CN114957479B - Anti-H1N 1 influenza virus bispecific neutralizing antibody Bis-Hu11-1 and application thereof - Google Patents
Anti-H1N 1 influenza virus bispecific neutralizing antibody Bis-Hu11-1 and application thereof Download PDFInfo
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- CN114957479B CN114957479B CN202210600393.4A CN202210600393A CN114957479B CN 114957479 B CN114957479 B CN 114957479B CN 202210600393 A CN202210600393 A CN 202210600393A CN 114957479 B CN114957479 B CN 114957479B
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Abstract
The invention discloses a bispecific neutralizing antibody Bis-Hu11-1 for resisting H1N1 influenza virus and application thereof, belonging to the technical field of genetic engineering antibodies. The invention uses an antibody ZJU11-01 targeting an antigen site Ca2 of an influenza H1N1 virus hemagglutinin protein and an antibody ZCMU-H1N1 targeting an antigen site Sb of an influenza H1N1 virus hemagglutinin protein as parent antibodies, and uses a gene engineering technology to exchange a bispecific IgG antibody Fab arm functional domain (CH 1-CL) according to a cross mab platform, so that a mammalian eukaryotic expression system expresses Bis-Hu11-1. The invention also provides a method for expressing and purifying the bispecific neutralizing antibody Bis-Hu11-1, and obtaining the target protein by means of CHO-S cell secretion expression and affinity chromatography purification. The bispecific neutralizing antibody Bis-Hu11-1 can be specifically combined with antigenic sites Ca2 and Sb of H1N1 influenza virus hemagglutinin protein at the same time, and the H1N1 influenza virus is inhibited by neutralization. The invention provides an effective tool for preventing and treating H1N1 subtype seasonal influenza virus infection, and can be popularized and applied to combined application with other medicines and other researches.
Description
Technical Field
The invention belongs to the technical field of genetic engineering antibodies, and particularly relates to a novel bispecific neutralizing antibody Bis-Hu11-1 for resisting H1N1 influenza virus, which can simultaneously target H1N1 influenza virus hemagglutinin protein antigenic sites Sb and Ca2, and application thereof. It can be used for preparing medicines for treating or preventing influenza A H1N1 virus infection.
Background
Seasonal influenza virus epidemics remain a public health concern of global public concern. Most humans are not immune to influenza a H1N1 virus and thus may result in vaccine inefficiency and pandemic when seasonal vaccines do not match the current year's epidemic strain. To date, H1N1 still causes an increase in death cases worldwide. The early symptoms of the traditional Chinese medicine are similar to those of common influenza, the patients are easy to be overlooked, the disease conditions can progress rapidly, the patients with sudden high fever and pneumonia can suffer from respiratory failure, multiple organ injury and even death, and the death rate can reach 6%.
The antiviral drugs for clinically treating influenza are mainly oseltamivir and amantadine at present, but researches have shown that the drug resistance rate of H1N1 influenza A virus to amantadine is over 90%. In addition, it was found in clinical studies that influenza virus developed drug resistance mutations during drug exposure, resulting in drug resistance to oseltamivir. Because of the increased rate of resistance and the limited therapeutic window (which must be administered within 48 hours after symptoms appear), it is imperative to seek new preventive and therapeutic interventions against influenza. Monoclonal antibodies are widely used because of their high specificity, limited off-target effects and good safety. Compared with monoclonal antibodies, bispecific antibodies have increased a specific antigen binding site, have stronger specificity, more accurate targeting, and lower off-target toxicity.
Based on the background, the invention designs a bispecific neutralizing antibody Bis-Hu11-1 for simultaneously targeting the H1N1 influenza virus hemagglutinin protein antigenic site Ca2 and Sb according to the cross mab platform, one end ZJU11-01 of the bispecific neutralizing antibody can identify the H1N1 influenza virus hemagglutinin protein antigenic site Ca2, and the other end ZCMU-H1N1 can identify the H1N1 influenza virus hemagglutinin protein antigenic site Sb, so that the virus is inhibited from entering cells through virus neutralization, and meanwhile, immune escape of the H1N1 influenza virus in treatment is avoided, and the antiviral effect is enhanced. The bispecific neutralizing antibody Bis-Hu11-1 can be combined with two different surface antigens, so that the combination specificity is increased, side effects caused by off-target and the like are reduced, and a new inspiration and thinking are provided for preventing and treating H1N1 influenza.
Disclosure of Invention
The invention aims to provide a bispecific neutralizing antibody Bis-Hu11-1 capable of simultaneously and specifically targeting H1N1 influenza virus hemagglutinin protein antigenic sites Sb and Ca2 for preventing and treating H1N1 influenza virus infection. The bispecific neutralizing antibody comprises variable region sequences of two parent full-length antibodies of an anti-H1N 1 influenza virus hemagglutinin protein antigen site Ca2 and an anti-H1N 1 influenza virus hemagglutinin protein antigen site Sb; the amino acid sequence of the heavy chain of the anti-H1N 1 influenza virus hemagglutinin protein antigen site Ca2 parent antibody ZJU11-01 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain of the anti-H1N 1 influenza virus hemagglutinin protein antigen site Ca2 parent antibody ZJU11-01 is shown as SEQ ID NO. 3; the amino acid sequence of the heavy chain of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO. 7.
The nucleotide sequence of the heavy chain of the anti-H1N 1 influenza virus hemagglutinin protein antigen site Ca2 parent antibody ZJU11-01 is shown as SEQ ID NO.2, and the nucleotide sequence of the light chain of the anti-H1N 1 influenza virus hemagglutinin protein antigen site Ca2 parent antibody ZJU11-01 is shown as SEQ ID NO. 4; the nucleotide sequence of the heavy chain of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO.6, and the nucleotide sequence of the light chain of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO. 8.
The sequence is as follows:
SEQ ID NO.1
Heavy chain:Amino acid sequence(124AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 EVQLQQSGAELVKPGASVKLSCTPSGFNIKDTYMHWVKQRPEQGLEWIGRIAPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCADGFYYYNTNYRDYFDHWGQGTTLTVSS
SEQ ID NO.2
Heavy chain:DNA sequence(372bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACACCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCC TGGAGTGGATTGGAAGGATTGCTCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG ACACTGCCGTCTATTACTGTGCTGACGGATTCTATTACTACAATACTAACTACAGAGACTACTTTGACCACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
SEQ ID NO.3
Light chain:Amino acid sequence(111AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ENVLTQSPASLAVSLGQRATISCKASQSVLYDGDNYMNWYQQKPGQSPKLLIYAASNLQSGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK
SEQ ID NO.4
Light chain:DNA sequence(333bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 GAAAATGTGCTCACCCAGTCTCCAGCCTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTCTTTATGATGGTGATAATTACATGAACTGGTACCAACAGAAACCAGGAC AGTCACCCAAACTCCTCATCTATGCTGCATCCAATCTACAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTAC TGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
SEQ ID No.5
Heavy chain:Amino acid sequence(120AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTYTGEPTYDDHFKGRF AFSLETSASTAYLQINNLKNEDMATYFCAREDNYAPSWFTHWGQGTLVTVSA
SEQ ID No.6
Heavy chain:DNA sequence(360bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATACACCTTCACAAATTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTT AAGGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGATGATCATTTTAAGGGACGATTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGAC ATGGCTACATATTTCTGTGCAAGGGAGGATAATTACGCCCCTTCCTGGTTTACTCACTGGGGCCAAGGG ACTCTGGTCACTGTCTCTGCA
SEQ ID No.7
Light chain:Amino acid sequence(108AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 QIVLSQSPTTMAASPGEKITITCSASSSISSNYLHWYQQRPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGHSIPYTFGGGTKLEIK
SEQ ID No.8
Light chain:DNA sequence(324bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 CAAATTGTTCTCTCCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAGGCCAGGATTCTCCCCTAA ACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTG GGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTCATAGTATACCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
the second object of the invention is to provide a preparation method of a bispecific neutralizing antibody Bis-Hu11-1, which is realized by the following steps and technical schemes:
(1) The mouse monoclonal antibody ZJU11-01 targeting the H1N1 influenza virus hemagglutinin protein antigenic site Ca2 and the mouse monoclonal antibody ZCMU-H1N1 targeting the H1N1 influenza virus hemagglutinin protein antigenic site Sb are obtained through hybridoma technology screening.
(2) The heavy and light chain variable region genes of murine monoclonal antibodies ZJU11-01 and ZCMU-H1N1 were sequenced.
(3) The bispecific neutralizing antibody Bis-Hu11-1 is constructed by adopting a molecular biological method: the light and heavy chain variable region amino acid sequences of ZJU11-01 and ZCMU-H1N1 are spliced with the light and heavy chain constant region amino acid sequence of the human IgG1 antibody to obtain four heavy and light chain sequences of the bispecific antibody, and cloning and recombination are carried out at the same time to construct the bispecific neutralizing antibody Bis-Hu11-1 recombinant vector. And (3) transforming the target plasmid into a competent cell of escherichia coli DH5 alpha, plating, selecting positive monoclonal to perform sequencing comparison, and selecting a bacterial protection with correct sequencing result to perform amplification and preservation.
(4) The four objective plasmids are co-transfected with CHO-S cells for expression, culture supernatant is obtained through low temperature centrifugation, antibodies in the supernatant are separated and purified by utilizing a Protein A affinity purification method, and whether expression products are expressed and assembled correctly is identified through polyacrylamide gel electrophoresis.
(5) The invention obtains a bispecific neutralizing antibody for resisting H1N1 influenza virus, namely Bis-Hu11-1, and analyzes the affinity of the antibody and antigen through ELISA experiments, and analyzes the antiviral ability of the antibody at the cellular level through in vitro neutralization experiments, and simultaneously tests the prevention and treatment effects of the antibody in mice. The results show that Bis-Hu11-1 can effectively bind to and neutralize H1N1 influenza virus.
The invention discloses a bispecific neutralizing antibody Bis-Hu11-1 for resisting H1N1 influenza virus and application thereof, belonging to the technical field of genetic engineering antibodies. The invention uses an antibody ZJU11-01 targeting an antigen site Ca2 of an influenza H1N1 virus hemagglutinin protein and an antibody ZCMU-H1N1 targeting an antigen site Sb of an influenza H1N1 virus hemagglutinin protein as parent antibodies, and uses a gene engineering technology to exchange a bispecific IgG antibody Fab arm functional domain (CH 1-CL) according to a cross mab platform, so that a mammalian eukaryotic expression system expresses Bis-Hu11-1. The invention also provides a method for expressing and purifying the bispecific neutralizing antibody Bis-Hu11-1, and obtaining the target protein by means of CHO-S cell secretion expression and affinity chromatography purification. The bispecific neutralizing antibody Bis-Hu11-1 can be specifically combined with antigenic sites Ca2 and Sb of H1N1 influenza virus hemagglutinin protein at the same time, and the H1N1 influenza virus is inhibited by neutralization. The invention provides an effective tool for preventing and treating H1N1 subtype seasonal influenza virus infection, and can be popularized and applied to combined application with other medicines and other researches.
Two parent monoclonal antibodies ZJU11-01 and ZCMU-H1N1 of the bispecific neutralizing antibody, wherein one end ZJU11-01 is characterized by being capable of specifically targeting an antigen site Ca2 of hemagglutinin protein of H1N1 influenza virus; the other end ZCMU-H1N1 can specifically target the H1N1 influenza virus hemagglutinin protein antigenic site Sb. Humanized bispecific antibody by using human IgG1 antibody as skeleton to reduce possible immunogenicity caused by murine antibody, and exchange light chain CL and heavy chain CH1 of ZCMU-H1N1 with Crossover to avoid heterogenous mismatch during the assembly process of bispecific antibody, and the structure of the antibody itself is not changed to maintain the affinity of the antibody and antigen; the mammalian cell expression system performs protein expression, guaranteeing the biological activity of the bispecific antibody.
It is another object of the present invention to provide the bispecific neutralizing antibody Bis-Hu11-1 capable of efficiently binding to, neutralizing H1N1 influenza virus and methods of use thereof.
Application of bispecific neutralizing antibody Bis-Hu11-1 for resisting H1N1 influenza virus in preparing medicine for preventing and treating H1N1 seasonal influenza virus is provided.
The invention has the advantages that the bispecific neutralizing antibody Bis-Hu11-1 for resisting the H1N1 influenza virus is provided, the antiviral effect of the antibody is verified in cells and animals, and a new reference scheme for preventing and treating the H1N1 influenza virus is provided.
Drawings
FIG. 1 is a schematic diagram showing the construction of a gene expression vector for the bispecific neutralizing antibody Bis-Hu 11-1.
FIG. 2 is a diagram showing the purification and identification of the expression of the bispecific neutralizing antibody Bis-Hu 11-1.
FIG. 3 shows the in vitro neutralization assay of the bispecific neutralizing antibody Bis-Hu 11-1.
FIG. 4 shows the in vivo preventive effect of the bispecific neutralizing antibody Bis-Hu 11-1.
FIG. 5 shows the in vivo therapeutic effect of the bispecific neutralizing antibody Bis-Hu 11-1.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1 construction of bispecific neutralizing antibody Bis-Hu11-1
(1) The mouse monoclonal antibody ZJU11-01 targeting the H1N1 influenza virus hemagglutinin protein antigen site Ca2 and the mouse monoclonal antibody ZCMU-H1N1 targeting the H1N1 influenza virus hemagglutinin protein antigen site Sb are obtained through hybridoma technology screening: ① Selecting BALB/C mice of 6 weeks of age, and immunizing the mice with purified H1N1 influenza virus hemagglutinin protein; ② Mice prepared in ① were sacrificed and spleen lymphocytes were obtained, and the mouse spleen lymphocytes were cell-fused with mouse myeloma cells by polyethylene glycol fusion. Properly diluting the fused cells, and inoculating the cells to a feeder cell culture plate for culture; ③ The above culture was cultured in a selective medium for hypoxanthine-phosphoribosyl transferase. When the cell colony grows to a proper size, sucking the cell culture supernatant for antibody identification, and screening positive clones; ④ Cloning hybridoma cells by a limiting dilution method, and taking culture supernatant to perform an enzyme-linked immunosorbent assay to identify positive clones. Limiting dilution cloning is repeated for a plurality of times until the positive porosity of the hybridoma cells reaches 100%. And (3) performing antibody identification and physicochemical property analysis on the cloned hybridoma cells by amplification culture. ⑤ Inoculating positive hybridoma cells into BALB/C mice, injecting paraffin oil into the abdominal cavity of the mice, 5×10 6 of positive hybridoma cells are inoculated into the mice, ascites are collected after 10 days for centrifugation, antibody titer is measured, and monoclonal antibodies are purified; ⑧ Monoclonal antibodies in ascites were purified by Protein G affinity purification.
(2) The heavy and light chain variable region genes of murine monoclonal antibodies ZJU11-01 and ZCMU-H1N1 were sequenced: ① 5X 10 6 target monoclonal hybridoma cells were collected, and RNA was extracted after cleavage with 1 ml Trizol; ② Synthesizing cDNA by using the extracted RNA as a template through a reverse transcription kit; ③ PCR amplification is performed using the cDNA obtained by reverse transcription as a template by specific primers, and the amplified fragment is inserted into an expression vector and sequenced. The amino acid sequence of the heavy chain of a parent antibody ZJU11-01 of the bispecific neutralizing antibody against the H1N1 influenza virus hemagglutinin protein antigen site Ca2 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain of the parent antibody ZJU11-01 of the bispecific neutralizing antibody against the H1N1 influenza virus hemagglutinin protein antigen site Ca2 is shown as SEQ ID NO. 3; the amino acid sequence of the heavy chain of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO. 7.
(3) Design and Synthesis of bispecific neutralizing antibody Bis-Hu11-1 four chains: ① The heavy chain variable region of ZJU11-01 and the heavy chain constant region (CH1+CH2+CH3) of human IgG1, the light chain variable region of ZJU11-01 and the light chain constant region (CL) of human IgG1, the heavy chain variable region of ZCMU-H1N1, the light chain constant region (CL) of human IgG1 and the heavy chain constant region (CH2+CH3) are connected by an overlap PCR technique, and the light chain variable region of ZJU11-01 and the heavy chain constant region (CH 1) of human IgG1 are constructed into a bispecific neutralizing antibody Bis-Hu11-1 heavy light chain gene; ② Cloning target genes into eukaryotic expression vectors pcDNA 3.4 respectively; ③ Respectively transforming plasmids into E.coli DH5 alpha competent cells, coating the transformed bacteria liquid on an LB plate, and culturing the transformed bacteria liquid in an incubator at 37 ℃ for overnight in an inverted manner; ④ 10 single clones are randomly selected and added into 4 milliliters of liquid LB culture medium respectively, shake amplification culture is carried out for 8 hours at 37 ℃,2 microliter bacterial liquid is absorbed for bacterial liquid PCR and sequencing is carried out to identify and screen recombinant plasmid DNA; ⑤ Performing amplification culture on bacterial liquid with correct sequencing, and preserving; ⑥ The plasmids are extracted by a plasmid extraction kit respectively to obtain four groups of recombinant plasmids pcDNA 3.4-ZJU11-01-H/L-Chain and pcDNA 3.4-ZCMU-H1N1-H/L-Chain.
The results are shown in FIG. 1.
Example 2 purification and identification of expression of bispecific neutralizing antibody Bis-Hu11-1
The recombinant plasmid expressing the four chains of the bispecific neutralizing antibody Bis-Hu11-1 is co-transfected, and the expression cells are CHO-S cells.
(1) Linearization of transfected plasmids: linearizing the constructed four groups of recombinant plasmids, carrying out enzyme digestion at 37 ℃ overnight, recovering plasmids, detecting the linearization degree of plasmid DNA by 1% agarose gel electrophoresis, detecting the concentration of the plasmid DNA by an ultraviolet spectrophotometer, and preserving at-20 ℃ for later use.
(2) And (3) cell subculture: CHO-S cells were cultured in FreeStyle TM CHO expression medium at 37 ℃,8% co 2, and the cells were grown to log phase for use.
(3) Four recombinant plasmids pcDNA 3.4-ZJU11-01-H/L-Chain and PcDNA 3.4-ZCMU-H1N1-H/L-Chain are co-transfected into CHO-S cells according to the ratio of 1:1:1:1 by electrotransfection method, liquid is changed the next day and the cells are passaged, the cell culture scale is gradually enlarged, the cell culture liquid is collected, a 0.22 micrometer filter membrane is used for filtering a sample, and then Protein A column affinity chromatography is carried out for purification, so that the target Protein is finally obtained in large quantity. And respectively carrying out 8% non-reduction and 12% reduction polyacrylamide gel electrophoresis to identify the molecular weight and assembly condition of the target protein, and carrying out preliminary verification on the target protein.
The results are shown in FIG. 2, and the bispecific neutralizing antibody Bis-Hu11-01 was successfully expressed and assembled correctly.
Example 3 bispecific neutralizing antibody Bis-Hu11-1 antiviral Effect
(1) Micro neutralization experiment: ① Half the histiocyte infection dose (TCID 50) titration was performed with H1N1 influenza virus (A/Michigan/45/2015); ② MDCK cells are inoculated in a 96-well culture plate, 2 multiplied by 10 4 cells are cultured in an incubator containing 5 percent of carbon dioxide saturated at 37 ℃ for 24 hours; ③ Diluting the virus with a virus culture medium containing 0.2% pancreatin to 100TCID50 per 50 μl; ④ 10 micrograms per milliliter of monoclonal antibody ZJU11-01 was diluted to different concentrations (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:521) with a virus broth doubling ratio in 96 well plates, 50 microliters per well; ⑤ 50 microliters of 100TCID50 per 50 microliters of virus solution are added into the wells with the antibodies, and the mixture is uniformly mixed, and 4 compound wells are formed at each dilution; penultimate row as virus back drop, virus from 100TCID50 per 100 microliter multiple dilution (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128), per well 100 microliters; the last column served as control, 4 wells served as negative cell control (100. Mu.l of virus culture medium was added per well) and 4 wells served as positive cell control (100. Mu.l of 100TCID50 per 100. Mu.l of virus solution was added per well), incubated for 2 hours in an incubator with 5% carbon dioxide saturation at 37 ℃; ⑥ Taking out the prepared 96-well MDCK cell culture plate, washing cells for 1 time by using phosphate buffer solution, transferring the prepared liquid in the 96-well plate in ⑤ into the cell culture plate, and incubating in an incubator with 5% carbon dioxide saturation at 37 ℃ for 2 hours; ⑦ Taking out the 96-well cell plate, and washing the cells with phosphate buffer solution for 2 times; 200 microliters of virus culture solution is added to each well, and the mixture is incubated in an incubator containing 5% carbon dioxide and saturated at 37 ℃ for 72 hours; ⑧ Taking 96-well cell plates after 72 hours of culture, taking 50 microliters of culture supernatant from each well, transferring to a hemagglutination plate, and adding 50 microliters of 1% chicken red blood cells into each well of the hemagglutination plate; ⑨ After 30 minutes the results were observed.
The result is shown in figure 3, and bis-Hu11-1 has good in vitro neutralization effect on H1N1 influenza virus
(2) Mouse prevention experiment: ① Half-lethal titration of H1N1 influenza virus (A/Michigan/45/2015) mice; ② Grouping mice: female BALB/C mice 7 weeks old, 5 in each group, five in total, numbered first to fifth respectively; ③ Each mouse was weighed and recorded; ④ The first and third groups of mice were intraperitoneally injected with 3 mg of the bispecific neutralizing antibody Bis-Hu11-1 per kg body weight, the second and fourth groups of mice were intraperitoneally injected with 30 mg of the bispecific neutralizing antibody Bis-Hu11-1 per kg body weight, and the fifth group was injected with 50 μl of phosphate buffer; ⑤ The H1N1 influenza virus was diluted to 5-fold half-lethal dose per 50. Mu.l, and the first, second, and fifth groups were inoculated with H1N1 influenza virus by intranasal administration for 6 hours after injection of the bispecific neutralizing antibody Bis-Hu11-1 or phosphate buffer, 50. Mu.l each; ⑥ The third and fourth groups were inoculated with H1N1 influenza virus by intranasal administration for 48 hours, 50 μl each; ⑥ Body weight was observed and recorded daily.
As a result, as shown in FIG. 4, the bispecific neutralizing antibody Bis-Hu11-1 can effectively prevent the infection of H1N1 influenza virus in mice, and can achieve 100% protection efficiency at a concentration of 30 mg per kg body weight.
(3) Mice treatment experiments: ① Grouping mice: 7 week old female BALB/C mice, 5 in each group, seven in total, numbered first to seventh groups, respectively; ② Each mouse was weighed and recorded; ② Diluting the H1N1 influenza virus to 10-fold half lethal dose per 50 microliters, and intranasally inoculating all mice in the first group to the seventh group with the H1N1 influenza virus, 50 microliters each; ③ 6 hours after infection, the first, second and third groups of mice were intraperitoneally injected with 3, 10 and 30 milligrams of bispecific neutralizing antibody Bis-Hu11-1 per kilogram of body weight, respectively, and the seventh group was intraperitoneally injected with 50 microliters of phosphate buffer; ④ 48 hours after infection, the fourth, fifth and sixth groups of mice were injected intraperitoneally with 3, 10 and 30 milligrams of bispecific neutralizing antibody Bis-Hu11-1 per kilogram of body weight, respectively; ⑤ Body weight was observed and recorded daily.
As a result, as shown in FIG. 5, the bispecific neutralizing antibody Bis-Hu11-1 can effectively treat H1N1 influenza virus infection in mice, and the treatment effect is closely related to the treatment time, and can still achieve 100% protection efficiency after 48 hours of infection at a concentration of 30 milligrams per kilogram of body weight.
It is to be understood that the present application has been described in conjunction with the preferred embodiments thereof, and that upon reading the foregoing, various changes and modifications may be made by one skilled in the art, and that these equivalents will fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Zhejiang university medical college affiliated first hospital
<120> Bispecific neutralizing antibody Bis-Hu11-1 against H1N1 influenza virus and use thereof
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 124
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Pro Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Ala Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asp Gly Phe Tyr Tyr Tyr Asn Thr Asn Tyr Arg Asp Tyr Phe Asp
100 105 110
His Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 2
<211> 372
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
gaggttcagc tgcagcagtc tggggcagag cttgtgaagc caggggcctc agtcaagttg 60
tcctgcacac cttctggctt caacattaaa gacacctata tgcactgggt gaagcagagg 120
cctgaacagg gcctggagtg gattggaagg attgctcctg cgaatggtaa tactaaatat 180
gacccgaagt tccagggcaa ggccactata acagcagaca catcctccaa cacagcctac 240
ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtgc tgacggattc 300
tattactaca atactaacta cagagactac tttgaccact ggggccaagg caccactctc 360
acagtctcct ca 372
<210> 3
<211> 111
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 3
Glu Asn Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Leu Tyr Asp
20 25 30
Gly Asp Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 4
<211> 333
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
gaaaatgtgc tcacccagtc tccagcctct ttggctgtgt ctctagggca gagggccacc 60
atctcctgca aggccagcca aagtgttctt tatgatggtg ataattacat gaactggtac 120
caacagaaac caggacagtc acccaaactc ctcatctatg ctgcatccaa tctacaatct 180
gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240
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<210> 5
<211> 120
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Asp Asp His Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Glu Asp Asn Tyr Ala Pro Ser Trp Phe Thr His Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 6
<211> 360
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgcaagg cttctggata caccttcaca aattatggaa tgaactgggt gaagcaggct 120
ccaggaaagg gtttaaggtg gatgggctgg ataaacacct acactggaga gccaacatat 180
gatgatcatt ttaagggacg atttgccttc tctttggaaa cctctgccag cactgcctat 240
ttgcagatca acaacctcaa aaatgaggac atggctacat atttctgtgc aagggaggat 300
aattacgccc cttcctggtt tactcactgg ggccaaggga ctctggtcac tgtctctgca 360
<210> 7
<211> 108
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 7
Gln Ile Val Leu Ser Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly
1 5 10 15
Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn
20 25 30
Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Phe Ser Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu
65 70 75 80
Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly His Ser Ile Pro
85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 8
<211> 324
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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caaattgttc tctcccagtc tccaaccacc atggctgcat ctcccgggga gaagatcact 60
atcacctgca gtgccagctc aagtataagt tccaattact tgcattggta tcagcagagg 120
ccaggattct cccctaaact cttgatttat aggacatcca atctggcttc tggagtccca 180
gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaattgg caccatggag 240
gctgaagatg ttgccactta ctactgccag cagggtcata gtataccata cacgttcgga 300
ggggggacca agctggaaat aaaa 324
Claims (6)
1. A bispecific neutralizing antibody Bis-Hu11-1 for resisting H1N1 influenza virus, which is characterized by comprising variable region sequences of two parent full-length antibodies of an antigen site Ca2 of hemagglutinin protein of the anti-H1N 1 influenza virus and an antigen site Sb of hemagglutinin protein of the anti-H1N 1 influenza virus; the amino acid sequence of the heavy chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Ca2 parent antibody ZJU11-01 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Ca2 parent antibody ZJU11-01 is shown as SEQ ID NO. 3; the amino acid sequence of the heavy chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO. 7.
2. The preparation method of the bispecific neutralizing antibody Bis-Hu11-1 as claimed in claim 1 is realized by the following steps and technical scheme:
(1) The bispecific neutralizing antibody Bis-Hu11-1 is constructed by adopting a molecular biological method: splicing the light and heavy chain variable region amino acid sequences of ZJU11-01 and ZCMU-H1N1 with the light and heavy chain variable region constant region amino acid sequence of the human IgG1 antibody to obtain four heavy and light chain variable region sequences of the bispecific antibody, and performing cloning recombination to construct a bispecific neutralizing antibody Bis-Hu11-1 recombinant vector; transforming the target plasmid into competent cells of the escherichia coli DH5 alpha, coating a plate, selecting positive monoclonal to perform sequencing comparison, and selecting the escherichia coli DH5 alpha with correct sequencing result to amplify and store;
(2) The four objective plasmids are co-transfected with CHO-S cells for expression, culture supernatant is obtained through low-temperature centrifugation, antibodies in the supernatant are separated and purified by using a Protein A affinity purification method, and whether expression and assembly of an expression product are correct or not is identified through polyacrylamide gel electrophoresis;
(3) The bispecific neutralizing antibody of the anti-H1N 1 influenza virus is obtained, namely Bis-Hu11-1, the affinity of the antibody with an antigen is analyzed through ELISA experiments, the antiviral capacity of the antibody at the cellular level is analyzed through in vitro neutralizing experiments, and meanwhile, the prevention and treatment effects of the antibody are tested in mice.
3. A nucleic acid molecule encoding a bispecific neutralizing antibody Bis-Hu11-1 according to claim 1, wherein the nucleotide sequence of the heavy chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Ca2 parent antibody ZJU11-01 is shown in SEQ ID No.2, and the nucleotide sequence of the light chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Ca2 parent antibody ZJU11-01 is shown in SEQ ID No. 4; the nucleotide sequence of the heavy chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO.6, and the nucleotide sequence of the light chain variable region of the anti-H1N 1 influenza virus hemagglutinin protein antigenic site Sb parent antibody ZCMU-H1N1 is shown as SEQ ID NO. 8.
4. An expression vector, which is derived from eukaryotic expression vector pcDNA3.4, comprises the nucleic acid molecule of claim 3, and can transfect eukaryotic cells CHO-S and express protein.
5. Use of the bispecific neutralizing antibody Bis-Hu11-1 against H1N1 influenza virus according to claim 1 for the preparation of a medicament for the prophylaxis of H1N1 seasonal influenza virus.
6. Use of the bispecific neutralizing antibody Bis-Hu11-1 against H1N1 influenza virus according to claim 1 for the preparation of a medicament for the treatment of H1N1 seasonal influenza virus.
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| "Influenza Human Monoclonal Antibody 1F1 Interacts with Three Major Antigenic Sites and Residues Mediating Human Receptor Specificity in H1N1 Viruses";Tshidi Tsibane 等;《Plos pathogens》;第8卷(第12期);第1-9页 * |
| "甲型流感病毒广谱中和抗体研究进展";陈雪晴 等;《江苏预防医学》;第28卷(第2期);第169-172页 * |
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