CN102850454A - Anti-RSV (respiratory syncytial virus) human monoclonal antibody, and its preparation method - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a virus monoclonal antibody and a preparation method thereof, more specifically to a human monoclonal antibody against respiratory syncytial virus RSV and a preparation method thereof. The antibody can be used to enhance the tolerance of human subjects against RSV infection, reduce the infection level in infected individuals or alleviate the symptoms caused by RSV infection, and the dosage is lower than that of current marketed products.

Description

Anti-respiratory syncytial virus human monoclonal antibody and preparation method thereof

technical field

The invention relates to the field of biotechnology, in particular to a virus monoclonal antibody and a preparation method thereof, more specifically to a human monoclonal antibody against respiratory syncytial virus RSV and a preparation method thereof. The antibodies can be used to increase the tolerance of human subjects against RSV infection and to reduce the level of infection in infected individuals or to alleviate the symptoms caused by RSV infection. the

Background technique

1. Overview and pathogenic mechanism

Respiratory syncytial virus pneumonia (respiratory syncytial virus pneumonia), referred to as syncytial virus pneumonia, is a negative-strand RNA virus with a capsule, belonging to the genus Pneumovirus of the Paramyxo-viridae family (Fenner, Virology 71 : 371-378, 1975; Huang et al., J. Virol. 43, 1982). RSV is a common interstitial pneumonia in children, which mostly occurs in infants and young children. Since maternally-inherited antibodies cannot prevent the occurrence of infection, the disease can occur in young infants shortly after birth, but it is rare in newborns. There are occasional reports of nosocomial infections leading to outbreaks in neonatal wards of maternity hospitals abroad. the

Respiratory syncytial virus (RSV, referred to as syncytial virus, also belongs to Paramyxoviridae) is the most common pathogen causing viral pneumonia in children, which can cause interstitial pneumonia and bronchiolitis. In Beijing, 48% of viral pneumonia and 58% of bronchiolitis were caused by syncytial virus (1980-1984); in Guangzhou, 31.4% of children's pneumonia and bronchiolitis were caused by syncytial virus (1973-1986) ; In the United States, 20% to 25% of infant pneumonia and 50% to 75% of bronchiolitis are caused by syncytial virus. the

Under the electron microscope, RSV is similar to parainfluenza virus. The size of virus particles is about 150nm, which is slightly smaller than parainfluenza virus. It is an RNA virus, sensitive to ether, and has no hemagglutination. It forms a unique syncytia in human epithelial tissue culture ( syncytium), the virus proliferates in the cytoplasm, and intracytoplasmic inclusion bodies can be seen. There is only one serotype of syncytial virus, and molecular biology methods have recently proved that there are two subtypes. the

The incubation period of syncytial virus infection is 2 to 8 days (mostly 4 to 6 days). A typical finding in syncytial virus pneumonia is an interstitial infiltration of monocytes. The main manifestations are widening of alveolar septa and interstitial infiltration dominated by monocytes, including lymphocytes, plasma cells, and macrophages. In addition, the alveolar cavity is filled with edema fluid, and the formation of hyaline membrane can be seen. In some cases, lymphocytic infiltration of the bronchiolar wall can also be seen. Edema with areas of necrosis occurs in the lung parenchyma, leading to alveolar tamponade, consolidation, and collapse. In a small number of cases, multinucleated fused cells can be seen in the alveolar cavity, which is similar in shape to measles giant cells, but no intranuclear inclusion bodies can be found. the

2. Epidemiology

Syncytial virus infection is very widespread. The results of immunofluorescence assay in Beijing (1978): the positive rate of umbilical cord blood was 93%, 89% from birth to 1 month, 40% from 1 to 6 months, and 70% at 2 and 3 years old. % or more, 80% positive from 4 years old until 14 years old (complement fixation assay is consistent with this). the

Syncytial virus pneumonia can occur at any time after birth because maternally-inherited antibodies do not completely prevent infection from occurring. It is more common in children under 3 years old, and severe cases can be seen in 1 to 6 months, and there are more males than females. It is more common in winter and spring in northern my country, while it is more common in spring and summer in Guangdong. Since antibodies cannot completely prevent infection, syncytial virus reinfection is extremely common. Some people observed that the incidence of reinfection was as high as 65% within 10 years. Syncytial virus is very contagious, and it has been reported that family members have been infected one after another. When it occurs in the family, older children and adults usually have upper respiratory tract infections. The literature reports that the secondary syncytial virus infection rate in the hospital is as high as 30% to 50%. the

Globally, including the United States, Europe, Australia and Japan, RSV infection has long been a major problem. RSV infection is especially troublesome for premature babies, young children and the elderly, as well as for adults with weakened immune systems. It is estimated that at least two-thirds of children under one year of age, and almost all children between one and four years of age, will develop chronic severe infection, a factor that is thought to predispose children to wheezing and asthma-like symptoms later on. the

In the past ten years, syncytial virus pneumonia and bronchiolitis accounted for the first viral pneumonia in infants and young children in my country, and their symptoms were almost indistinguishable clinically from parainfluenza virus pneumonia, mild influenza virus pneumonia and mild adenovirus pneumonia. Severe influenza virus pneumonia and severe adenovirus pneumonia have persistent high fever, severe poisoning symptoms and respiratory symptoms, and the clinical manifestations are far more serious than syncytial virus pneumonia. the

3. Product R&D and development trend

RSV has two major surface glycoproteins, F and G. Two glycoproteins (90KD and 68KD) are exposed on the virion surface. The highly glycosylated G protein of 90KD is responsible for the binding of viral particles to target cells (Walsh et al., J. Gen. Virol. 65:761-767, 1984). The 68KDF F protein mediates fusion of the viral envelope with cells and syncytium formation (Walsh et al., J. Gen. Virol. 66:409-415, 1985). The F and G surface glycoproteins are the main protective antigens, and nucleoprotein N and envelope protein M2 have less protective activity. Neutralizing and fusion-inhibiting monoclonal antibodies have also been identified in specific regions of the F glycoprotein (Walsh et al., Infect, Immun. 43:756-758, 1984; Trudel et al., J.Gen.Virol.68: 2273-80, 1987; Beeler et al., J.Virol.63:2941-50, 1989; Lopez et al., J.Virol.64:927-30, 1990; Paradiso et al., Vaccine 9:231- 7, 1991). Monoclonal antibodies against the G glycoprotein are less likely to neutralize the virus than those against the F glycoprotein and do not have fusion inhibitory activity (Norrby et al., proc. Natl. Acad. Sci. USA 84:6572-6576 , 1987; Garcia-Barreno et al., J. Virol.63: 925-932, 1989; Walsh et al., J. Gen. Virol. 70: 2953-2961, 1989). The amino acid sequence of the F glycoprotein is approximately 90% conserved among RSV subgroups associated with human infection (Toms, FEMS Micro-biol. Immunol. 76:243-256, 1991). the

MedImmune制造)，其作用广谱，但目前每次给药所需帕利珠单抗的剂量为15mg/kg(体重)，由于单克隆抗体生产工艺要求高，且制作昂贵，故目前市售产品价格偏高，在我国难以大批量推广生产。由此，需要一种防止或治疗RSV疾病的有效方法，且提供特异性更高、生产工艺更经济，给药剂量更低，价格更低廉的产品以满足目前广大的社会需求。本发明探索满足此需求。  Efforts in the past have focused on developing live attenuated virus vaccines. However, recent efforts have focused on RSV surface glycoproteins F and G due to poor efficacy, insufficient attenuation, or genetic instability. The only anti-RSV monoclonal antibody currently on the market is only approved for the prevention of RSV infection in premature infants, and it is an antibody against the F protein. The name of the antibody is palivizumab (

Manufactured by MedImmune), it has a broad spectrum of action, but the current dosage of palivizumab required for each administration is 15 mg/kg (body weight). Due to the high requirements for the production process of monoclonal antibodies and the expensive production, the current commercially available products The price is high, and it is difficult to promote production in large quantities in our country. Therefore, it is necessary to provide an effective method for preventing or treating RSV disease, and provide products with higher specificity, more economical production process, lower dosage and lower price to meet the current social needs. The present invention seeks to meet this need.

Contents of the invention

The technical problem to be solved by the present invention is to provide new human monoclonal antibody and preparation method thereof to respiratory syncytial virus RSV fusion protein, namely a kind of new anti-RSV human monoclonal antibody and preparation method thereof, to overcome The above-mentioned defective that prior art exists. the

That is to say, the present invention aims at the deficiencies in the prior art, and through practical research such as a large number of biotechnology experiments, as well as literature search and theoretical research, one of the purposes is to provide a class of anti-RSV human monoclonal antibodies with higher affinity, that is, to provide a class of A low-dose high-efficiency monoclonal antibody for treating RSV diseases, especially a human monoclonal antibody that can be used for preventing and/or treating RSV diseases, which is specific to the F glycoprotein of RSV. Preferably, the monoclonal antibody is in substantially pure form, free of other immunological substances. the

The present invention provides an anti-RSV human monoclonal antibody, which includes a human constant region, its heavy chain variable region is selected from SEQ ID NO: 1; its light chain variable region is selected from SEQ ID NO: 2. Preferably, the homology between the monoclonal antibody and the sequence is 98% or 95%. the

In another aspect, the invention provides compositions comprising one or more monoclonal antibodies and a suitable carrier or diluent. the

The present invention also provides a composition comprising the cell line, the composition comprising the cell line and a nutrient medium capable of maintaining the cell line. Suitable media contain a carbon source, a nitrogen source and, if desired, vitamins and/or organic salts. the

The second object of the present invention is to provide a method for treating or preventing RSV infection in a host, the method comprising administering the antibody of the present invention to the host in an amount sufficient to treat or prevent the disease. the

In another aspect, the present invention also includes a pharmaceutical composition for the prevention or treatment of RSV, including reducing inflammatory responses, which composition comprises a single antibody or immunoreactive fragment of the present invention as an active agent, or no more than two antibodies of the present invention. an antibody or fragment as the active agent. the

Other aspects of the invention include methods of using the antibodies to treat RSV infection in human subjects or to induce resistance to RSV in such subjects. the

The third object of the present invention is a preparation method comprising the human monoclonal antibody or its conservative mutant or its active fragment. the

The monoclonal antibodies of the invention can be produced using recombinant methods, and thus this invention also includes recombinant materials used to produce the monoclonal antibodies as well as cell lines or immortalized cells used to produce these antibodies. the

In another aspect, the invention provides a method of propagating a hybridoma cell line of the invention comprising culturing the cells in a nutrient medium. Propagation methods also represent methods for producing antibodies of the invention that can be isolated from the culture medium. Preferably, propagation of the hybridoma cell lines of the invention is performed in vitro. Wherein the cell line is cultivated in a nutrient medium. A suitable nutrient medium for the cells of the invention contains a carbon source, a nitrogen source and, if desired, vitamins and/or inorganic salts. For example, RPMI 1640 medium supplemented with 10% fetal bovine serum (SIGMA) can be used. Another suitable medium is Sigma serum- and protein-free hybridoma medium. the

(1) Technical conception

Due to technical limitations, the price of monoclonal antibody drugs currently on the market has been high, limiting their use and promotion. Therefore, it is of great significance to improve and perfect the existing monoclonal antibody technology, especially to explore low-dose and high-efficiency anti-RSV human monoclonal antibody and its preparation method. the

According to this thought and train of thought, the inventor has successfully obtained the expected research result and the anti-RSV human monoclonal antibody of the application product through repeated experimental research and analysis and theoretical exploration. the

(2) Preparation method of monoclonal antibody

The preparation method of anti-RSV human monoclonal antibody, comprises the steps:

1. Preparation of hybridomas, including:

A. Mouse immunization;

B. Hybridoma cultivation and screening;

2. Comparison and screening of the ability of monoclonal antibodies to neutralize antigens;

3. Cloning and humanization of monoclonal antibodies;

4. Construction of monoclonal antibody expression plasmids;

5. Expression and purification of monoclonal antibodies. the

Wherein, the method of described mouse immunization is:

A. obtain the cell strain expressing anti-RSV F protein by hybridoma technology screening. Add 120ul of complete Freund's adjuvant (CFA) to 120ul of RSV antigen-containing phosphate buffer solution (PBS) commercially available from Capricon, mix and emulsify to prepare 240ul of CFARSV antigen solution. The same method utilizes Freund's incomplete adjuvant (IFA) to make the IFA RSV antigen solution of 240ul;

B. Inject 240ul of FCA RSV antigen solution intraperitoneally to BALB/C female mice aged 7-8 weeks for the first immunization. After the first immunization, booster immunization with FIA RSV antigen solution was performed every 2-3 weeks. 240ul RSV antigen (manufactured by Capricon) was intravenously injected 10 days and 3 days before the splenocytes were isolated. On the 42nd day after the immunization, the spleen was taken out, the splenocytes were separated, and the splenocytes and SP2-0 mouse myeloma cells were fused 1/1 by the PEG method to prepare hybridomas. the

The method for cultivating and screening described hybridomas is:

A. Under sterile conditions, the hybridomas were suspended in HAT medium to adjust the concentration to 3×10 ⁶ /ml, and plated in each well of a 96-microwell plate (Croming Company), 3×10 ⁵ /well, 100ul/well. Place the 96-microwell plate in a carbon dioxide incubator at 37°C and 8% CO2 for static culture until hybridoma clones appear;

B. Prepare RSV antigen-coated ELISA screening 96-well plate: RSV antigen (Capricon company) is diluted with phosphate buffer solution (PBS) containing 0.1% (w/v) NaN3 with pH 7.0, to concentration 0.5ug/ml , and dispensed into blank ELISA plates (NUNC Company), 100ul/well, and incubated overnight at 2-8°C. After incubation, the ELISA plate was washed 3 times with PBS buffer solution containing 0.05% TWEEN 20 (washing solution for short), and the residual solution was controlled to dry. Then add PBS buffer solution (coating solution for short) containing 1% (w/v) BSA to the ELISA plate, 300ul/well, and incubate at 2-8°C for more than 6 hours before use. Store at 2-8°C;

C. Remove the coating solution in the ELISA plate before detection, prepare fresh coating solution, and add 60ul to each well. Take out the hybridoma culture 96-well plate to be tested, mark and code each well where the clone has grown, absorb 40ul/well cell culture supernatant under sterile conditions, add it to the coated ELISA plate, Mix well and label well. Gently shake the ELISA plate at room temperature for 1-2 hours to allow the immune reaction to fully proceed. Remove the reaction liquid, wash the ELISA plate 3 times with washing solution and drain the residual liquid. The horseradish peroxidase (POD)-labeled anti-mouse IgG polyclonal antibody (SANTA CRUZ) was diluted with the freshly prepared coating solution, and the dilution factor was 1×10 ⁴ . Add 120ul of diluted detection antibody to each well of the ELISA plate, and react for 30 minutes at room temperature in the dark. Remove the reaction liquid to stop the reaction, wash the ELISA plate 3 times with the cleaning solution and dry the residual liquid, add 100ul of the reaction substrate o-phenylenediamine (OPD) to each well, react at room temperature for 15 minutes in the dark, then add the stop solution to stop reaction. ELISA plate detects the absorbance value of 492nm wavelength with microplate reader (MD company);

D. Three hybridoma cell lines with high expression were screened and numbered BW01-4H13, BW01-7B05 and BW01-10E02, and the cells were expanded and cultured and then frozen and stored in liquid nitrogen until use. the

The comparison and screening method of the neutralizing antigen ability of the monoclonal antibody is as follows:

The antigen neutralization ability of three anti-RSV monoclonal antibodies obtained by screening hybridoma cell lines was confirmed. Three RSV strains were purchased from the American Standard Biological Collection (ATCC): B1 wild-type strain (10[4.5]TCID(50)/ml), A2 (10[5.5]TCID(50)/ml) and For Long strain (10[6.7]TCID(50)/ml), the obtained RSV was diluted 10 times with phosphate buffered saline (abbreviated as diluent) containing bovine serum albumin (BSA). Antigen extraction reagents were prepared: 0.4M NaCl, 0.1M citric acid, 10mM dithiothreitol and 0.1% polyoxyethylene novel phenyl ether. The diluted RSV virus and the antigen extraction reagent are mixed in equal volumes, and the antigen extraction solution is obtained after reacting for 5-10 minutes. Agarose gel (GE) coated with anti-mouse IgG polyclonal antibody (SANTA CRUZ) was prepared, and the colloidal concentration was 15% (v/v) of the agarose gel solution. The gel solution is mixed with the extracted antigen to prepare a mixed reagent (detection solution) for antibody titer detection. Add the above-mentioned uniformly mixed detection solution into a 96-microwell plate (Croming Company, referred to as a reaction plate), aliquot 50 ul into each well, and mark it for later use. At the same time, the iso-conditioned culture supernatants of BW01-4H13, BW01-7B05 and BW01-10E02 cell lines were added to each well, 150ul/well. At the same time, a positive control group was set up, that is, an equal volume of cleaning solution was used to replace the three kinds of antibodies and the detection solution to react, which were respectively three kinds of positive controls. Then shake and mix gently at room temperature and incubate for 1-2 hours, so that the antigens extracted from the three virus strains and the three antibodies can fully react. Prepare the positive detection ELISA plate the day before the experiment: coat the goat anti-RSV polyclonal antibody (CHEMICON Company) on a blank ELISA plate (NUNC Company), dilute the antibody to 10ug/ml with coating solution, and pack 100ul/well in the ELISA Plates were incubated overnight at 2-8°C. Wash the positive detection ELISA plate with washing solution before use, and control the residual liquid. Aspirate the reaction mixture from the reaction plate, and transfer it to the positive detection ELISA plate in turn, 100ul per well, strictly avoid aspirating the gel. After the corresponding markers are made, shake the positive detection ELISA plate continuously and gently to mix, and react at room temperature for 1-2 hours, so that the coated antibody can fully capture the free antigen molecules. At the same time, a negative control is set up, that is, the reaction of the cleaning solution and the coated antibody is used as the background value of the detection. The immune reaction was terminated by three washes with washing solution. The horseradish peroxidase (POD)-labeled anti-mouse IgG polyclonal antibody (SIGMA) was diluted with the freshly prepared coating solution, and the dilution factor was 1×10 ² . Add 100ul of diluted detection antibody to each well of the ELISA plate, and react for 30 minutes at room temperature in the dark. Remove the reaction liquid to stop the reaction, wash the ELISA plate 3 times with the cleaning solution and dry the residual liquid, add 100ul of the reaction substrate o-phenylenediamine (OPD) to each well, react at room temperature for 20 minutes in the dark, then add the stop solution to stop reaction. The ELISA plate uses a microplate reader (MD company) to detect the absorbance value of 492nm wavelength; from the absorbance value results, it can be concluded that, except for 4H13, the other two antibodies have a certain neutralization effect on the three virus strains. The order of neutralization ability is 7B05>10E02>4H13.

The cloning and humanization methods of the monoclonal antibody are:

A. Total mRNA was extracted from 2×107 BW01-7B05 hybridoma cells according to the instructions of the RNesay kit (Qiagen). Single-stranded cDNA was prepared using oligo-dT primers and reverse transcriptase, and an aliquot of the cDNA was used as the starting material for the polymerase chain reaction (PCR) to amplify the gene of the variable region;

B. Preparation of single-stranded cDNA Using 1ug of total mRNA as a template, in the buffer system of 50mM Tris-cl, 8mM Mg2Cl, 30mM KClPH8.5, add 1mM dithiothreitol (DTT), 1mM dNTP, 25 units of Human placental ribonucleic acid inhibitor, 33uM random hexamer and 10 units of AMV reverse transcriptase, the reaction system is placed at 42°C for 1-2 hours. The heavy chain variable region (VH) of the 7B05 monoclonal antibody was amplified by polymerase chain reaction (PCR) with primers P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2), and the light chain variable region (VL ) obtained by PCR amplification with primers P3 (SEQ ID NO: 3) and P4 (SEQ ID NO: 4);

C. The PCR amplification of DNA fragments uses 1ug single-stranded CRNA as a template, in the buffer system of 10mM Tris-cl, 1.5mM Mg2Cl, 50mMKCl PH8.5, add 1mM dithiothreitol (DTT), 0.5mM dNTPs, 1uM corresponding primer and 2.5 units of Taq enzyme (TAKARA), the reaction system was placed in a PCR instrument (THERMO) at 94°C for 1 minute annealing, 55°C primer binding for 2 minutes, 72°C amplification for 2 minutes, 25 cycles of reaction. The amplified DNA fragments were extracted with phenol-chloroform and then dissolved and diluted with sterile purified water. The DNA fragment was digested with EcoR I and BamH I (TAKARA) and integrated into the plasmid pUC18 (TAKARA). For the operation instructions, see the pUC18 kit. Use primer T7 to sequence the amplified product DNA (Shanghai Meiji Biotechnology Company) to confirm the accuracy of its sequence;

D. Submit the VH and VL sequences of 7B05 to the NCBI database for sequence comparison, compare with multiple submitted human VH and VL sequences, and select the sequence with the highest similarity for sequence humanization. By comparison, the VH sequence of 7B05 has the highest similarity with the human IgG Cor sequence, reaching 82%; the VL sequence has the highest similarity with the human IgG K102 sequence, reaching 75%. As a result of humanization, the degree of humanization should be improved as much as possible while maintaining the specificity and high affinity of the antibody to the antigen recognition site as much as possible. Therefore, the three hypervariable regions (CD1, CD2 and CD3) of VH and VL are retained The sequence of the mouse 7B05 antibody is used, and the human IgG Cor sequence and K102 sequence are used for the rest of the region. the

The construction method of described monoclonal antibody expression plasmid is:

A. The constant region sequence (CH and CL) of the 7B05 monoclonal antibody adopts the human IgG Cor sequence and the corresponding fragment of the K102 sequence. The amplification template of the CH fragment is the IgG Cor gene, and the amplification template of the CL fragment is the IgG K102 gene. CH and CL fragments are obtained by sequence splicing;

B. The synthesis of fragments uses primer splicing overlap extension PCR, such as fragments 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10 are paired and mixed respectively, and overlap extension PCR is performed under conventional PCR conditions. Annealing, primer recognition pairing and fragment amplification can obtain overlapping and extended fragments such as 1-2, 3-4, 5-6, 7-8, 9-10, etc. In the same way, 1-2 and 3-4, 5-6 and 7-8 are spliced and extended, and 9-10 fragments are bye. Fragments 1-10 were obtained after multiple rounds of PCR. Finally, the light and heavy chain fragments were obtained after multiple rounds of splicing. The humanized heavy chain fragment was integrated into the MCSA region of the pIRES plasmid (Clontech, plasmid map shown in Figure 1) between Nhe I and Mlu I, and the light chain fragment was integrated into the pIRES plasmid between Not I and Sal I. MCSB interval. After large-scale culture, the plasmid was prepared with a plasmid extraction kit (QIAGEN) and frozen for use. the

The method for the expression and purification of the monoclonal antibody is:

A. The acquisition of 7B05 monoclonal antibody is obtained by transfecting CHO-K1 cells (ATCC), and in the case of drug screening, a cell line stably expressing the antibody protein is obtained;

B. Transfection The lipofectamineTM 2000 liposome transfection kit produced by Invitrogen was used, and the operation was performed according to the instructions. In the blank control of the transfection experiment, CHO-K1 cells were transfected with pIRES vector. G418 (200uM) was added to the medium for combined pressurized screening, the cells were cultured under 8% CO2 and 37°C, and the medium was changed every 3 days to remove dead floating cells. After 20 days of continuous culture, cell clones dispersed into a single colony appeared in the cell culture dish. After removing the medium, the cells were digested and separated with trypsin (INVITROGEN), and then transferred to a 96-well plate for culture and screening by limiting dilution. single cell. The 96-well plate was cultured at 8% CO2 and 37°C for 1 week, and then the culture supernatant was taken for ELISA to determine the protein expression level;

C. Mouse anti-human TNFR primary antibody (SIGMA) was diluted with PBS (diluent) with pH 7.0 and containing 0.1% (w/v) NaN3, and the dilution factor was 1×10 3 . Add the diluted antibody to the ELISA plate (CORING) to be coated, 100ul/well, overnight at 2-8°C. The ELISA plate was washed three times with PBS buffer solution containing 0.05% TWEEN 20 (cleaning solution for short), and 100ul 5% (w/v) BSA phosphate buffer solution was added to each well for 1-2 hours at room temperature, and the cell culture supernatant to be tested Add it to the ELISA plate, 100ul/well, let it stand at room temperature and incubate for 1-2 hours. Discard the reaction solution, wash three times with the cleaning solution, add mouse anti-human IgG-Fc/HRP secondary antibody (SIGMA) diluted 1000 times with the diluent, 150ul/well, incubate at room temperature for 1-2 hours and then TMB reagent (PERCE) Color development, OD value measured at 450nm. Use fresh culture medium as negative control;

D. Multiple clones with the highest expression levels were selected and amplified to a 24-well plate, and the protein expression was detected after 3 days of culture, and the above-mentioned ELISA was used for detection. Select 10 clones with high expression levels for continuous expansion and culture, and keep the cell selection conditions unchanged (G418 (200uM) and MSX (50ug/ml)); after multiple rounds of amplification, select the 6 clones with the highest expression level to expand to T75 In culture flasks, cell lines were established as cell banks and stored at low temperature. At the same time, the clone BW-7B05-3 with the highest expression level was inoculated into a 2L spinner bottle, cultivated with EX-CELL 302 medium (SIGMA) containing 5% calf serum (INVITROGEN), and when the cells covered the wall of the bottle, replace It is the serum-free medium EX-CELL 302, and the liquid is collected every other day, and the liquid is collected continuously for 3-5 times;

E. Using the specific adsorption of protein A to IgG, use the separation medium rProteinA Sepharose 4Fast Flow (GE) to perform affinity chromatography on the harvested cell culture supernatant to purify the expressed protein. See the product description for the operation method. After purification, the A260 and A280 values were measured with a UV spectrophotometer. Protein quantification formula: protein content (mg/ml) = OD280 value × 1.45-OD260 value × 0.74. the

(3) Main purpose and technical expertise

The invention provides a new anti-RSV human monoclonal antibody and a preparation method thereof for industries such as medicine, thereby expanding the application of new treatment or prevention products for respiratory syncytial virus pneumonia. the

The anti-RSV human monoclonal antibody of the present invention is safe and can be used for large-scale production and application in industries such as medical treatment and diagnosis. the

Compared with the existing anti-RSV disease products, the anti-RSV human monoclonal antibody of the present invention can greatly reduce the unit dosage and production cost in practical application and other aspects. the

Therefore, the successful development of the method of the present invention is of great significance for the detection of the effective component of the antigenic epitope in the research and development of the vaccine, and the method also provides accurate, accurate and accurate information for the prevention and treatment, early screening and diagnosis of RSV diseases. It is a simple and effective monitoring method with a wide range of applications and social needs. the

In a word, the present invention actively adapts to the needs of work in the fields of modern biology, preventive health care, clinical diagnosis, etc. and the needs of humanized services. RSV disease products are of great value. the

Description of drawings

Figure 1 is the gene map of pIRES expression vector. the

Figure 2 is a map of the multiple cloning sites and restriction enzyme cut points of the PIRES expression vector. the

Figure 3 shows the absolute number of plaques per microgram of humanized 7B05 monoclonal antibody and Synagis antibody. the

Figure 4 is a comparison chart of the neutralization effect of the humanized 7B05 monoclonal antibody and Synagis on the RSV-B1 virus strain. the

Detailed ways

The present invention studies the existing RSV disease treatment or prevention methods, and provides a new anti-RSV human monoclonal antibody, which is convenient for safe use in the fields of modern biology, preventive health care, clinical diagnosis and the like. the

The present invention finally needs to be prepared into an anti-RSV human monoclonal antibody for application, and the following examples are given for further illustration. If you have any questions, you can contact the inventor directly. Some experimental data provided above and the following examples provide several anti-RSV human monoclonal antibodies and their preparation methods and some experimental research contents according to the foregoing invention contents, but it should be understood that the present invention is not limited to the research contents listed here , it should also be understood that the terminology used herein is only used to describe specific embodiments, but not to limit the present invention. the

That is to say, in the present invention, the specific implementation methods described above and the examples described below are all to better illustrate the present invention, especially these embodiments are only exemplary descriptions of the present invention, and cannot be interpreted as Restrictions on the present application may be used to limit the protection scope of the present invention. the

In order to illustrate the present invention more clearly, the present invention will be described in detail below in conjunction with the following examples. the

The preparation of embodiment 1 hybridoma

1. Immunization of mice

Cell lines expressing anti-RSV F protein were obtained by hybridoma technology screening. Add 120ul of complete Freund's adjuvant (CFA) to 120ul of RSV antigen-containing phosphate buffer solution (PBS) commercially available from Capricon, mix and emulsify to prepare 240ul of CFARSV antigen solution. In the same way, 240 ul of IFARSV antigen solution was prepared using incomplete Freund's adjuvant (IFA). the

BALB/C female mice aged 7-8 weeks were intraperitoneally injected with 240ul FCARSV antigen solution for the first immunization. After the first immunization, booster immunization with FIARSV antigen solution was carried out every 2-3 weeks. 240ul RSV antigen (manufactured by Capricon) was intravenously injected 10 days and 3 days before the splenocytes were isolated. On the 42nd day after the immunization, the spleen was taken out, the splenocytes were separated, and the splenocytes and SP2-0 mouse myeloma cells were fused 1/1 by the PEG method to prepare hybridomas. the

2. Hybridoma culture and screening

Under sterile conditions, the hybridomas were suspended in HAT medium to adjust the concentration to 3×10 ⁶ /ml, and plated in each well of a 96-microwell plate (Croming Company), 3×10 ⁵ /well, 100ul/well. The 96-microwell plate was placed in a carbon dioxide incubator at 37°C and 8% CO2 for static culture until hybridoma clones appeared.

Prepare the RSV antigen-coated ELISA screening 96-well plate: the RSV antigen (Capricon company) is diluted with phosphate buffer solution (PBS) containing 0.1% (w/v) NaN3 to a concentration of 0.5ug/ml with pH 7.0, and Dispense to a blank ELISA plate (NUNC Company), 100ul/well, and incubate overnight at 2-8°C. After incubation, the ELISA plate was washed 3 times with PBS buffer solution containing 0.05% TWEEN 20 (washing solution for short), and the residual solution was controlled to dry. Then add PBS buffer solution (coating solution for short) containing 1% (w/v) BSA to the ELISA plate, 300ul/well, and incubate at 2-8°C for more than 6 hours before use. Store at 2-8°C. the

Remove the coating solution in the ELISA plate before detection, prepare fresh coating solution, and add 60ul to each well. Take out the hybridoma culture 96-well plate to be tested, mark and code each well where the clone has grown, absorb 40ul/well cell culture supernatant under sterile conditions, add it to the coated ELISA plate, Mix well and label well. Gently shake the ELISA plate at room temperature for 1-2 hours to allow the immune reaction to fully proceed. Remove the reaction liquid, wash the ELISA plate 3 times with washing solution and drain the residual liquid. The horseradish peroxidase (POD)-labeled anti-mouse IgG polyclonal antibody (SANTA CRUZ) was diluted with the freshly prepared coating solution, and the dilution factor was 1×10 ⁴ . Add 120ul of diluted detection antibody to each well of the ELISA plate, and react for 30 minutes at room temperature in the dark. Remove the reaction liquid to stop the reaction, wash the ELISA plate 3 times with the cleaning solution and dry the residual liquid, add 100ul of the reaction substrate o-phenylenediamine (OPD) to each well, react at room temperature for 15 minutes in the dark, then add the stop solution to stop reaction. The ELISA plate was detected with a microplate reader (MD Company) to detect the absorbance at a wavelength of 492 nm.

Three hybridoma cell lines with high expression were screened and numbered BW01-4H13, BW01-7B05 and BW01-10E02, and the cells were expanded and cultured and then frozen and stored in liquid nitrogen until use. the

Example 2 Monoclonal Antibody Neutralizing Antigen Ability Comparison

The antigen neutralization ability of three anti-RSV monoclonal antibodies obtained by screening hybridoma cell lines was confirmed. Three RSV strains were purchased from the American Standard Biological Collection (ATCC): B1 wild-type strain (10[4.5]TCID(50)/ml), A2 (10[5.5]TCID(50)/ml) and For Long strain (10[6.7]TCID(50)/ml), the obtained RSV was diluted 10 times with phosphate buffered saline (abbreviated as diluent) containing bovine serum albumin (BSA). the

Prepare antigen extraction reagents: 0.4M NaCl, 0.1M citric acid, 10mM dithiothreitol and 0.1% polyoxyethylene new phenyl ether. The diluted RSV virus and the antigen extraction reagent are mixed in equal volumes, and the antigen extraction solution is obtained after reacting for 5-10 minutes. the

Prepare agarose gel (GE) coated with anti-mouse IgG polyclonal antibody (SANTA CRUZ), and the colloidal concentration is 15% (v/v) agarose gel solution. The gel solution is mixed with the extracted antigen to prepare a mixed reagent (detection solution) for antibody titer detection. For the concentration and virus titer of the antigen extract, the mitigation plan is shown in the following table (Table 1):

Table 1 Mitigation schemes for each antigen extract

virus strain Antigen extract (ml) Diluent (ml) Agarose gel solution (ml) B1wild-type strain 0.3 1.2 1.5 A2 0.09 1.41 1.5 Long strain 0.7 0.8 1.5

Add the above-mentioned uniformly mixed detection solution into a 96-microwell plate (Croming Company, referred to as a reaction plate), aliquot 50 ul into each well, and mark it for later use. At the same time, the iso-conditioned culture supernatants of BW01-4H13, BW01-7B05 and BW01-10E02 cell lines were added to each well, 150ul/well. At the same time, a positive control group was set up, that is, an equal volume of cleaning solution was used to replace the three kinds of antibodies and the detection solution to react, which were respectively three kinds of positive controls. Then shake and mix gently at room temperature and incubate for 1-2 hours, so that the antigens extracted from the three virus strains and the three antibodies can fully react. the

Prepare the positive detection ELISA plate the day before the experiment: coat the goat anti-RSV polyclonal antibody (CHEMICON Company) on a blank ELISA plate (NUNC Company), dilute the antibody to 10ug/ml with coating solution, and distribute 100ul/well in the ELISA Plates were incubated overnight at 2-8°C. the

Wash the positive detection ELISA plate with washing solution before use, and control the residual liquid. Aspirate the reaction mixture from the reaction plate, and transfer it to the positive detection ELISA plate in turn, 100ul per well, strictly avoid aspirating the gel. After the corresponding markers are made, shake the positive detection ELISA plate continuously and gently to mix, and react at room temperature for 1-2 hours, so that the coated antibody can fully capture the free antigen molecules. At the same time, a negative control is set up, that is, the reaction of the cleaning solution and the coated antibody is used as the background value of the detection. The immune reaction was terminated by three washes with washing solution. the

The horseradish peroxidase (POD)-labeled anti-mouse IgG polyclonal antibody (SIGMA) was diluted with the freshly prepared coating solution, and the dilution factor was 1×10 ² . Add 100ul of diluted detection antibody to each well of the ELISA plate, and react for 30 minutes at room temperature in the dark. Remove the reaction liquid to stop the reaction, wash the ELISA plate 3 times with the cleaning solution and dry the residual liquid, add 100ul of the reaction substrate o-phenylenediamine (OPD) to each well, react at room temperature for 20 minutes in the dark, then add the stop solution to stop reaction. The ELISA plate was detected with a microplate reader (MD Company) to detect the absorbance at a wavelength of 492 nm.

Use the following formula to calculate the capture ability (neutralization rate) of the three antibodies (referred to as 4H13, 7B05 and 10E02) to the virus:

Neutralization rate%=[1-(absorbance value-negative control)/(positive control-negative control)]100%

The evaluation of the neutralization rate of the three antibodies to the virus is shown in the following table (Table 2). Among them, "+++" indicates that the neutralization rate is above 80%, "++" indicates that it is above 60%, "+" indicates that it is above 30%, and "-" indicates that it is below 30%. the

Table two three kinds of antibodies to RSV virus neutralization rate

It can be seen from the results in the above table that, except for 4H13, the other two antibodies have a certain neutralizing effect on the three virus strains. In comparison, the order of neutralizing ability is 7B05 > 10E02 > 4H13. the

Cloning and humanization of embodiment 37B05 monoclonal antibody

Total mRNA was extracted from 2×10 ⁷ BW01-7B05 hybridoma cells according to the instructions of the RNesay kit (Qiagen). Single-stranded cDNA was prepared using oligo-dT primers and reverse transcriptase, and an aliquot of the cDNA was used as a starting material for polymerase chain reaction (PCR) to amplify the gene of the variable region.

The preparation of single-stranded cDNA uses 1ug of total mRNA as a template, in the buffer system of 50mM Tris-cl, 8mM Mg2Cl, 30mM KCl PH8.5, add 1mM dithiothreitol (DTT), 1mM dNTP, 25 units of human Placental ribonucleic acid inhibitor, 33uM random hexamer and 10 units of AMV reverse transcriptase, the reaction system is placed at 42°C for 1-2 hours. The heavy chain variable region (VH) of the 7B05 monoclonal antibody was amplified by polymerase chain reaction (PCR) with primers P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2), and the light chain variable region (VL) was obtained by PCR amplification with primers P3 (SEQ ID NO: 3) and P4 (SEQ ID NO: 4). The primers were synthesized by Shanghai Meiji Biotechnology Co., Ltd., and the primer sequences are as follows:

SEQ ID NO: 1: AGCGGATCCA GGGGCCAGTG GATAGAC

SEQ ID NO: 2: TGGATGGTGG GAAGATG

SEQ ID NO: 3: GGCCAGTGGA TAGAC

SEQ ID NO: 4: TACAGTTGGT GCAGCA

For the PCR amplification of DNA fragments, 1ug single-stranded CRNA was used as a template, and 1mM dithiothreitol (DTT), 0.5mM dNTPs, 1uM corresponding primers and 2.5 units of Taq enzyme (TAKARA), the reaction system was placed in a PCR instrument (THERMO) at 94°C for 1 minute annealing, 55°C primer binding for 2 minutes, 72°C amplification for 2 minutes, 25 cycles of reaction. The amplified DNA fragments were extracted with phenol-chloroform and then dissolved and diluted with sterile purified water. The DNA fragment was digested with EcoR I and BamH I (TAKARA) and integrated into the plasmid pUC18 (TAKARA). For the operation instructions, see the pUC18 kit. The DNA of the amplified product was sequenced using primer T7 (Shanghai Meiji Biotechnology Co., Ltd.) to confirm the accuracy of the sequence. the

The VH and VL sequences of 7B05 were submitted to the NCBI database for sequence comparison, compared with multiple submitted human VH and VL sequences, and the sequence with the highest similarity was selected for sequence humanization. By comparison, the VH sequence of 7B05 has the highest similarity with the human IgG Cor sequence, reaching 82%; the VL sequence has the highest similarity with the human IgG K102 sequence, reaching 75%. As a result of humanization, the degree of humanization should be improved as much as possible while maintaining the specificity and high affinity of the antibody to the antigen recognition site, so the three hypervariable regions (CD1, CD2 and CD3) of VH and VL are retained The sequence of the mouse 7B05 antibody is used, and the human IgG Cor sequence and K102 sequence are used for the rest of the region. The comparison and humanization results of the VH and VL of murine 7B05 with human IgG Cor sequence and K102 sequence are as follows:

VH of humanized 7B05

1 5 10 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Human IgG VH(Cor)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly VH of humanized 7B05

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly VH of Mouse 7B05

20 25 30

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys

Ala Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys

35 40 45

Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

Asp Tyr Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

Asp Tyr Tyr Ile Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr

Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Val Phe

Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Val Phe

65 70 75

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser

Asp Pro Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser

Asp Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ser Asp Thr Ser

80 85 90

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp

95 100 105

Thr Ala Val Tyr Tyr Cys Ala

Thr Ala Val Tyr Tyr Cys Ala Tyr Tyr Gly Thr Ser Ser Phe Asp

Thr Ala Val Tyr Tyr Cys Ala Tyr Tyr Gly Thr Ser Ser Phe Asp

110 115

Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser VH of humanized 7B05

VH of Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Rat 7B05

VL of humanized 7B05

1 5 10 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Human IgG VL(K102)

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Humanized VL of 7B05

VL of Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Val Ser Leu Rat Source 7B05

20 25 30

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser

Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn

Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn

35 40 45

Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

Arg Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

Arg Tyr Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys

50 55 60

Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

Leu Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser

Thr Leu Ile His Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser

65 70 75

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile

Arg Phe Ser Gly Ser Gly Ser Gly Gln Glu Tyr Ser Leu Thr Ile

80 85 90

Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

Phe His Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu

Phe His Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu

107

Ile Lys VL of humanized 7B05

Ile Lys VL of Rat Source 7B05

The construction of embodiment 47B05 monoclonal antibody expression plasmid

The constant region sequences (CH and CL) of the 7B05 monoclonal antibody adopt the corresponding fragments of human IgG Cor sequence and K102 sequence. The amplification template of the CH fragment is the IgG Cor gene, and the amplification template of the CL fragment is the IgG K102 gene. The CH and CL fragments are obtained by sequence splicing, and the primers are designed as follows:

The synthesis of fragments uses primer splicing overlap extension PCR. For example, fragments 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10 are paired and mixed respectively, and overlap extension PCR is performed. Under conventional PCR conditions, annealing, Primer recognition pairing and fragment amplification yield overlapping and extended fragments such as 1-2, 3-4, 5-6, 7-8, 9-10, etc. In the same way, 1-2 and 3-4, 5-6 and 7-8 are spliced and extended, and 9-10 fragments are bye. Fragments 1-10 were obtained after multiple rounds of PCR. Finally, the light and heavy chain fragments were obtained after multiple rounds of splicing. The humanized heavy chain fragment was integrated into the MCSA region of the pIRES plasmid (Clontech, plasmid map shown in Figure 2) between Nhe I and Mlu I, and the light chain fragment was integrated into the pIRES plasmid between Not I and Sal I. MCSB interval. After large-scale culture, the plasmid was prepared with a plasmid extraction kit (QIAGEN) and frozen for use. the

Example 5 Expression and purification of 7B05 monoclonal antibody

The 7B05 monoclonal antibody was obtained by transfecting CHO-K1 cells (ATCC), and in the case of drug screening, a cell line stably expressing the antibody protein was obtained. the

For transfection, lipofectamine ^TM 2000 liposome transfection kit produced by Invitrogen was used, and the operation was performed according to the instructions. In the blank control of the transfection experiment, CHO-K1 cells were transfected with pIRES vector. G418 (200uM) was added to the medium for combined pressurized screening, the cells were cultured under 8% CO2 and 37°C, and the medium was changed every 3 days to remove dead floating cells. After 20 days of continuous culture, cell clones dispersed into a single colony appeared in the cell culture dish. After removing the medium, the cells were digested and separated with trypsin (INVITROGEN), and then transferred to a 96-well plate for culture and screening by limiting dilution. single cell. The 96-well plate was cultured at 8% CO2 and 37°C for 1 week, and then the culture supernatant was taken for ELISA to measure the protein expression level.

The mouse anti-human TNFR primary antibody (SIGMA) was diluted with PBS (diluent) with pH 7.0 and containing 0.1% (w/v) NaN3 (diluent), and the dilution factor was 1×10 ³ . Add the diluted antibody to the ELISA plate (CORING) to be coated, 100ul/well, overnight at 2-8°C. The ELISA plate was washed three times with PBS buffer solution containing 0.05% TWEEN 20 (cleaning solution for short), and 100ul 5% (w/v) BSA phosphate buffer solution was added to each well for 1-2 hours at room temperature, and the cell culture supernatant to be tested was Add it to the ELISA plate, 100ul/well, let it stand at room temperature and incubate for 1-2 hours. Discard the reaction solution, wash three times with the cleaning solution, add mouse anti-human IgG-Fc/HRP secondary antibody (SIGMA) diluted 1000 times with the diluent, 150ul/well, incubate at room temperature for 1-2 hours and then TMB reagent (PERCE) Color development, OD value measured at 450nm. Fresh medium was used as negative control.

Multiple clones with the highest expression levels were selected and amplified to 24-well plates, and the protein expression was detected after 3 days of culture, and the above-mentioned ELISA was used for detection. Select 10 clones with high expression levels for continuous expansion and culture, and keep the cell selection conditions unchanged (G418 (200uM) and MSX (50ug/ml)); after multiple rounds of amplification, select the 6 clones with the highest expression level to expand to T75 In culture flasks, cell lines were established as cell banks and stored at low temperature. At the same time, the clone BW-7B05-3 with the highest expression level was inoculated into a 2L spinner bottle, cultivated with EX-CELL 302 medium (SIGMA) containing 5% calf serum (INVITROGEN), and when the cells covered the wall of the bottle, replace It is serum-free medium EX-CELL 302, and the liquid is collected every other day, and the liquid is collected continuously for 3-5 times. the

Utilizing the specific adsorption of protein A to IgG, the harvested cell culture supernatant was subjected to affinity chromatography using the separation medium rProtein A Sepharose 4 Fast Flow (GE) to purify the expressed protein. See its product description for the operation method. After purification, the A260 and A280 values were measured with a UV spectrophotometer. Protein quantification formula: protein content (mg/ml) = OD280 value × 1.45-OD260 value × 0.74. the

RSV neutralization experiment of embodiment 6 7B05 monoclonal antibody

The ability of 7B05 monoclonal antibody to neutralize virus in vitro was determined by standard plaque assay, and a commercially available RSV antibody was used as a parallel control in the experiment. The concentration of Hep 2 cells was adjusted with RPMI 1640 medium (SIGMA) containing 10% fetal bovine serum (SIGMA) and inoculated into 12-well plates (CRONING) at a seeding concentration of 2×10 ⁵ /well, 5% CO2, 37 Cultivate overnight at ℃. The 7B05 monoclonal antibody was diluted in a concentration gradient with medium, and about 200 PFU/well of RSV-B1 was added to the diluted antibody solution, and rabbit complement serum (SIGMA) was added at the same time, and incubated at room temperature for 1-2 hours. Add 200ul/well antibody-virus mixed solution to the culture medium of Hep 2 cells and incubate at room temperature for 2-3 hours to infect the cells with the virus. All medium was removed, fresh medium containing 1% (w/v) methylcellulose (MERCK) was added, and the culture dish was incubated at 35° C. for 6 days for cell fixation and staining.

Remove the medium containing methylcellulose in the culture dish, fix the cells with 100% methanol at room temperature for 30 minutes, and then wash the culture dish three times with washing solution. Add the primary antibody diluted 1000 times with the diluent—goat anti-RSV polyclonal antibody (CHEMICON Company), 100ul/well, react at room temperature for 1-2 hours, add washing solution to wash three times, and terminate the reaction. Add the secondary antibody diluted 800 times with the diluent—horseradish peroxidase-labeled rabbit anti-goat polyclonal antibody (MERCK), 100ul/well, react at room temperature for 1-2 hours, add washing solution to wash three times, and terminate the reaction. Add 200 ul/well of chlorinated naphthol reagent (PERCE), react at room temperature for 10 minutes, rinse with water to remove the reagent, and count the number of plaques in a single well after drying. the

Figure 3 shows the results of the absolute number of plaques per microgram of humanized 7B05 antibody, which also includes the results of Synagis antibody. The results showed that the IC50 of 7B05 was 15ng/ml, corresponding to an affinity of 100pM, while the IC50 of the Synagis antibody was 300ng/ml, corresponding to an affinity of 2nM. the

Figure 4 shows the neutralization effect of 7B05 antibody on RSV-B1 strain compared with that of Synagis. Neutralization data (% of control) were calculated from the absolute number of 160-180 plaques per experiment. The EC50 value of the antibody of the present invention with an in vitro affinity of 1 pM to 5 nM is between 10-100 ng/ml. the

Claims (22)

1. An anti-RSV human monoclonal antibody or its conservative mutant or its active fragment, comprising human constant region, its heavy chain variable region is selected from SEQ ID NO: 1; its light chain variable region is selected from SEQ ID NO: 2. 2. The monoclonal antibody or its conservative mutant or its active fragment according to claim 1, which has 98% or 95% homology with the sequence of claim 1. 3. monoclonal antibody as claimed in claim 1 or its conservative mutant or its active fragment is the antibody specific for RSV F albumen. 4. The anti-RSV human monoclonal antibody of claim 1 in a substantially pure form, free of other immunological substances. 5. A DNA molecule encoding the heavy chain variable region sequence SEQ ID NO:1 according to claim 1. 6. A DNA molecule encoding the light chain variable region sequence SEQ ID NO:2 according to claim 1. 7. A composition of carrier or diluent, comprising one or more monoclonal antibodies or conservative mutants or active fragments thereof as claimed in claim 1. 8. A hybridoma cell line from which the monoclonal antibody or its conservative mutant or its active fragment as claimed in claim 1 can be obtained. 9. The cell line of claim 8, further comprising the cell line and a nutrient medium capable of maintaining the cell line. 10. The cell line according to claim 9, wherein the nutrient medium can be a carbon source or a nitrogen source. 11. The cell line according to claim 8, wherein said nutrient medium further contains vitamins and/or organic salts. 12. A method for treating or preventing RSV infection in a host, the method comprising administering to the host an antibody or conservative mutant or active fragment thereof as claimed in claim 1 in an amount sufficient to treat or prevent the disease. 13. A pharmaceutical composition for preventing or treating RSV, which comprises the single antibody or immunoreactive fragment according to claim 1 as an active agent. 14. The composition of claim 13, further comprising no more than two antibodies or fragments of claim 1 as active agents. 15. The composition of claim 13, which acts to reduce inflammatory responses. 16. A preparation method comprising the human monoclonal antibody or its conservative mutant or its active fragment according to claim 1. 17. The method according to claim 16 is using gene recombination method. 18. A method of propagating the hybridoma cell line of claim 8 comprising culturing the cell line in a nutrient medium. 19. The method of claim 18, comprising the method of isolating the antibody of claim 1 or a conservative mutant or active fragment thereof from a culture medium. 20. The method of claim 18, wherein propagation of said hybridoma cell line is performed in vitro. 21. The method according to claim 18, wherein said nutrient medium contains carbon source and nitrogen source. 22. The method of claim 18, wherein said nutrient medium further contains vitamins and/or inorganic salts.

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