CN107266566B - The anti-EV71 virus neutrality antibody E1 of source of people and its application - Google Patents

The anti-EV71 virus neutrality antibody E1 of source of people and its application Download PDF

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CN107266566B
CN107266566B CN201710418696.3A CN201710418696A CN107266566B CN 107266566 B CN107266566 B CN 107266566B CN 201710418696 A CN201710418696 A CN 201710418696A CN 107266566 B CN107266566 B CN 107266566B
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金奇
陈哲
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Institute of Pathogen Biology of CAMS
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Abstract

The present invention provides a kind of anti-EV71 virus neutrality antibody E1 of source of people obtained using display technique of bacteriophage screening, the amino acid sequence of light chain and heavy chain variable region is respectively as shown in SEQ ID No.1 and SEQ ID No.2.The antibody can specific recognition EV71 virion antigen, significant enzyme linked immunoassay can occur with EV71 virus, and have the function of the neutralization activity of anti-EV71 virus infection.In addition, antibody of the invention can be made to the specific antibodies medicine for preventing and treating hand-foot-and-mouth disease, thus for preventing and treating the hand-foot-and-mouth disease as caused by EV71 virus in clinic.

Description

Humanized anti-EV 71 virus neutralizing antibody E1 and application thereof
Technical Field
The invention relates to the technical field of genetic engineering and phage display, in particular to a humanized neutralizing antibody E1 against EV71 virus and application thereof.
Background
Hand-foot-mouth disease (HFMD) is an infectious disease caused by enteroviruses, mostly occurs in infants under 5 years old, and can cause fever, rash and ulcer of hands, feet, oral cavity and other parts, and individual patients can cause complications such as myocarditis, pulmonary edema, aseptic meningoencephalitis and the like. There are more than 20 enteroviruses causing hand-foot-and-mouth disease, of which coxsackie virus (Cox ackievirus) type a16 (Cox a16) and enterovirus type 71 (enterovirus71.ev71) are the most common. In 1974 Schmidt et al first reported the isolation of EV71 from patients presenting with neurological symptoms disease in U.S. California outbreaks (1969-1973), and subsequently, in many countries of the world, the prevalence of EV71 virus in different regions was reported in succession, and it was gradually recognized that EV71 virus is the main cause of hand-foot-and-mouth disease.
Because the hand-foot-and-mouth disease does not have corresponding vaccine and specific medicine at present, the preparation of the passive immune preparation with high efficiency, low price and small side reaction becomes a hot point of research. The use of human or animal serum immunoglobulins containing specific antibodies for the prevention and treatment of infectious diseases has long been known. In vitro antiviral neutralizing activity of monoclonal antibodies and in vivo protection of the body against viral challenge a number of experiments have been carried out to demonstrate that neutralizing monoclonal antibodies such as hantavirus, measles virus, RSV virus, rabies virus, etc. can protect 100% of animals against viral challenge in vivo.
Immunoglobulin (Ig) as an antibody component is mainly derived from immune serum of a donor (convalescent patient), and it takes a long time from obtaining positive serum to passing safety test, and a lot of manpower and financial resources are required, so that mass production thereof is limited, and infection of blood-borne diseases is easily caused because the antibody is mainly derived from serum. The human genetic engineering product is used for replacing blood products to overcome the defects, and with the continuous and deep research of human genetic engineering antibodies, the method brings new hope and broad prospect for the development of biological products in the field.
The development of Phage Display technology (see Barbas, C.F., 1991) and the whole research field of genetic engineering antibody technology, which was emerging in the early 90 s, has greatly promoted the development and research of humanized or genetic engineering antibodies, and has stepped from the basic research stage into the substantial application research and development stage. The research success of the human anti-virus genetic engineering antibody, in particular the human full antibody opens up a new idea for the specific prevention and treatment of various viral infectious diseases, and a new anti-virus medicine is gradually developed in the field of anti-virus infection biomedicine.
Disclosure of Invention
The invention aims to provide a human anti-EV 71 virus neutralizing antibody E1 and application thereof.
To achieve the object of the present invention, the amino acid sequences of the light and heavy chain hypervariable regions CDR1, CDR2 and CDR3 of the antibody E1 or an active fragment thereof derived from the neutralizing antibody E1 against EV71 virus of the present inventors are shown in the following table:
the amino acid sequence of the light chain variable region of the antibody E1, i) is shown in SEQ ID No.1, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence. For example, the substitution of Arg at position 15 with Lys does not affect the function of the protein.
ii) the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.2, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence. For example, replacement of Lys at position 8 to Ala does not affect protein function.
The invention also provides a gene encoding the antibody E1. Wherein, the nucleotide sequences of the coding light chain variable region and the heavy chain variable region are respectively shown as SEQ ID No.3 and SEQ ID No. 4.
The invention also provides an expression cassette, an expression vector or a cloning vector comprising a nucleic acid comprising a gene sequence encoding said antibody E1.
The invention also provides a host cell containing the encoding gene of the antibody E1, or the expression cassette and the vector.
The invention also provides a single-chain antibody ScFv or Fab antibody or whole antibody immunoglobulin IgG obtained by modifying the antibody E1 or an active fragment thereof.
The human neutralizing antibody E1 against EV71 can be prepared by the following method: a phage display technology is utilized to collect a plurality of EV71 patient convalescent peripheral blood lymphocytes, a humanized EV71 virus resistant gene engineering antibody library is constructed by a gene engineering method, and a specific EV71 virus resistant gene engineering antibody Fab fragment is obtained by screening.
The antibody is determined by the specific gene sequences of hypervariable regions (CDRs) existing in the variable regions of the antibody light chain and heavy chain genes, and can obtain an effectively expressed functional antibody which is specifically combined with EV71 virus in prokaryotic cells. The recombinant EV71 virus particle antigen specifically recognizes EV71 virus particle antigen, and has obvious enzyme-linked immunosorbent assay (ELISA) reaction and an activity of neutralizing the EV71 virus infection.
The specific light chain and heavy chain variable region genes of the antibody E1 are derived from specific enrichment screening of a human anti-EV 71 virus antibody gene library, and the antibody library is established from peripheral blood lymphocyte genes of Chinese EV71 virus patients. The combination of three CDR sequences corresponding to the light chain and heavy chain variable regions and the framework region sequences between the CDR regions form the variable region sequence characteristics of the antibody, and E1 belongs to the antibody light chain family VL 1.The function of the antibody protein is determined by specific nucleotide sequences and complementary sequences thereof in determinant complementary regions CDR1, CDR2 and CDR3 of the variable regions of the light chain and the heavy chain of an antibody gene, and 6 corresponding amino acid sequences of the CDR regions form a specific antigen binding region of the antibody, thereby determining the antigen binding characteristic of the antibody and the anti-EV 71 virus functional characteristic.
In addition, in consideration of the degeneracy of codons, the gene sequence encoding the above-mentioned Fab fragment antibody can be modified, for example, in the coding region thereof without changing the amino acid sequence, to obtain a gene encoding an antibody having the same function. One skilled in the art can artificially synthesize and modify genes according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.
Further, the present invention provides a single chain antibody (ScFv) having a smaller molecular weight, which specifically recognizes the surface antigen of EV71 virus and has an intracellular immunity effect, by recombining the light chain variable region and the heavy chain variable region of the Fab antibody. The single-chain antibody has strong penetrating power and is easy to enter local tissues to play a role.
The Fab antibody-encoding gene and ScFv gene can be cloned into an expression vector, and the Fab antibody and single-chain antibody can be obtained by transforming a host and inducing expression.
In addition, the light chain encoding gene and the heavy chain encoding gene of the Fab antibody can be cloned into a full-antibody expression vector and introduced into a host cell to obtain the full-antibody immunoglobulin for expressing the anti-EV 71 virus.
The obtained E1 antibody is subjected to functional identification by using methods such as ELISA, SDS-PAGE, neutralization experiments and the like, the result shows that the human antibody E1 has specific binding to the FUYANG-0805 strain EV71 virus particles, and the result shows that the E1 has better neutralization activity.
The invention also provides application of the antibody E1 or the active fragment thereof in preparing a medicament for preventing or treating hand-foot-and-mouth disease caused by EV71 virus.
The invention also provides application of the antibody E1 or the active fragment thereof in preparation of an EV71 virus antigen detection reagent or detection kit.
The invention further provides a medicament, a detection reagent or a detection kit containing the antibody E1 or the active fragment thereof.
According to the invention, a humanized neutralizing antibody E1 specific to EV71 virus is successfully obtained by using a phage display technology; the obtained humanized neutralizing anti-EV 71 virus genetic engineering antibody variable region gene, Fab antibody gene and whole antibody gene under the antibody gene characteristic can be used for expressing and producing the antibody in prokaryotic cells, yeast cells, eukaryotic cells and any recombination system or any other gene containing the antibody gene after modification based on the expression and production to obtain an antibody product capable of neutralizing EV71 virus infection, and the antibody product is prepared into a specific antibody medicament for clinically preventing and treating hand-foot-and-mouth disease.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of the Fab fragment of the E1 antibody after purification in example 1 of the present invention; wherein, 1 is a purified antibody Fab E1, and M is a protein Marker.
FIG. 2 shows the results of ELISA detection of Fab fragment of anti-EV 71 virus E1 antibody in example 1 of the present invention; the amino acid sequences of the FabE1 light chain and heavy chain variable regions are respectively shown in SEQ ID No.1 and 2, FabE 1' is a mutant of FabE1 (a mutant formed by replacing Arg at position 15 of a FabE1 light chain variable region with Lys and replacing Lys at position 8 of a FabE1 heavy chain variable region with Ala), a positive control is commercial murine antibody, and a negative control is a Coxsackie A4 virus antibody.
FIG. 3 is a SDS-PAGE electrophoresis of full antibody IgG constructed from EV71 virus E1 antibody in example 3 of the present invention; wherein, 1 is EV71 virus E1 whole antibody IgG, and M is protein Marker.
FIG. 4 shows the neutralization test results of human E1 whole IgG antibody against EV71 virus in example 3; among them, the negative control was coxsackie a4 virus antibody.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 construction of humanized anti-EV 71 Virus antibody library and screening of Fab antibody
1.1 materials and methods
1.1.1 sources of viruses, cells and vectors: the strain FUYANG-0805 of EV71 virus has been disclosed in the literature (Yang F, Jin Q, He Y, Li L, Hou Y.2001.the complete genome of Enterovirus71 Chinese strain SciChina C Life Sci and Wu Z, Yang F, ZHao R, ZHao L, Guo D, Jin Q.2009. identifying effective small interfering RNAs whihich inhibit the replication of viral Enterovirus71 strain in China J virons Methods) and is provided by the institute of pathogenic biology of Chinese academy of medicine sciences. Cells used for the in vitro neutralization experiment of EV71 virus were RD cells, purchased from ATCC. The strain was XL1-Blue (Stratagene, USA), vector pComb3H (supplied by the Scripps research, USA).
1.1.2 antigen preparation
Purification of EV71 virus: the EV71 virus FUYANG-0805 strain infected RD cells were harvested, the culture supernatant was subjected to formaldehyde inactivation and safety inspection, and virus particles were purified by centrifugation at 35000g of 20% sucrose density gradient for 3h at 4 ℃ (Beckman SW 28).
1.1.3 construction of phage antibody libraries
Lymphocytes were separated from recovered anticoagulated blood of EV71 patient with lymphocyte separating medium (Sigma, USA), total cellular RNA was extracted with RNeasy Mini Kit (QIAGEN, Germany), and cDNA was generated by reverse transcription using Oligo-dT as primer and RNA as template using first Strand synthesis Kit (SuperScriptTMIIIFirst-Strand Synthesis System for RT-PCR. Cat No. 18080-. A set of primers for amplifying the heavy chain Fd and light chains Kappa and Lambda of the human antibody IgG1 was used, and the sequences of the primers used were as follows:
heavy chain Fd region primer
5' end
VH1a 5'-CAG GTG CAG CTC GAG CAG TCT GGG-3'
VH1f 5'-CAG GTG CAG CTG CTC GAG TCT GGG-3'
VH2f 5'-CAG GTG CAG CTA CTC GAG TCG GG-3'
VH3a 5'-GAG GTG CAG CTC GAG GAG TCT GGG-3'
VH3f 5'-GAG GTG CAG CTG CTC GAG TCT GGG-3'
VH4f 5'-CAG GTG CAG CTG CTC GAG TCG GG-3'
VH6f 5'-CAG GTG CAG CTA CTA GAG TGG GG-3'
VH6a 5'-CAG GTA CAG CTC GAG CAG TCA GG-3'
3' end
CG1Z 5'-GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG-3'
(II) light chain primer
5' -end of kappa chain variable region
VK1a 5'-GAC ATC GAG CTC ACC CAG TCT CCA-3'
VK2a 5'-GAT ATT GAG CTC ACT CAG TCT CCA-3'
VK3a 5'-GAA ATT GAG CTC ACG CAG TCT CCA-3'
3-terminal of the kappa chain variable region
CK1d 5'-GCG CCG TCT AGA ATT AAC ACT CTC CCC TGT TGA AGC TCT
TTG TGA CGG GCG AAC TCA-3'
5-terminal of the lambda chain variable region
VL1 5'-AAT TTT GAG CTC ACT CAG CCC CAC-3'
VL2 5'-TCT GCC GAG CTC CAG CCT GCC TCC GTG-3'
VL3 5'-TCT GTG GAG CTC CAG CCG CCC TCA GTG-3'
VL4 5'-TCT GAA GAG CTC CAG GAC CCT GTT GTG TCT GTG-3'
VL5 5'-CAG TCT GAG CTC ACG CAG CCG CCC-3'
VL6 5'-CAG ACT GAG CTC ACT CAG GAG CCC-3'
3-terminal of the lambda chain variable region
CL2 5'-CGC CGT CTA GAA TTA TGA ACA TTC TGT AGG-3'
Human light and heavy chain Fab genes were amplified by PCR. The PCR conditions were 94 ℃ for 1min, 54 ℃ for 1min, and 72 ℃ for 2min for 35 cycles. The library construction method is essentially carried out according to the literature (Barbas, C.FIII., Kang, A.S., and girder, R.A.Assembly of combinatorial antibody libraries on phase surface: the genetic III. Proc.Natl.Acad.Sci.USA.1991; 88(18): 7978-. The method comprises the following specific steps:
all different heavy and light chain PCR products were first mixed in separate clusters. 1.5-2. mu.g of pComb3H vector DNA digested with XbaI and SacI and purified by electrophoresis was ligated with 500 ng of light chain mixture, 2. mu.l of high concentration ligase (NEB 2000U/. mu.l), ligation buffer and l6 ℃ overnight. Adding 100 μ l purified water the next day, adding 3M NaAc 16 μ l, adding 2.5-3 times of anhydrous ethanol to precipitate DNA, centrifuging, precipitating, and addingAfter 20 mul of pure water is used for resuspension, the pellet is added into the competent bacteria XL1-Blue, the voltage is 2.5kv, and the electric shock is carried out for 1 minute. Immediately after the electroporation, 2ml of SOC culture medium was added, and immediately transferred to a bacterial incubator, followed by shaking culture at 37 ℃ for 1 hour. The bacterial liquid is completely coated on an LB plate containing ampicillin and cultured overnight at 37 ℃. Adding 10-15ml of culture solution into a plate the next day, scraping bacterial plaque, subpackaging in a centrifuge tube, centrifuging at 12000rpm, discarding supernatant, extracting plasmid pComb3H-L by using QIAGEN large quality-improving granule kit, mixing and freezing at-20 ℃ for later use. PCR products were purified from pComb3H-L and Fd strands cloned into L strand, and digested with XhoI and SpeI for 3-4h at 37 ℃. Corresponding bands are recovered by electrophoresis, and the recovered products are quantified after the digestion of the plasmids and Fd. Taking 2 mu g of recovered and purified vector DNA after digestion, adding 2 mu l of high-concentration ligase into about 600ng of heavy chain PCR product, adding corresponding ligation buffer solution, and connecting overnight at 16 ℃. The next day, 100. mu.l of purified water, 3M NaAc 16. mu.l, 2.5-3 times of absolute ethanol were added to precipitate DNA, the DNA was centrifuged and precipitated, 20. mu.l of purified water was used to resuspend the precipitate, and the precipitate was added to allelochemicals XL1-Blue at a voltage of 2.5kv and electrically shocked for 1 minute. Immediately after the electric transfer, 2ml of SOC culture solution was added, and immediately transferred to a bacterial incubator, followed by shaking culture at 37 ℃ and 200rpm for 1 hour. The culture solution was transferred to a flask, 10ml of SB-A + culture medium containing 20. mu.g/ml ampicillin was added thereto, and shaking culture was carried out at 37 ℃ and 200rpm for 1 hour. 100ml of SB medium (containing 100. mu.g/ml Amp and 20. mu.g/ml Tet) was added and cultured with shaking for 1 hour. Adding 1012The pfu helper phage VCSM13 was left to stand at 37 ℃ for 20min for infection, and then cultured at 37 ℃ for 2h with Kan added to a final concentration of 70. mu.g/ml, and cultured overnight at 37 ℃. To be OD600When the bacterial strain is about 1 hour, the bacterial strain is centrifuged at 4000rpm at 4 ℃ for 15min, the supernatant is transferred to a sterile triangular flask, 4% (w/v) PEG8000 and 3% (w/v) NaCl are added, and after complete dissolution, the phage is precipitated by ice bath for more than 30 min. Centrifuging at 4 deg.C and 9000rpm for 20-30min, discarding supernatant, resuspending the precipitate with 2ml PBS, and centrifuging instantaneously to obtain supernatant as Fab phage antibody library.
1.1.4 enrichment screening of phage antibody library and inducible expression of Fab fragment antibodies
The inactivated virus particle FUYANG-0805 strain purified by ultracentrifugation is used as a screening antigen. Make itUsing 0.1M NaHCO3(pH8.6) solution dilution, coating immune tube, using 4% skim milk PBS, at 37 degrees C after 2h blocking, adding the phage antibody library, each tube of 1ml, 37 degrees C incubation for 2h, using 5% Tween-20 TBS solution repeatedly 20 times, finally each tube of 1ml pH2.2 glycine-hydrochloric acid elution, and using pH9.6 Tris solution neutralization. The eluted phage was further infected with 2ml of fresh OD600About 1.0 of XL1-Blu bacteria were infected with the helper phage VCSM13(Stratagene, USA) and subjected to the next round of selection. The screening is repeated for 3-4 times. Specific enrichment screening methods and the inducible expression of Fab fragments were performed essentially as described in the literature (Barbas, C.FIII., Kang, A.S., and Large, R.A.Assembly of combinatorial antibodies on phase surface: the genetic III site, Proc.Natl.Acad.Sci.USA 1991; 88(18): 7978-.
Enrichment of phage antibody library: with 0.1M NaHCO3(pH8.6) the purified FUYANG-0805 strain was diluted 1:100 and coated on 96-well plates overnight at 4 ℃. The next day, unadsorbed antigen was washed with PBS-T (20mM PBS plus 0.05% Tween-20) and blocked with 3% skim milk at 37 ℃ for 1 h. The blocking solution was discarded, and 60. mu.l phage antibody library was added to each well and incubated at 37 ℃ for 2 h. Unbound phage were discarded from the wells, and each well was washed with TBS-T (50mM Tris-HCl, 150mM NaCl, 0.5% Tween-20, pH7.5) wash solution, which was repeatedly pipetted and washed 20 times in total to sufficiently wash away the non-adsorbed phage. Finally using ddH2And washing twice by using O, and completely absorbing residual liquid in the holes. Mu.l of Glycine-HCl (pH2.2) eluent was added to each well and incubated at room temperature for 10min, during which time the pipette tip was repeatedly blown (note that no air bubbles were blown). The eluates were pooled in a centrifuge tube and 2M Tris was added at a rate of 3. mu.l per well to bring the pH of the solution to about 7 to neutralize the eluted phage. The eluted phage was immediately added to 2ml of fresh XL1-Blue broth (OD)6001), incubate for 20min at room temperature. Transfer to a 250ml Erlenmeyer flask, add 10ml SB (containing ampicillin 20 u g/ml and tetracycline 20 u g/ml), immediately take 10 u l coated ampicillin plate, for titration of phage. The rest liquid was cultured with shaking at 37 ℃ for 1 hour. 100ml SB (containing ampicillin 100. mu.g/ml and tetracycline 20. mu.g/ml) was addedAnd carrying out shaking culture at 37 ℃ for 2 h. Then the helper phage VCSM13 (9X 10) was added12pfu/ml)1ml, standing at 37 ℃ for 20min, and performing shake culture at 37 ℃ for 2 h. Kanamycin (final concentration 70. mu.g/ml) was added and cultured overnight at 37 ℃ with shaking. When the bacteria grow to OD600When the bacterial strain is about 1 hour, the bacterial strain is centrifuged at 6500rpm at 4 ℃ for 15min, the supernatant is transferred to a sterile triangular flask, 4% (w/v) PEG8000 and 3% (w/v) NaCl are added, and the phage is fully precipitated after complete dissolution in ice bath for more than 30 min. Centrifuging at 9000rpm at 4 deg.C for 20-30min, and discarding the supernatant. And (3) resuspending the precipitate with 2ml of PBS, and performing instantaneous centrifugation to obtain a supernatant, namely a first round enrichment screening Fab phage antibody library.
Expression of Fab positive clones: 2000 single colonies after 3 enrichment screens were randomly picked in 96 deep well plates with 800. mu.l of medium per well and incubated overnight at 37 ℃. The following day, the cells were transferred to a 96-well plate containing 800. mu.l of SB medium (containing Amp 100. mu.g/ml) at a ratio of 1:20, and cultured at 37 ℃ to OD600When the concentration is 0.2-0.3, IPTG is added to the mixture to a final concentration of 1mM, and the expression is induced at 30 ℃ for 8-10 hr. The mixture was centrifuged at 4000rpm at 4 ℃ for 15min, and the supernatant was used for detection.
1.1.5 ELISA detection of humanized anti-EV 71 Virus Fab antibody
(1) Detecting Fab expression
With 0.1M NaHCO3(pH9.6) solution anti-human Fab antibody (1:2000 diluted for use, Sigma, USA) was coated on the microplate overnight at 4 ℃; sealing with 4% skimmed milk, adding expressed Fab antibody at 37 deg.C for 1 hr, and sealing at 37 deg.C for 1 hr; adding enzyme-labeled anti-human Fab secondary antibody (1:2000 diluted for use, Sigma, USA) at 37 deg.C for 1 h; color developing solution for color development, 2M H2SO4The reaction is stopped, and the absorbance A value is detected by a microplate reader.
(2) Indirect enzyme-linked immunosorbent assay for detecting binding activity of Fab and EV71 virus
Purified inactivated EV71 virions were used as coating antigens, and the rest of the procedure was as above.
1.1.6 analysis of nucleic acid sequence of variable region Gene of humanized Fab antibody
Plasmid DNA was prepared using the Qiagen Miniprep Kit (QIAGEN, Germany) for nucleic acid sequence analysis. The sequencing primers for the light and heavy chains are 5 '-AAACTAGCTAGTCGCC AAGGA-3' and 5 '-CCGCGGTGGCGGCCGCAAAT-3', respectively. And comparing the sequencing result with the IgG gene sequence in the Internet V-Base gene library.
1.1.7 purification of humanized Fab antibodies against EV71 Virus
A2 ml affinity column was prepared with an anti-human Fab antibody (Sigma, USA), and the filtered Fab antibody-containing suspension was applied to the column and repeated cycles were carried out for 30min to 1 h. PBS prewashing buffer solution with pH value of 6.8 is added to wash off non-specific adsorbed protein. The bound Fab antibody protein was eluted by adding glycine-HCl buffer solution of pH 2.7, and the eluted solution was neutralized by adding Tris-HCl buffer solution of pH 9.0, followed by centrifugation and concentration using a concentration column. And performing SDS-PAGE electrophoresis on 10-20 mu g of the purified and concentrated Fab fragment protein.
1.1.8 neutralization assay detection of humanized anti-EV 71 virus Fab antibody
(1) Experimental procedure
Will 100TCID50EV71 virus 25. mu.l and dilution-by-fold antibody 25. mu.l were incubated at 37 ℃ for 2 hours and then added at a concentration of 2X 105Each RD cell/ml cell culture solution was 50. mu.l. The observation was carried out for 7 days, and the cytopathic results were recorded.
(2) Determination of results
When 1 hole of 2 holes of the antibody with the highest dilution has cytopathy, and the other 1 hole does not have cytopathy, the reciprocal of the dilution is the neutralizing antibody titer of the antibody; when the high dilution 2 hole is completely diseased and the adjacent low dilution 2 hole is not diseased, the reciprocal of the average dilution of the two is the neutralizing antibody titer of the antibody; when the antibodies of two adjacent dilutions have cytopathy of 1 well and no cytopathy occurs in the other 1 well, the reciprocal of the average dilution of the two dilutions is the titer of the neutralizing antibody of the antibody.
1.2 results
1.2.1 screening of the humanized anti-EV 71 Virus antibody library
Enrichment screening is carried out on a phage antibody library by using a purified EV71 virus particle FUYANG-0805 strain, and 1000 clones are randomly picked after 3 rounds of screening. A96-well plate was coated with an anti-human Fab antibody (diluted 1:2000 for use, Sigma, USA) and an EV71 virus particle FUYANG-0805 strain antigen, and the supernatant of the sample to be tested was added and detected with an enzyme-labeled anti-human Fab secondary antibody (diluted 1:2000 for use, Sigma, USA). The results showed that 425 human Fab expression positive clones were obtained altogether, of which 231 were able to specifically bind to the EV71 virion FUYANG-0805 strain (table 1).
TABLE 1 enrichment screening result of FUYANG-0805 strain on phage antibody library
1.2.2 sequence analysis of humanized Fab antibody against EV71 Virus
The Fab fragments were analyzed and processed by DNASTAR sequence analysis software, and the IgG sequences in the Internet V-Base gene library were compared, and among the 231 human Fab monoclonal antibodies specifically binding to EV71 virus, the sequences of 11 Fab fragments were different. Therefore, the invention successfully screens and clones 11 antibodies with different antibody light-heavy chain variable region sequences and combinations thereof, wherein the heavy chain variable region mainly belongs to IgG VH3 and VH4 families, and the light chain variable region mainly belongs to IgG VL1, VK1 and VK3 families. Wherein, E1 belongs to antibody light chain family VL1, the amino acid sequences of its light chain variable region and heavy chain variable region are shown as SEQ ID No.1 and SEQ ID No.2, respectively, and the nucleotide sequences of encoding light chain variable region and encoding heavy chain variable region are shown as SEQ ID No.3 and SEQ ID No.4, respectively.
1.2.3 purification of humanized Fab antibodies against EV71 Virus
A2 ml affinity column was prepared using an anti-human Fab antibody (Sigma, USA), the Fab antibody expression supernatant was purified, and the expression and purification of the Fab antibody were examined by SDS-PAGE, which confirmed that a relatively pure protein was obtained, and that the light chain and Fd chain of the antibody after melting were clearly observed (FIG. 1).
1.2.4 ELISA detection of humanized anti-EV 71 Virus Fab antibody
The binding activity of the antibody was measured using purified inactivated EV71 virion FUYANG-0805 strain as the coating antigen, and the results are shown in FIG. 2.
ELISA assay for antigen binding activity: with 0.1M NaHCO3(pH9.6) solution coated with purified inactivated EV71 virus particle FUYANG-0805 strain, 4 ℃ overnight, PBS-T washing liquid washed plate 3 times, fully removing liquid and adding 5% skimmed milk 100 μ l, 37 ℃ temperature 1h incubation, PBS-T washing liquid washed plate 3 times, fully removing liquid, adding prokaryotic expression antibody supernatant 50 μ l, adding 5% PBS-skimmed milk 50 μ l to each well, slightly shaking and mixing, 37 ℃ temperature 1h incubation. Washing the plate with PBS-T washing solution for 6 times, removing liquid completely, adding 100 μ l enzyme-labeled anti-human Fab antibody (Sigma, diluted at 1: 2000), incubating at 37 deg.C for 1h, washing the plate with PBS-Tween20 washing solution for 6 times, removing liquid completely, adding developing solution A, B, developing at room temperature for 10min, and developing at 2M H2SO4Termination of the reaction, OD450And (6) reading.
1.2.5 neutralization assay of humanized Fab antibodies against EV71 Virus
The neutralizing activity of the antibody was examined in RD cells using strain EV71 virus FUYANG-0805 as challenge strain, and the results are shown in Table 2.
The experimental steps are as follows: neutralizing activity of the antibody was detected in RD cells using EV71 virus Fuyang-0805 strain as challenge strain.
50 μ l of 100TCID50EV71 virus (Fuyang-0805 strain) and 50. mu.l of antibody diluted in two fold (prepared in example 1) were incubated at 37 ℃ for 2 hours, and then added at a density of 2X 105Pieces/ml of cell suspension in 100. mu.l. The observation was carried out for 7 days, and the cytopathic results were recorded.
The EV71 virus (Fuyang-0805 strain) used was treated as follows: the viral particles prepared in example 1 were diluted to 10-fold gradient-1To 10-10VirusSolutions, each added to the cell plate at 50 μ l per well, 4 wells of cells per dilution; adding 50 μ l of cell suspension into each well, setting cell control (50 μ l of diluent +50 μ l of cell suspension), culturing at 37 deg.C for 7d, and observing cytopathy; calculating TCID of separated virus strain according to Karber formula50;log TCID50L-d (S-0.5). Wherein: l ═ log of the lowest dilution used in the experiment; d is the log of the dilution gradient; s is the sum of the positive fractions at the end of the call (i.e. the sum of the proportion of the cell pores in which CPE appears).
1.2.6 Effect of antibodies after mutation of the non-hypervariable region on EV71 Virus resistance
The Arg at position 15 of the light chain VL sequence of E1 is replaced by Lys and the Lys at position 8 of the heavy chain VH sequence is replaced by Ala by gene site-directed mutagenesis. After synthesizing the mutated light chain and heavy chain encoding nucleic acid sequences respectively, the light chain and heavy chain genes were cloned into pComb3H to obtain mutant E1'. The mutant is subjected to immunological detection, and an ELISA experiment shows that E1' can be specifically bound with EV71 virus. Neutralization experiments were used to detect the neutralization reaction of the antibody with FUYANG-0805 strain in vitro, and the results showed that the properties were substantially the same as those of E1 (data in table 2 are the dilution factor of the antibody).
TABLE 2 neutralizing Activity of antibody E1 and its mutant E1' on FUYANG-0805 Strain
Example 2 use of a human neutralizing antibody E1 against EV71 virus
At present, no specific vaccine or medicine exists for preventing and treating the hand-foot-and-mouth disease, and clinically used immunoglobulin is derived from positive serum of human anti-EV 71, so that the preparation of the immunoglobulin is limited in large quantity, and the immunoglobulin is derived from the serum so that the infection of blood-borne diseases is easy to occur. The humanized EV71 virus resistant gene engineering antibody obtained by the invention is used for replacing blood-borne immunoglobulin, and provides a new way for treating hand-foot-and-mouth disease.
Example 3 method for preparing Whole antibody immunoglobulin IgG Using neutralizing antibody E1
1.1 expression and purification of Whole antibody IgG
1.1.1 construction of recombinant expression plasmid for full antibody the light chain fragment of Fab antibody was first amplified with primers (upstream 5'-cccAAGCTTGTTGCTCTGGATCTCTGGTGCCTACGGGgaaattgtgttgacccagtctcc-3', downstream 5'-ctagTCTAGAATTAACACTCTCCCCTG-3'), digested with XbaI/HindIII, cloned into PIgG vector (supplied by Scripps research USA) (Christoph radiogram, Mikhail Popkov, John. Neves, and Carlos F. Barbas III. Integrin α v β -targeted therapy for Kaposi. ssarcomaw with an in vitro-expressed antibody FAS EB Journal, (October 18,2002)10.1096/fj.02-0281 fje), amplified with primers (upstream 5'-gagGAGCTCACTCCgaggtgcagctgttggagtctgggggaggcttggtac-3', downstream 5'-gagGGGCCCTTGGTGGAGGCTGAGGAGACGGT-3') and digested with SacI/ApaI into Fab antibody expression vector.
1.1.2 transfection: using a transfection kit available from Invitrogen, USA, the procedure is outlined below: after 5. mu.g of recombinant plasmid DNA was mixed with the transfection reagent, 293T cells with a growth density of 70% were transfected with 5% CO at 37 ℃2And (5) culturing.
1.1.3 Whole antibody IgG purification: after 3 days of culture, the supernatant was collected and the expression supernatant was directly purified using Protein-A affinity chromatography column purchased from Amersham (Harlow E, Lane D.antibodies: A Laboratory Manual [ M ]. New York: Cold Spring Harbor Laboratory Press, 1988). The functional properties of the purified IgG antibodies obtained were characterized by ELISA and IFA.
The SDS-PAGE electrophoresis results of the constructed whole antibody IgG are shown in FIG. 3.
1.2 Whole antibody IgG neutralization assay
To further verify the neutralizing activity of whole antibody IgG, neutralizing activity of antibody was detected in RD cells using strain EV71 virus FUYANG-0805 as challenge strain, and the results are shown in fig. 4.
The experimental steps are as follows: neutralizing activity of the antibody was detected in RD cells using EV71 virus Fuyang-0805 strain as challenge strain.
50 μ l of 100TCID50EV71 virus (Fuyang-0805 strain) and 50. mu.l of antibody diluted in two fold (prepared in example 3) were incubated at 37 ℃ for 2 hours, and then added at a density of 2X 105Pieces/ml of cell suspension in 100. mu.l. The observation was carried out for 7 days, and the cytopathic results were recorded.
The EV71 virus (Fuyang-0805 strain) used was treated as follows: the viral particles prepared in example 1 were diluted to 10-fold gradient-1To 10-10Virus solutions are added into the cell plate respectively, 50 mu l of virus solution is added into each hole, and 4 holes of virus solution are added into each dilution; adding 50 μ l of cell suspension into each well, setting cell control (50 μ l of diluent +50 μ l of cell suspension), culturing at 37 deg.C for 7d, and observing cytopathy; calculating TCID of separated virus strain according to Karber formula50;log TCID50L-d (S-0.5). Wherein: l ═ log of the lowest dilution used in the experiment; d is the log of the dilution gradient; s is the sum of the positive fractions at the end of the call (i.e. the sum of the proportion of the cell pores in which CPE appears).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of pathogenic biology of Chinese academy of medical sciences
<120> humanized anti-EV 71 virus neutralizing antibody E1 and application thereof
<130> KHP171113303.0
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 109
<212> PRT
<213> amino acid sequence of light chain variable region of antibody E1
<400> 1
Glu Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val
1 5 10 15
Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp
20 25 30
Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asn Tyr Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala
65 70 75 80
Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu Ser
85 90 95
Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 2
<211> 116
<212> PRT
<213> amino acid sequence of heavy chain variable region of antibody E1
<400> 2
Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
1 5 10 15
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His Trp
20 25 30
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn
35 40 45
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val
50 55 60
Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser
65 70 75 80
Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Glu
85 90 95
Trp Gln Leu Asp Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 3
<211> 327
<212> DNA
<213> variable region nucleic acid sequence of antibody E1 light chain
<400> 3
gagctcacgc agccgccctc agtgtctggg gccccagggc agagggtcac catctcctgc 60
actgggagca gctccaacat cggggcaggt tatgatgtac actggtacca gcagcttcca 120
ggaacagccc ccagactcct catctataat tacagcaatc ggccctcagg ggtccctgac 180
cgattctctg gctccaagtc tggcacctca gcctccctgg ccatcactgg gctccaggct 240
gaggatgagg ctgattatta ctgccagtcc tatgacagca gcctgagtgg ttcggtattc 300
ggcggaggga ccaagctgac cgtccta 327
<210> 4
<211> 348
<212> DNA
<213> antibody E1 heavy chain variable region nucleic acid sequence
<400> 4
ctcgagtctg gggctgaggt gaagaagcct ggggcctcag tgaaggtctc ctgcaaggct 60
tctggataca ccttcaccgg ctactatatg cactgggtgc gacaggcccc tggacaaggg 120
cttgagtgga tgggatggat caaccctaac agtggtggca caaactatgc acagaagttt 180
cagggcaggg tcaccatgac cagggacacg tccatcagca cagcctacat ggagctgagc 240
aggctgagat ctgacgacac ggccgtgtat tactgtgcga gaggagaatg gcagttggac 300
gggtggttcg acccctgggg ccagggaacc ctggtcaccg tctcctca 348

Claims (11)

1.The human anti-EV 71 virus neutralizing antibody E1 is characterized in that the amino acid sequences of the light chain hypervariable regions CDR1, CDR2 and CDR3 of the antibody E1 are as follows: SSNIGAGYD, NYS and QSYDSSLSGSV, the amino acid sequences of the hypervariable regions CDR1, CDR2 and CDR3 of the heavy chain of the antibody E1 are: GYTFTGYY, inpsggt and ARGEWQLDGWFDP.
2. The antibody E1 of claim 1, wherein the amino acid sequence of its light chain variable region is represented by SEQ ID No.1 and the amino acid sequence of its heavy chain variable region is represented by SEQ ID No. 2; or,
an antibody comprising Arg at position 15 of the sequence shown in SEQ ID No.1 substituted with Lys and Lys at position 8 of the sequence shown in SEQ ID No.2 substituted with Ala.
3. A gene encoding the antibody E1 of claim 2.
4. The gene of claim 3, wherein the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are shown in SEQ ID No.3 and SEQ ID No.4, respectively.
5. An expression cassette, an expression vector or a cloning vector comprising a nucleic acid comprising the gene sequence of claim 3 or 4.
6. A host cell comprising the gene of claim 3 or 4, or the expression cassette or vector of claim 5.
7. The single chain antibody ScFv or Fab antibody or whole antibody IgG which is obtained by modifying the antibody E1 of claim 1 or 2.
8. Use of the antibody E1 of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of hand-foot-and-mouth disease caused by EV71 virus.
9. Use of the antibody E1 of claim 1 or 2 in preparation of EV71 virus antigen detection reagent or detection kit.
10. A detection reagent or a detection kit comprising the antibody E1 according to claim 1 or 2.
11. A medicament comprising the antibody E1 of claim 1 or 2.
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