100 μg protein per first injection and 50 μg protein per last three booster injections. The 7d mouse tail vein was bled to isolate antisera after the fourth immunization.
More preferably, the specific steps of step d are: BALB/c mice with high antiserum titers were selected for booster immunization with Freund's incomplete adjuvant at a dose of 50 μg protein/mouse; the mice are sacrificed the next day, spleen cells are collected and counted, and myeloma cells and spleen cells are mixed according to the proportion of 1:3-1:10 for cell fusion; detecting whether an antibody aiming at the AdC68-Hexon protein is secreted in cell supernatant by adopting an indirect ELISA method by taking the AdC68-Hexon protein as a coating antigen, selecting positive clones, and continuously performing two rounds of subcloning on the positive clones by adopting a limiting dilution method; the wild chimpanzee 68 type adenovirus and the human 5 type adenovirus are taken as screening strains, and single monoclonal hybridoma cell strains which are stable and positive and are used for identifying the wild chimpanzee 68 type adenovirus are screened by a cell immunostaining method, and the cell strains are expanded to be cultured and frozen for seed preservation.
More preferably, the step e is specifically: c, selecting the cells obtained in the step d for expansion culture, injecting the cells into the abdominal cavity of the Balb/C mice which are sensitized by Freund incomplete adjuvant in advance, observing that the abdominal cavity of the mice is obviously raised after 10 days, and extracting ascites; performing affinity purification on ascites obtained by cell immunized mice by using a Protein A column; packing Protein A into a gravity purification column, and washing three times by using 10 times of PBS solution; adding the ascites into the column, mixing and incubating for 4 hours at 4 ℃; discharging the effluent, and washing three times by using PBS solution with the volume of 10 times of the column volume; eluting the antibody with pre-chilled HCl-Glycine (pH 3.0), and immediately neutralizing the collected antibody with 10-X PBS neutralizing solution; the concentration of antibodies in the collection tubes was measured by OD280, the high concentration collection tubes were pooled and dialyzed overnight at 4 ℃ with PBS solution.
In order to achieve one of the above objects, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the above antibody.
In order to achieve one of the above objects, the present invention provides a recombinant vector comprising any one of the above nucleotides.
In order to achieve one of the above objects, the present invention provides a cell line comprising the recombinant vector described above, or a cell line having the nucleotide described above integrated in its genome.
In order to achieve one of the above objects, the present invention provides the use of a monoclonal antibody for detecting chimpanzee type 68 adenovirus.
The antibody can be specifically combined with chimpanzee 68 virus Hexon protein, so that the content of the Hexon protein can be effectively detected, the sensitivity is high, the linear range is 104.56-109.57 IFU/mL, and the requirement of quantitative detection of vaccines can be met.
Compared with the prior art, the invention uses chimpanzee 68 type adenovirus Hexon protein as antigen to immunize mice to obtain hybridoma cells secreting anti-AdC 68 Hexon protein monoclonal antibody, sequences the antibody variable region to obtain corresponding sequences, constructs the corresponding sequences to expression vector plasmids, obtains recombinant monoclonal antibodies with stable and efficient expression, uses the recombinant antibodies for virus titration and verification of chimpanzee 68 type adenovirus, and adopts the obtained recombinant monoclonal antibodies to detect the specificity, linear range, repeatability, intermediate precision and durability of the chimpanzee 68 type adenovirus to meet the requirements. The cell immunostaining method in the verification experiment of the embodiment calculates the titer of the infectious adenovirus by adopting a mode of staining and counting cells infected with the virus by adopting a specific monoclonal antibody after inoculating the gradient diluted chimpanzee adenovirus type 68 to the cells for 48 hours, and the whole experiment period only needs 3 days, the stained spots are accurately counted, and the high-throughput full-automatic spot counting determination can be realized by matching with a cell imaging counter. The recombinant anti-chimpanzee adenovirus 68 type monoclonal antibody is used for staining and counting, so that the chimpanzee adenovirus 68 type can be distinguished from other adenovirus vectors, especially human adenovirus type 5 vectors, specific, accurate and efficient detection of the infectious titer of the chimpanzee adenovirus 68 type can be realized, and the process research, development and production process monitoring of the chimpanzee adenovirus 68 type gene therapy medicine and vaccine are supported.
Compared with the current commercial adenovirus antibody, the recombinant monoclonal antibody obtained by the invention has the advantages of good specificity, clear and definite antibody sequence, large-scale production by adopting cells, good consistency among batches and the like, and has important significance for qualitative identification and quantitative analysis of gene medicines and vaccines by taking chimpanzee 68 adenovirus as a vector.
Drawings
FIG. 1 is a diagram showing the SDS-PAGE identification of the AdC68 Hexon protein;
FIG. 2 is a graph showing the results of cell immunostaining by subcloning hybridoma cell lines obtained in the present invention, wherein A to E are respectively the results of virus immunostaining by 20#, 22#, 36#, 42# and commercial adenovirus antibodies against wild type human adenovirus type 5, and a to E are respectively the results of virus immunostaining by 20#, 22#, 36#, 42# and commercial adenovirus antibodies against wild type chimpanzee type 68;
FIG. 3 is a Western Blot analysis result of the specificity investigation of the monoclonal antibody obtained by the invention, lane 1, wild chimpanzee adenovirus type 68; lane 2, wild type human adenovirus type 5;
FIG. 4 is a diagram showing the result of a reduction SDS-PAGE method of the purity of the recombinant monoclonal antibody.
Detailed Description
The method for producing the monoclonal antibody of the present invention and its application are described in further detail and fully below with reference to examples. The following examples are illustrative only and are not to be construed as limiting the invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below were all commercially available unless otherwise specified.
The main biological materials related to the embodiment of the invention are as follows: wild chimpanzee type 68 adenovirus, wild type human adenovirus type 5, HEK293A cells and myeloma cells SP2/0 are all derived from ATCC, BL21 DE3 competent cells are derived from Thermo Scientific company, and pET-32a plasmid is stored by Wobkwon Biotechnology Co.Ltd.
The reagents and buffers involved in the examples of the present invention are as follows: ampicillin, IPTG, PMSF and complete and incomplete freund's adjuvant were manufactured by Sigma company, protein a column, nickel column was manufactured by cytova company, and commercial adenovirus antibody was manufactured by Cell Biolabs company. HRP-labeled goat anti-mouse IgG antibody, BCA protein quantification reagent and Trizol were purchased from Thermo Fisher Scientific, TMB substrate solution was purchased from SeraCare, and monoclonal antibody subtype identification reagent was purchased from Southern Biotech. Other common biochemical reagents are all domestic analytically pure reagents.
Examples
1. Preparation of monoclonal antibodies
1. Construction of vectors
According to chimpanzee 68 type adenovirus Hexon protein sequence, according to the codon preference of colibacillus, synthesizing target gene and inserting into expression vector pET-32a, converting colibacillus, picking positive clone, and sequencing to verify that the synthetic sequence is correct.
2. Antigen expression
BL21 DE3 competent cells were transformed with the constructed plasmids, inoculated with a resistant LB plate medium (ampicillin), and cultured overnight at 37 ℃. The monoclonal colonies were picked up, inoculated into 3ml of a resistant liquid LB medium (ampicillin), cultured at 37℃at 220rpm until OD reached 1.0, and transferred to 60. Mu.l of an Erlenmeyer flask containing 200ml of the resistant liquid LB medium, and cultured at 37℃at 200rpm overnight. 800mL of fresh resistant LB liquid medium is added into the Erlenmeyer flask in the next day, and the culture is carried out for 1-2 h until the OD600nm is 0.5-0.6. 200 μl of 1M IPTG was added to the Erlenmeyer flask and the mixture was induced at 37℃for 3.5h.
3. Antigen purification
The cells were collected by centrifugation at 4000rpm at 4℃for 15min, the supernatant was discarded, 30ml of PBST was added to resuspend the cells, PMSF was added to a final concentration of 1mM, and the cells were sonicated at 200W under ice bath conditions for 6min. Centrifuging at 8000rpm for 15min at 4deg.C, collecting supernatant, purifying target protein by nickel column affinity chromatography, and dialyzing the purified target protein to obtain PBS buffer. SDS-PAGE (SDS-PAGE) is used for identifying the molecular weight and purity of the AdC68-Hexon protein, and the result is shown in figure 1; protein concentration was determined by BCA method and total target antigen was 3mg.
4. Immunization of animals
BALB/c mice were immunized four times with equal volumes of purified AdC68-Hexon protein and complete and incomplete Freund's adjuvant, 2 weeks apart, with a dose of 100 μg protein per first injection and 50 μg protein per last three booster injections. The antiserum was collected from 7d tail vein after the fourth immunization (supernatant was obtained by centrifugation at 4000rpm for 10min after blood collection at 4 ℃ overnight), the titer of the antiserum was measured by indirect ELISA using AdC68-Hexon protein as the coating antigen, and the highest titer was 1:32000 as shown in Table 1.
TABLE 1 mouse serum anti-Hexon protein IgG antibody titres
5. Determination of anti-AdC 68-Hexon antibody titers by indirect ELISA method
AdC68-Hexon protein was diluted with coating solution to 2. Mu.g/ml, 100. Mu.L/well was added to the ELISA plate and allowed to stand overnight at 4 ℃. The next day the wells were discarded, the plates were washed 3 times with PBST wash, blocking solution was added, L50. Mu.L/well, and blocked at room temperature for 0.5h. The washing was performed 3 times with PBST wash. The antisera to be tested were diluted in a 1:2000 initial fold ratio and 100. Mu.L of the antisera were added to each well and incubated at 37℃for 1h. Negative controls were made with blank serum. Wash 3 times with PBST wash, add HRP-labeled anti-mouse IgG secondary antibody, 100 μl/well, incubate at 37 ℃ for 40min.
The plates were washed 5 times with PBST wash and 2 times with distilled water. After adding 100. Mu.l/well of TMB substrate solution and incubating at room temperature for 15min in the dark, 50. Mu.l/Kong Zhongzhi solution was added, and the absorbance of each well at 450nm was measured with an ELISA reader. The negative control OD value is 2.1 times of the negative control OD value, and the maximum dilution of the antiserum OD value to be detected exceeding the negative control OD value is taken as the antiserum titer.
6. Cell fusion and subcloning
BALB/c mice with high antiserum titers were selected for booster immunization with Freund's incomplete adjuvant at a dose of 50. Mu.g protein/mouse. The following day mice were sacrificed, spleens were harvested, spleen cells were collected and counted, and myeloma cells and spleen cells were mixed at a ratio of 1:3 to 1:10 for cell fusion. Detecting whether an antibody aiming at the AdC68-Hexon protein is secreted in cell supernatant by using the AdC68-Hexon protein as a coating antigen by adopting an indirect ELISA method, selecting positive clones, and continuously performing two rounds of subcloning on the positive clones by adopting a limiting dilution method.
And (3) screening out stable positive monoclonal hybridoma cell strains by adopting a cell immunostaining method and taking wild chimpanzee 68 type adenovirus as a positive control and wild human 5 type adenovirus as a negative control. The 293A cells forming the monolayer are digested by pancreatin to prepare suspension with the cell concentration of 2.5X105-3.0X105/ml, and the suspension is inoculated into a 24-hole culture plate for culture. After one day a monolayer was formed and the culture medium in the 24-well plate was removed. Serial 10-fold dilution of the test sample, inoculating 293A cells at a proper dilution ratio of 0.1 ml/hole, and setting cell control and antibody control holes; placing the culture plate in a 37 ℃ and 5% CO2 incubator to adsorb for 2 hours (shaking the plate 1 time every 15 minutes), removing the virus liquid in the 24-pore plate, adding 1ml of the culture liquid into each pore, continuously culturing for 2 days in the 37 ℃ and 5% CO2 incubator, adding methanol to fix for 10 minutes, washing the plate, adding the recombinant monoclonal antibody to incubate for 1 hour, washing the plate, adding the enzyme-labeled secondary antibody to incubate for 1 hour, washing the plate, developing for 15-20 minutes, counting the staining spots under a microscope, and calculating the IFU according to the dilution corresponding to the average staining spot number of each dilution.
The results are shown in FIG. 2, wherein A-E are the results of virus immunostaining of wild-type human adenovirus type 5 with the commercial adenovirus antibodies 20#, 22#, 36#, 42#, respectively. a-e are the results of virus immunostaining of wild chimpanzee type 68 adenovirus with the 20#, 22#, 36#, 42# and commercial adenovirus antibodies, respectively; in cell supernatants secreted by the four monoclonal hybridoma cell lines, except 42# can simultaneously identify wild chimpanzee 68 type adenovirus and wild human 5 type adenovirus, the rest 20# and 22# and 36# can specifically identify wild chimpanzee 68 type adenovirus, three stable and positive monoclonal hybridoma cell lines which singly identify wild chimpanzee 68 type adenovirus are screened out, expanded culture is carried out, and the seeds are preserved by freezing.
7. Monoclonal antibody subtype identification
Antibody subtypes of hybridoma cell supernatants were detected using a monoclonal antibody subtype identification kit from Southern Biotech. Cell supernatants were added to each of 6 wells of 50ng Hexon protein coated enzyme-labeled plate, 100 ul/well. Incubation at 37℃for 1h, washing the plates with PBST three times, adding diluted antibodies of the secondary anti-mouse IgM, igA, igG1, igG2a, igG2b and IgG3 into 6 wells, respectively, incubating at 37℃for 1h, washing the plates with PBST three times, and developing TMB. The subtype of the antibody is identified as the subtype of the antibody by the identification of the secondary antibody corresponding to the positive hole, and the antibody subtype of the positive hybridoma cell supernatant is detected by adopting a monoclonal antibody subtype identification kit, and the results are shown in Table 2 and are lgG1 subtypes.
TABLE 2 determination of positive hybridoma cell antibody subtypes
8. Ascites preparation and antibody purification
A positive cell line was selected for expansion culture and injected into the abdominal cavity of Balb/C mice which had been pre-sensitized with Freund's incomplete adjuvant, after 10 days the mice were observed for apparent abdominal distension and ascites was extracted.
The positive cells are immunized with mice to obtain ascites, and affinity purification is carried out by using a Protein A column. Protein a packing was loaded into a gravity purification column and washed three times with 10 column volumes of PBS solution. Ascites is added to the column and incubated for 4h with mixing at 4 ℃. The effluent was discharged and washed three times with 10 column volumes of PBS solution. The antibody was eluted with a pre-chilled pH3.0 HCl-Glycine eluate and the collected antibody was immediately neutralized with 10xPBS neutralization solution. The concentration of antibodies in the collection tubes was measured by OD280, the high concentration collection tubes were pooled and dialyzed overnight at 4 ℃ with PBS solution.
The concentration of the dialyzed monoclonal antibody is detected by adopting a BCA method, the titer of the monoclonal antibody is measured by adopting an indirect ELISA method, the wild chimpanzee adenovirus and the human adenovirus type 5 are taken as antigens, and the specificity of the monoclonal antibody for recognizing the chimpanzee adenovirus is examined by adopting Western Blot.
Monoclonal antibody specificity evaluation
Preparation of 10% isolation gel and 5% concentration gel, respectively mixing 30 μl of wild chimpanzee 68 type adenovirus and wild human adenovirus type 5 with 10 μl of 4×reduced loading buffer, boiling water bath for 5min, loading 3 μl, and pre-dyeing Marker loading 15 μl. Electrophoresis was performed at 90V for 30min and then at 150V for 60min. The membrane is transferred for 1h by wet transfer method at 90V, and is washed three times by PBST for 5min each time, and is sealed for 1h by 10% skimmed milk at 37 ℃. The PBST was washed three times for 5min each. AdC68-Hexon monoclonal antibodies were diluted 10000-fold with 10% skim milk, and the membranes were immersed in primary anti-dilution and incubated overnight at 4 ℃. The membrane was removed, washed four times with PBST for 3min each, and HRP-labeled goat anti-mouse IgG antibody was diluted 10000-fold with 10% skim milk, and the membrane was immersed in the secondary antibody dilution and incubated for 1h at 37 ℃. The chemiluminescent substrate solution is evenly dripped on the membrane, and after standing for 1min, color development is carried out on a chemiluminescent imager, and the exposure time is 3s, and the result is shown in figure 3, the monoclonal antibody can specifically identify wild chimpanzee 68 adenovirus and does not identify the Hexon protein of wild human adenovirus type 5.
Monoclonal antibody variable region sequencing
Extracting anti-AdC 68-Hexon monoclonal hybridoma cell strain RNA by using a Trizol method, carrying out reverse transcription by using a 5'RACE method to obtain cDNA, designing primer amplification and TSO primer sequences according to a constant region at the 3' end of the antibody, and obtaining heavy chain and light chain variable regions. Cloning to pCE2 vector, and obtaining variable region sequences VH and VL of anti-AdC 68-Hexon monoclonal antibody by adopting a generation sequencing method.
Specifically, the heavy chain and light chain variable region genes are amplified from total RNA of hybridoma cell strain secreting anti-AdC 68-Hexon antibodies by RT-PCR method and sequenced. According to Blast comparison results, the heavy chain variable region gene VH is 345bp long, codes 115 amino acids, the light chain variable region gene VL is 336bp long, and codes 112 amino acids.
The antibody is an anti-AdC 68 antibody, and the sequence of the heavy chain of the antibody is:
EVQLVETGGGLVQPKGSLKLSCAVSGLPFNTKAMDWVRQAPGEGLEWVARIRTKS NNYATYYADSVKDRFTISRDDAQSMLYLQMTNLKTEDTAMYYCVRDEASWGHGTLVTV SA(SEQ ID NO:15)；
the sequence of the light chain is:
DIVMTQIPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFPWTFGGGTKLEIK(SEQ IDNO:16)。
9. recombinant monoclonal antibody plasmid construction, expression and purification
The heavy chain constant region sequence of the mouse IgG1 (IgG 1CH, genBank No.: NC-000078.7) and the light chain constant region sequence of the mouse IgG kappa (IgG kappa CL, genBank No.: NC-000072.7) are synthesized, and the anti-AdC 68-Hexon mouse monoclonal antibody VH (VL) and the mouse IgG1CH (IgG kappa CL) are spliced by adopting recombinant PCR to obtain the full-length sequences of the anti-AdC 68-Hexon mouse monoclonal antibody heavy chain and the anti-mouse monoclonal antibody light chain. The PCR products obtained were introduced into the commercial eukaryotic expression vectors pcDNA3.1 (+) and pcDNA3.1/zeo (+). The positive anti-AdC 68-Hexon heavy and light chain eukaryotic expression vectors MHpcDNA3.1 (+) and ML pcDNA3.1/zeo (+) (wherein MH prefix represents mouse heavy chain and ML prefix represents mouse light chain) were identified by enzyme digestion, and were sent to sequencing company for sequencing.
And (3) splicing the anti-AdC 68-Hexon mouse monoclonal antibody VH and the mouse IgG1CH, and the anti-AdC 68-Hexon mouse monoclonal antibody VL and the mouse IgG kappa CL by adopting recombinant PCR to obtain an anti-AdC 68-Hexon mouse monoclonal antibody heavy chain full-length sequence (4328 bp) and a light chain full-length sequence (656 bp). The full-length sequences of the heavy chain and the light chain are respectively introduced into eukaryotic expression vectors pcDNA3.1 (+) and pcDNA3.1/zeo (+), and the sequencing result shows that the connection is successful.
After eukaryotic cell HEK293 is co-transfected by anti-AdC 68-Hexon heavy and light chain eukaryotic expression vector, the expressed mouse monoclonal antibody heavy and light chain molecules are secreted outside cells after being assembled into IgG1 k. Cell supernatants were collected and purified using Protein A columns to obtain recombinant anti-AdC 68-Hexon mouse monoclonal antibodies. The concentration of the recombinant anti-AdC 68-Hexon murine monoclonal antibody was determined by BCA method, and the purity thereof was determined to be >90% by reduced SDS-PAGE method, and the results are shown in FIG. 4; an indirect ELISA method was used to identify whether the parent murine antibody retained the binding specificity to the AdC6-Hexon protein, with a titre of 6561000 for the AdC6-Hexon protein.
2. Monoclonal antibody application validation
The monoclonal antibody obtained in this example can effectively and specifically recognize chimpanzee adenovirus type 68, and does not recognize human adenovirus type 5 and wild type respiratory syncytial virus, and the following experiment was used to verify the application of the monoclonal antibody.
(1) Experimental procedure
The 293A cells forming the monolayer are digested by pancreatin to prepare suspension with the cell concentration of 2.5X105-3.0X105/ml, and the suspension is inoculated into a 24-hole culture plate for culture. After one day a monolayer was formed and the culture medium in the 24-well plate was removed. Serial 10-fold dilution of the test sample, inoculating 293A cells at a proper dilution ratio of 0.1 ml/hole, and setting cell control and antibody control holes; placing the culture plate in a 37 ℃ and 5% CO2 incubator to adsorb for 2 hours (shaking the plate 1 time every 15 minutes), removing the virus liquid in the 24-pore plate, adding 1ml of the culture liquid into each pore, continuously culturing for 2 days in the 37 ℃ and 5% CO2 incubator, adding methanol to fix for 10 minutes, washing the plate, adding the recombinant monoclonal antibody to incubate for 1 hour, washing the plate, adding the enzyme-labeled secondary antibody to incubate for 1 hour, washing the plate, developing for 15-20 minutes, counting the staining spots under a microscope, and calculating the IFU according to the dilution corresponding to the average staining spot number of each dilution.
(2) Methodological verification
2.1 specificity
Wild type respiratory syncytial virus and human type 5 adenovirus are used as negative control, wild type chimpanzee type 68 adenovirus (test 1) and recombinant chimpanzee type 68 adenovirus modified by using the wild type chimpanzee type 68 adenovirus as a vector (test 2) are used as test, wherein the recombinant chimpanzee type 68 adenovirus is prepared by inserting a gene fragment GFP for expressing green fluorescent protein (Green Fluorescence Protein, GFP) into the wild type chimpanzee type 68 adenovirus, and the Wohbo biotechnology limited company. The specific recognition ability of the antibody against chimpanzee type 68 adenovirus was verified by observing the presence or absence of stain formation. The test sample is diluted 10 times by using the virus diluent and various matrixes in the test sample as diluents, the titer is measured, and the titer value variation coefficient of the test sample measured by adopting the two dilution modes is compared to verify that the matrix components in the test sample have no influence on the measurement of the chimpanzee adenovirus titer by using the antibody.
The experimental results are shown in table 4, the test samples are diluted 10 times by using the virus diluent and the matrix of each test sample as the diluent, the titer of the test samples is measured by using the recombinant monoclonal antibody, and the titer value variation coefficient of each test sample measured by using the two dilution modes is less than 1%, which indicates that the antibody is not interfered by the matrix component in the test sample on the chimpanzee 68 adenovirus titer measurement, and the specificity is good.
TABLE 4 chimpanzee 68 adenovirus titration (cytoimmunostaining) specificity
2.2 Linear sum Range
Recombinant chimpanzee type 68 adenovirus reference was subjected to 10-fold gradient dilution with virus dilution to determine 6 different dilutions of the reference (dilution: 10 0 、10 1 、10 2 、10 3 、10 4 10 5 Doubling; the theoretical titres are respectively: 9.50, 8.50, 7.50, 6.50, 5.50, and 4.50lg IFU/mL). Each dilution reference was assayed 3 times in parallel. The titer of the same dilution reference and the corresponding dilution factor are plotted as regression curves. Regression coefficients (r 2) were calculated for the measured titer of each dilution reference and the coefficient of variation of the titer measurements.
The test sample titer is good in linear relation within the range of 4.56-9.57 lg IFU/mL, and the linear equation is y= -0.436 xn (x) +9.5849, R 2 1.0000; the titer CV values of the reference products with the concentration of 6 are all less than or equal to 1 percent; the titer of the 6 reference substances is within the theoretical titer of +/-0.10 lgIFU/ml, which shows that the titer of chimpanzee 68 adenovirus detected by the antibody is within the range of 4.56-9.57 lgIFU/ml, and the antibody has good linearity, accuracy and precision.
2.3 precision
A batch of recombinant chimpanzee type 68 adenovirus was taken as a test. The same panel of laboratory workers performed 6 titer determinations on the same day and calculated the coefficient of variation of the 6 determinations to verify their reproducibility. The titre measurement was performed 24 times on each day for this batch of test samples by 2 groups of experimenters, and the coefficient of variation of the measurement results was calculated 6 times to verify the intermediate precision.
As shown in Table 5, the same group of experimenters measured the same batch of test articles in parallel for 6 times in the same day with a variation coefficient of 0.18% -0.27%, and different experimenters measured the same batch of test articles in parallel for 6 times in different days with a variation coefficient of 0.39%, which indicates that the antibody has good repeatability and intermediate precision for measuring the titer of chimpanzee type 68 adenovirus.
TABLE 5 chimpanzee 68 adenovirus titration (cytoimmunostaining) precision
2.4 durability
The titer of the same batch of test samples was determined by respectively selecting different primary antibody incubation times (incubation for 30min, incubation for 60min, incubation for 90 min), each incubation time was measured 3 times in parallel. The coefficient of variation of titer measurements at different incubation times was calculated to demonstrate the durability of the primary incubation time. The titres of the same batch of test samples were determined using different antibody concentrations (1:1500, 1:2000 and 1:2500), respectively, and each staining concentration was measured 3 times in parallel. The coefficient of variation of titer measurements for different antibody concentrations was calculated to demonstrate the durability of staining time.
The results are shown in Table 6, the same batch of test samples are parallelly measured for 3 times by using the primary antibody incubation time of 30min, 60min and 90min, the variation coefficient is 0.28%, the same batch of test samples are parallelly measured for 3 times by using the primary antibody concentration of 1:1500, 1:2000 and 1:2500, and the variation coefficient is 0.14%, so that the test results show that the titer of chimpanzee 68 adenovirus measured by using the antibody has good durability.
TABLE 6 chimpanzee 68 adenovirus titration (cytoimmunostaining) durability
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In summary, the invention uses chimpanzee 68 type adenovirus Hexon protein as antigen to immunize mice to obtain hybridoma cells secreting anti-AdC 68 Hexon protein monoclonal antibody, and sequences the antibody variable region to obtain corresponding sequences, constructs the corresponding sequences to expression vector plasmids, obtains recombinant monoclonal antibodies with stable and efficient expression, uses the recombinant antibodies for virus titration and verification of chimpanzee 68 type adenovirus, and adopts the recombinant monoclonal antibodies obtained by the invention to detect specificity, linear range, repeatability, intermediate precision and durability of chimpanzee 68 type adenovirus to meet the requirements. The cell immunostaining method verified in the experiment calculates the titer of the infectious adenovirus by adopting a specific monoclonal antibody to dye and count cells infected with the virus after the gradient diluted chimpanzee adenovirus type 68 is inoculated to the cells for 48 hours, the whole experiment period only needs 3 days, the count of the dyed spots is accurate, and the high-flux full-automatic spot counting measurement can be realized by matching with a cell imaging counter. The recombinant anti-chimpanzee adenovirus 68 type monoclonal antibody is used for staining and counting, so that the chimpanzee adenovirus 68 type can be distinguished from other adenovirus vectors, especially human adenovirus type 5 vectors, specific, accurate and efficient detection of the infectious titer of the chimpanzee adenovirus 68 type can be realized, and the process research, development and production process monitoring of the chimpanzee adenovirus 68 type gene therapy medicine and vaccine are supported.
Compared with the current commercial adenovirus antibody, the recombinant monoclonal antibody has the advantages of good specificity, clear and definite antibody sequence, large-scale production by adopting cells, good batch-to-batch consistency and the like, and has important significance for qualitative identification and quantitative analysis of gene medicines and vaccines by taking chimpanzee 68 adenovirus as a vector.
Finally, what is necessary here is: the above embodiments are only for further detailed description of the technical solution of the present invention, and should not be construed as limiting the scope of the present invention, and any person skilled in the art should make some changes, modifications, substitutions, combinations and simplifications using the technical solution disclosed above without departing from the scope of the technical solution of the present invention, all of which are included in the scope of the present invention.
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Claims (10)
1. A chimpanzee type 68 adenovirus based monoclonal antibody, characterized in that: the antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise 3 CDR regions and 4 FR regions, the amino acid sequence of the CDR1 region of the heavy chain is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 region of the heavy chain is shown as SEQ ID NO. 2, the amino acid sequence of the CDR3 region of the heavy chain is shown as SEQ ID NO. 3, the amino acid sequence of the FR1 region of the heavy chain is shown as SEQ ID NO. 4, the amino acid sequence of the FR2 region of the heavy chain is shown as SEQ ID NO. 5, the amino acid sequence of the FR3 region of the heavy chain is shown as SEQ ID NO. 6, the amino acid sequence of the FR4 region of the heavy chain is shown as SEQ ID NO. 7, the amino acid sequence of the CDR1 region of the light chain is shown as SEQ ID NO. 8, the amino acid sequence of the CDR2 region of the light chain is shown as SEQ ID NO. 9, and the amino acid sequence of the CDR3 region of the light chain is shown as SEQ ID NO. 10; the amino acid sequence of the FR1 region of the light chain is shown as SEQ ID NO. 11, the amino acid sequence of the FR2 region of the light chain is shown as SEQ ID NO. 12, the amino acid sequence of the FR3 region of the light chain is shown as SEQ ID NO. 13, and the amino acid sequence of the FR4 region of the light chain is shown as SEQ ID NO. 14.
2. The antibody of claim 1, wherein: the antibody is an anti-AdC 68 antibody, the sequence of the heavy chain of the antibody is shown as SEQ ID NO. 15, and the sequence of the light chain is shown as SEQ ID NO. 16.
3. The antibody of claim 1, wherein: the Hexon protein sequence of chimpanzee type 68 adenovirus is shown in SEQ ID:17.
4. A method for preparing the antibody of any one of claims 1 to 3, characterized in that: the chimpanzee 68 type adenovirus hexon protein is used as antigen to immunize and obtain hybridoma cells, and the hybridoma cells are constructed to an expression vector plasmid to obtain the recombinant monoclonal antibody.
5. The method of manufacturing according to claim 4, wherein: the method comprises the following steps:
a) Adopting chimpanzee 68 adenovirus Hexon protein sequence, synthesizing target gene according to the codon preference of colibacillus, inserting into expression vector pET-32a, converting colibacillus, and picking positive clone;
b) Antigen expression and purification;
c) Immunizing animals;
d) Cell fusion and subcloning screening monoclonal hybridoma cell strains;
e) The monoclonal hybridoma cell line was expanded and purified.
6. The method of manufacturing according to claim 5, wherein: the preparation method also comprises an identification step, wherein the antibody subtype of the hybridoma cell supernatant is detected by adopting a monoclonal antibody subtype kit.
7. A nucleic acid, characterized in that: a nucleotide sequence comprising a sequence encoding an antibody according to any one of claims 1-3.
8. A recombinant vector, characterized in that: the plasmid comprises the nucleic acid of claim 7.
9. A cell line, characterized in that: comprising the recombinant vector of claim 8, or having integrated into its genome the nucleic acid of claim 7.
10. A method for detecting chimpanzee adenovirus 68 for non-disease diagnostic or therapeutic purposes, comprising: detection using the antibody of any one of claims 1 to 3.
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