CN103951749B - The preparation and its application of a kind of monoclonal neutrality antibody E4 2 of anti-Staphylococcus aureus eLtaS albumen - Google Patents

Abstract

The invention discloses a kind of monoclonal neutrality antibody E4 2 of the anti-Staphylococcus aureus eLtaS albumen for belonging to immune antibody production techniques field.The present invention has cloned antibody light and heavy chain variable region gene, gained light chain and the correct mouse antibody variable region of heavy chain variable region gene codified from the hybridomas of eLtaS neutralizing monoclonal antibodies E4 2 for preparing.Light and heavy chain variable region gene based on said monoclonal antibody, can build and express various small molecule genetic engineering antibodies, based on polypeptide or protein coded by said gene, multiple biological activities molecule can be crosslinked, medicine for preparing anti-Staphylococcus aureus infection, has broad application prospects.

Description

A monoclonal neutralizing antibody E4-2 against Staphylococcus aureus eLtaS protein preparation and application of

technical field

The invention belongs to the technical field of antibody preparation for immunization, and in particular relates to a monoclonal neutralizing antibody E4-2 against Staphylococcus aureus eLtaS protein, its preparation method and its application in the preparation of drugs against Staphylococcus aureus infection .

Background technique

Staphylococcus aureus (Staphylococcus aureus) belongs to the genus Staphylococcus and is an important pathogenic bacterium of humans, which can cause various serious diseases such as pneumonia, endocarditis, burn and war wound infection, sepsis, toxic shock, etc. Infection (Lowy FD.New Engl J Med.1998; 339,520-532), it is the main pathogenic bacteria of Gram-positive bacteria sepsis, and also an important pathogenic bacteria of burn wound infection and acute liver failure (Yao Yongming et al. , Some New Developments in Sepsis Research 2000;13, 517-519). It can spread on a large scale through droplets and contact in crowded areas. What's more serious is that the drug resistance of Staphylococcus aureus continues to increase. For example, the methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to β-lactam antibiotics, was discovered in 1961, and it was discovered in October 2002. Vancomycin-resistant Staphylococcus aureus (VRSA). In October, 2007, surveys such as Klevens R M showed that the number of people infected with MRSA in the United States exceeded 90,000 in 2005, and the death rate of patients infected with MRSA was 19.8%, and the number of deaths exceeded the number of deaths of HIV infection (Klevens RM, etal. 298:1763–1771). What's more serious is that there is no clinically effective antibiotics to deal with multidrug-resistant MRSA, and new and more effective treatment strategies for anti-Staphylococcus aureus infections are urgently needed.

LtaS (LTA synthetase) is one of the cell membrane proteins of Staphylococcus aureus, with a full length of 646aa, the N-terminus is located inside the cell, the C-terminus is located outside the cell, and there are 5 transmembrane regions, and its functional region is located outside the cell, which is similar to the golden yellow The synthesis of LTA, the main component of Staphylococcus cell wall, is closely related. Type I peptidase SpsB can hydrolyze full-length LtaS and release its extracellular domain eLtaS (218-646aa) in the supernatant of bacterial growth. During the experiment, the applicant confirmed that eLtaS can significantly aggravate the infection of Staphylococcus aureus in mice in acute peritonitis model and pneumonia model, thus confirming that it is one of the important virulence factors of Staphylococcus aureus; The neutralizing monoclonal antibody of eLtaS protein can significantly slow down the infection of Staphylococcus aureus in mice in the peritonitis model and pneumonia model, and it is confirmed that its anti-infection function depends on its ability to synthesize LTA of Staphylococcus aureus inhibition. At present, there is no relevant report on the research on the use of specific antibodies against bacterial virulence proteins for anti-bacterial infection treatment.

Contents of the invention

The purpose of the present invention is to provide a monoclonal neutralizing antibody E4-2 against Staphylococcus aureus eLtaS protein.

The object of the present invention is also to provide the preparation method of the above-mentioned monoclonal neutralizing antibody E4-2 and its application in the preparation of anti-Staphylococcus aureus infection medicine.

The light and heavy chain protein molecules of the anti-eLtaS monoclonal antibody E4-2 of the present invention, the amino acid sequences of their variable regions are respectively shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing, and their coding genes are respectively shown in Shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. The amino acid sequences of CDR1, CDR2, and CDR3 of the variable region of the light chain protein molecule of the monoclonal antibody are respectively shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 in the sequence listing. The amino acid sequences of CDR1, CDR2, and CDR3 of the variable region of the heavy chain protein molecule of the monoclonal antibody are respectively shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 in the sequence listing.

The preparation method of the above-mentioned monoclonal antibody E4-2, the main contents are as follows:

1. Construction of anti-eLtaS monoclonal antibody hybridoma cell line.

Firstly, Balb/c mice were immunized with eLtaS protein, and cell fusion was carried out by conventional methods. Screening by ELISA method, positive cell clones were subcloned repeatedly until all hybridoma cell culture supernatants were detected as 100% positive.

2. Screening of anti-eLtaS monoclonal antibody hybridoma cell line E4-2.

The eLtaS neutralizing antibody was screened by detecting the effect of anti-eLtaS monoclonal antibody on the LTA synthesis ability of Staphylococcus aureus. Cultivate Staphylococcus aureus under conventional conditions in brain heart infusion medium, add anti-eLtaS monoclonal antibody and control IgG to 100 μg/ml respectively in the culture system, continue to culture for 6 hours, collect bacteria, and use glass beads to grind to obtain bacterial cell wall LTA , SDS-PAGE separated the obtained LTA and transferred to PVDF membrane, using LTA-specific antibodies to detect bacterial LTA levels under different treatment conditions. The results showed that the anti-eLtaS monoclonal antibody E4-2 could effectively inhibit the LTA synthesis of Staphylococcus aureus.

3. Identification of anti-eLtaS monoclonal antibody hybridoma cell line E4-2.

The immunoglobulin subtype secreted by E4-2 hybridoma cells was IgG2a by biphasic agar diffusion experiment.

4. Detection of affinity and specificity of anti-eLtaS monoclonal antibody hybridoma cell line E4-2

The ascites of Balb/c mouse hybridoma cells was purified by an affinity column, measured by an ultraviolet spectrophotometer, and the protein content was calculated. The affinity between E4-2 and eLtaS was detected by ELISA method, and the results showed that EC50=31ng/ml. The specificity of E4-2 was evaluated by western-blot, and the results showed that E4-2 could specifically recognize eLtaS linear epitope.

5. Detection of the protective effect of anti-eLtaS monoclonal antibody E4-2 on BALB/C mice infected with Staphylococcus aureus.

The mouse model of acute peritonitis was prepared by intraperitoneal injection of Staphylococcus aureus and the monoclonal antibody E4-2 was injected to record the survival of the mice. The results showed that the monoclonal antibody E4-2 could significantly reduce the lethality of Staphylococcus aureus to mice. The pneumonia model was prepared by perfusing the mouse trachea with Staphylococcus aureus and injected with the monoclonal antibody E4-2. After 72 hours, the lung pathological sections were prepared and stained with HE. The results showed that E4-2 could significantly reduce the impact of Staphylococcus aureus on the lungs of mice Ministry of infection.

6. Anti-eLtaS monoclonal antibody hybridoma E4-2 light and heavy chain gene capture

Take neutral monoclonal antibody E4-2 cells in the logarithmic growth phase, extract RNA, and use two pairs of specific primers MuLC4 and MuCKκ, MuHC1 and MuIgG2a to catch the light and heavy chain genes of the antibody by RT-PCR. Connect into the vector by conventional method, transform competent bacteria, pick a single colony after culture, extract the plasmid for PCR identification, and then conduct DNA sequencing analysis. By this part of the experiment, the carrier of anti-eLtaS monoclonal antibody E4-2 light and heavy chain genes was constructed, and through sequence analysis and comparison, the coding sequence was mouse immunoglobulin light and heavy chain genes (SEQ ID NO in the sequence listing : 1 and SEQ ID NO: 2).

7. Determination of gene sequence and amino acid sequence of light and heavy chain variable regions of monoclonal antibody E4-2

Use the www.expasy.org online software to translate the nucleotide sequence of the light and heavy chain variable regions of the monoclonal antibody E4-2 encoding eLtaS into its encoded amino acid sequence, and the amino acids of the light and heavy chain variable regions of the monoclonal antibody E4-2 The sequence is shown as SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing. According to the Kabat database (ElvinA.Kabat. "Sequences of Proteins of Immunological Interest". 1991), the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 in the light chain variable region sequence are determined as SEQ ID NO: 5, SEQ ID NO: 5 in the sequence listing, respectively. ID NO:6 and SEQ ID NO:7. The amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 in the heavy chain variable region sequence are respectively shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing.

Application of the above monoclonal neutralizing antibody E4-2 against Staphylococcus aureus eLtaS protein in the preparation of medicaments against Staphylococcus aureus infection.

Beneficial effects of the present invention: the present invention uses a set of designed primers to successfully clone antibody light and heavy chain variable region genes from cultured anti-eLtaS monoclonal antibody E4-2 hybridoma cells. The resulting light and heavy chain variable region genes encode the correct mouse antibody variable regions. The monoclonal antibody of the present invention is based on the light and heavy chain variable region genes of the anti-eLtaS monoclonal antibody E4-2 that has been cloned above, and can construct and express a variety of small molecule genetically engineered antibodies, such as single chain antibodies, single domain antibodies, Chimeric antibodies, Fab antibodies, antibody fusion proteins, etc., are used as therapeutic drugs against Staphylococcus aureus infection, and have very broad application prospects.

Description of drawings

Figure 1 is an agarose electrophoresis analysis diagram of the eLtaS gene fished product; where lane 1 is the DNA molecular weight standard, and lane 2 is the eltaS gene PCR fished product.

Figure 2 is the SDS-PAGE results pattern of eLtaS for immunization; where lane 1 is the protein molecular weight standard, and lane 2 is the purified eLtaS protein.

Figure 3 is the SDS-PAGE result pattern of the monoclonal antibody purified by Protein G; where lane 1 is the protein molecular weight standard, lane 2 is the neutralizing monoclonal antibody purified by Protein G, A is the heavy chain, B is the light chain .

Figure 4 Western-blot detection of the effect of E4-2 on the LTA synthesis of Staphylococcus aureus; where lanes 1, 2, and 3 are the addition of PBS, E4-2, and control IgG during bacterial culture.

Figure 5 ELISA identification monoclonal antibody E4-2 affinity line graph.

Figure 6 Western-blot identification pattern of the binding properties of monoclonal antibody E4-2; where lane 1 is the detection result of monoclonal antibody E4-2, and swimming lane 2 is the detection result of control mouse IgG, the results show neutralizing monoclonal antibody E4- 2 can recognize eLtaS linear epitope.

Figure 7: The effects of E4-2 against Staphylococcus aureus infection in the acute peritonitis model; the results show that the neutralizing monoclonal antibody E4-2 can effectively resist the infection of Staphylococcus aureus in mice.

Figure 8 is a graph showing the effect of E4-2 against Staphylococcus aureus infection in pneumonia models; the results show that the neutralizing monoclonal antibody E4-2 can effectively resist the infection of Staphylococcus aureus in mice.

Fig. 9 Electropherogram of the RNA extraction results of the anti-eLtaS monoclonal antibody E4-2 hybridoma cells.

Figure 10 is a PCR result map of the light and heavy chain genes of eLtaS neutralizing monoclonal antibody E4-2 hybridoma cells, wherein lane 1 is DL2000; lane 2 is the gene of the light chain; lane 3 is the gene of the heavy chain. The gene sizes are about 690bp and 780bp, respectively.

detailed description

The present invention will be further described below by accompanying drawing and specific embodiment, can understand content of the present invention more easily by referring to following embodiment, and these embodiments are only for further illustration, do not limit protection scope of the present invention.

Embodiment 1 Construction of anti-eLtaS monoclonal antibody hybridoma cell line

Materials: Freund's complete adjuvant and incomplete adjuvant, TMB is a product of Sigma, 20% fetal bovine serum is a product of Beijing Yuanheng Shengma Biotechnology Research Institute, serum-free RPMI1640 is a product of Gibco, SP2/0 cells are from ATCC Imported and kept in our laboratory, Balb/c mice and Kunming mice were purchased from the Experimental Animal Center of Academy of Military Medical Sciences. The rest of the reagents were purchased from the market.

Method result:

1. Expression and purification of eLtaS protein. The eLtaS gene in the genome of Staphylococcus aureus 8325-4 was fished out with primers specific to the eLtaS coding region (as shown in Figure 1 for electrophoresis detection), and cloned into the pET-28a vector to induce, express, and purify the eLtaS protein (its SDS-PAGE results are shown in Figure 2).

2. Immunization of Balb/c mice. Select 6 female Balb/c mice aged 4-6 weeks, subcutaneously immunize with 100 μg eLtaS protein in the groin, add Freund's complete adjuvant for the first injection, add Freund's incomplete adjuvant for the second injection, and immunize once every 3 weeks, A total of 3 times of immunization. Blood was collected from the tail vein after the third immunization, and the antibody production was detected by ELISA. Three days before fusion, 100 μg of eLtaS protein was administered intraperitoneally for a booster immunization, and fusion was performed on the third day.

3. Cell fusion. The immunized mice were sacrificed by decapitating the eyeballs, and the splenocytes of the mice were aseptically removed, and cell fusion was carried out according to conventional methods. Specific methods: ①The immunized mice were sacrificed by taking off the eyeballs and bleeding, soaked in 75% alcohol for 3 minutes, aseptically removed the spleen, and ground the single cell suspension with 200-mesh steel mesh, washed twice with serum-free RPMI1640 and counted; ② Collect SP2/0 cells in the logarithmic growth phase, wash twice with serum-free RPMI1640 and count; ③ mix the two kinds of cells according to the ratio of SP2/0 cells: splenocytes = 1:5, wash once with RPMI1640, and discard Supernatant, gently break up the cells; ④ Slowly add 1ml of 50% PEG (MW1500) solution within 1min, put in 37℃ water bath for 1min; ⑤ Add serum-free RPMI16401ml, 5ml, 10ml within 1min, 2min, 2min, 5min , 10ml; ⑥Centrifuge at 800r/min for 7min, discard the supernatant, and suspend the cells as gently as possible; ⑦Add HAT(Sigma)-RPMI1640 culture medium containing 20% FCS, adjust the cell concentration to 2×106/ml, mix well Afterwards, add it dropwise to a 96-well culture plate (Gibco) with trophoblast cells (1×104 cells/well), 100 μl/well, and culture in a 5% CO ₂ incubator at 37°C.

4. Anti-eLtaS monoclonal antibody hybridoma cells were screened by ELISA. ELISA plates were coated with 10 μg/ml eLtaS, overnight at 4°C and blocked. The culture supernatant of the cells to be tested (1h at 37°C, washed four times with PBST), and 50 μl of HRP-GAM diluted at 1:40000 (45 minutes at 37°C, washed four times with PBST) were added sequentially. After developing color with TMB substrate, the absorbance was measured at a wavelength of 450 nm.

5. Cloning of hybridoma cells. Neutralizing antibody cell clones were screened and subcloned repeatedly until all hybridoma cell culture supernatants were detected as 100% positive. Cloning of hybridoma cells using limiting dilution method: (1) Prepare trophoblasts on the day of cloning or 1 day before: Kunming mice are treated by denecking, skin is soaked in 75% alcohol to disinfect the skin, abdominal skin is aseptically peeled off, and 5ml1640 is extracted with a syringe The culture solution was injected into the peritoneal cavity of the mouse, after repeated washing, the peritoneal washings were sucked out, diluted with 20% fetal bovine serum 1640 culture solution, and dropped into a 96-well plate, about 0.1ml per well. (2) Take a few hybridoma cells to be cloned and transfer to another sterile test tube, and count them accurately. (3) Subcloning by limiting dilution. (4) Place the culture plate in a 5% CO ₂ incubator at 37°C for culture, and cell clones can be observed under a microscope in about 5 days. Change the medium at the right time, test, take the positive monoclonal cell line for expansion culture, and freeze the cell line in time.

Through this part of the work, eight anti-eLtaS monoclonal antibody hybridoma cell lines were constructed.

Example 2 Screening and Identification of Anti-eLtaS Neutral Monoclonal Antibody Hybridoma Cell Lines

Material: Protein G Sepharose CL4B column: product of GE healthcare company; Ibid.

Method result:

1. Purification of eLtaS monoclonal antibody. Add 1 ml of pH8.0, 0.1moL/L phosphate buffer to 2 ml of mouse ascites and adjust the pH to 9 with pH9.0, 1moL/L TRIS-HCL. Add the mouse ascites to the Protein G Sepharose CL4B column protein column that has been equilibrated with 0.1moL/L phosphate buffer solution pH8.0, and wash the column with the above buffer solution until no foreign protein can be detected in the effluent. Elute with citric acid buffer of pH 3.0, collect the effluent, and immediately neutralize with 1moL/L TRIS-HCL pH 8.5 buffer, and dialyze with pH 7.2, 0.01M PBS for 72h. Samples were taken for SDS-PAGE detection (Figure 3).

2. Screening of neutralizing antibodies. Use brain heart infusion medium to culture Staphylococcus aureus 8325-4, 37°C, 200rpm, add 1640 medium and 100ul supernatant of different cell lines to 1ml of culture supernatant, culture the bacteria for 6 hours and then centrifuge to collect the bacteria. Add 4 times the volume of glass beads to shake and break the bacteria, centrifuge to collect the supernatant and use 15% SDA-PAGE to separate the obtained lysate, use the semi-dry transfer method to transfer the separated product to PVDF membrane (20V, 30min), use LTA specific antibody The differences in bacterial LTA levels after different treatments were detected, among which E4-2 had the activity of blocking bacterial LTA production ( FIG. 4 ).

3. Determination of E4-2 immunoglobulin subtypes of hybridoma cells. Using goat anti-mouse IgG1, IgG2a, IgG2b and IgG3, the hybridoma cells concentrated culture supernatant was used for biphasic agar diffusion experiments, which proved that the immunoglobulin subtype secreted by E4-2 hybridoma cells was IgG1.

4. Affinity identification of eLtaS monoclonal antibody E4-2. ELISA plates were coated with 10 μg/ml eLtaS, overnight at 4°C and blocked. Add different concentrations of E4-2 to be tested (1h at 37°C, wash the plate 4 times with PBST), and 50 μl of HRP-GAM diluted 1:40000 (45 min at 37°C, wash the plate 4 times with PBST). After color development with TMB substrate, the absorbance was measured at a wavelength of 450 nm (Figure 5).

5. Specific identification of eLtaS monoclonal antibody E4-2. The eLtaS protein was separated by SDS-PAGE (Figure 5) and transferred to a nitrocellulose membrane, then blocked overnight at 4°C by adding PBS containing 5% milk powder dropwise, and washing the membrane 3 times with PBS containing 0.5ml/L Tween-20. Cut the membrane to the same width, add eLtaS monoclonal antibody E4-2 dropwise (37°C for 1 hour, wash the membrane 3 times with PBST, then wash 3 times with TBS containing 0.5ml/L Tween-20) and HRP-GAM IgG (Combine at 37°C for 1 hour, wash the membrane 3 times) After color development with DAB, observe the results. The results showed that E4-2 was an eLtaS-specific monoclonal antibody ( FIG. 6 ).

Through this part of the work, anti-eLtaS neutralizing monoclonal antibody E4-2 was screened, and the secreted immunoglobulin subtype was IgG1. The monoclonal antibody E4-2 has a very high affinity to the eLtaS protein, and the monoclonal antibody E4-2 can specifically recognize the eLtaS linear epitope.

Example 3 The protective experiment of monoclonal antibody E4-2 to CD1 mice

Materials: Same as Examples 1 and 2 above.

Method result:

1. The acute peritonitis model was used to detect the effect of anti-eLtaS monoclonal antibody E4-2 against Staphylococcus aureus infection. 6-week-old female CD1 mice were treated with intraperitoneal injection of PBS, control IgG (100 μg) and E4-2 (100 μg); the single clone of Staphylococcus aureus 8325-4 was picked and cultured in brain heart infusion medium , 37°C, 200rpm for 12hr. After the bacteria were collected, the bacteria were washed once with PBS, and the concentration of the bacteria was adjusted to OD600=1.5, and 500 μl was injected intraperitoneally into mice, and the number of surviving mice at different time points was observed ( FIG. 7 ).

2. The pneumonia model was used to test the anti-eLtaS monoclonal antibody E4-2 against Staphylococcus aureus infection. 6-week-old female CD1 mice were treated with intraperitoneal injection of PBS, control IgG (100 μg) and E4-2 (100 μg); single clones of Staphylococcus aureus 8325-4 were picked and cultured in brain heart infusion medium , 37°C, 200rpm for 12hr. After collecting the bacteria, wash the bacteria once with PBS, adjust the bacterial concentration to OD600=1, draw 100 μl through the trachea and inject it into the lungs of the mice, kill the mice 72 hours later, take the lung tissue to prepare slices, and observe the inflammation by HE staining Response levels (Figure 8).

Example 4ELtaS Neutralizing Monoclonal Antibody Hybridoma E4-2 Light and Heavy Chain Gene Capture

Materials: Primers: MuLC4: see sequence 11 in the sequence listing; MuCκ: see sequence 12 in the sequence listing; MuHC1: see sequence 13 in the sequence listing; MuIgG1: see sequence 14 in the sequence listing. DNA fragment purification kit: product of OMEGA Biotechnology Company; T4DNA ligase: product of New England Biolabs; vector PGEM Teasy: product of Promega Company; TRIzol: product of Invitrogen Company; competent bacteria JM109: purchased from Promega Company. Yu ditto.

Method result:

Take 5×106-107 cells of neutralizing monoclonal antibody E4-2 in the logarithmic growth phase, centrifuge to remove the supernatant, and pop the cells evenly. Add 1ml TRIzol and pipette repeatedly to fully lyse the cells. After shaking for 5 minutes, add 0.2ml chloroform, shake for 15 seconds, place at room temperature for 2-3 minutes, centrifuge at 12000r/min at 2-8°C for 15 minutes, and take the supernatant into another new tube Add 500 μl of isopropanol to mix well, then place at room temperature for 10 minutes, and centrifuge at 12,000 r/min at 2-8°C for 10 minutes. The precipitate was washed with 75% ethanol, and after drying, the precipitate was dissolved with 20 μl RNase-free deionized water ( FIG. 9 ).

Take a solution containing 1 μg of total RNA, add AMV5×buffer 4 μl, Oligo (dT) (500ng/μl) 0.5 μl, 2.5 mmol/L dNTP 2 μl, Rnasin (50U/μl) 0.5 μl, deionized water to 20 μl, Reverse transcriptase 2-5U, extended at 42°C for 1 hour. Denature at 95°C for 5 minutes, place in an ice bath, and the product is the first strand of cDNA. Use 2 pairs of specific primers MuLC4 and MuCκ, MuHC1 and MuIgG1, add 2 μl of reverse transcription product, 10×buffer 2 μl of Taq enzyme, 1 μl of upstream and downstream primers, 1 μl of 2.5 mmol/L dNTP, and add Taq enzyme in a 20 μl PCR reaction system 1-2U, make up to 20μl with deionized water. Denaturation at 95°C for 2 minutes, cycle parameters: 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C, a total of 30 cycles, and extension at 72°C for 10 minutes (Figure 10).

Use to separate the DNA fragments to be recovered, cut off the gel blocks containing the target DNA fragments under long-wave ultraviolet light, put them into a centrifuge tube, add three times the gel volume of gel solution, and dissolve the gel blocks completely in a 55°C water bath. DNA fragments were recovered with a DNA fragment purification kit and the purified DNA fragments were placed in an aqueous solution, and the recovered PCR product was mixed with the carrier PGEM Teasy in a 2:1 ratio (molar ratio) in T4 DNA ligase buffer, and then added with 0.5 U's T4 DNA ligase was ligated overnight at 16°C, and the total volume of the ligation reaction was 10 μL.

Take 10 μl of the connection solution, add 200 μl of competent bacteria JM109 and mix gently, place in ice bath for 30 minutes, heat shock in 42°C water bath for 90 seconds, quickly transfer to ice bath for 2 minutes, add 800 μl LB medium, transfer to 37°C for constant temperature shaking Bed, shake at a speed of 150 rpm for 45 minutes, centrifuge at 4000r/min for 1 minute, discard 800 μl supernatant, take the precipitate and apply it to a solid LB plate containing Amp (final concentration is 100 μg/ml), and place the plate upside down Incubate at 37°C for 12-18 hours.

A single clone was picked from the above plate and inoculated in LB medium containing ampicillin (100 μg/ml). 37 ℃ constant temperature shaker 170rpm, shaking culture overnight. Take 3ml of the bacterial solution and add it to a 1.5ml Eppendorf tube, centrifuge at 10000rpm for 1min, and discard the supernatant. Resuspend the precipitated bacteria in 100 μL of solution I, add 200 μL of freshly prepared solution II, and gently invert several times until the liquid becomes clear. Then, add 150 μL of solution III, and gently invert the solution several times to mix the liquid evenly, at this time a large amount of white flocculent precipitates appear. Centrifuge at 12000rpm for 5min at 4°C, add the supernatant to another Eppendorf tube, add an equal volume of Tris-HCl saturated phenol, shake vigorously, centrifuge at 12000rpm for 5min, and transfer the upper aqueous phase to a new tube. Then add 500 μL chloroform and extract again. Thereafter, carefully absorb the upper aqueous phase, transfer it to a new tube, add 2 times the volume of absolute ethanol to mix, and place it at -20°C for 3 hours. Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, wash the precipitate twice with 70% ethanol, dry at room temperature for 20 min, dissolve in 40 μL sterile double distilled water, and perform PCR identification and DNA sequencing analysis.

The vector containing the light and heavy chain genes of the eLtaS neutralizing antibody E4-2 was constructed, and after sequencing analysis and sequence alignment, it was the mouse immunoglobulin light and heavy chain genes (SEQ ID NO: 1 and SEQ ID NO: 2).

Claims (2)

1. a kind of monoclonal neutrality antibody E4-2 of anti-Staphylococcus aureus eLtaS albumen, it is characterised in that the Dan Ke SEQ ID NO in the light chain protein matter molecule variable region encoding gene of grand neutrality antibody E4-2 such as sequence table：Shown in 1, heavy chain SEQ ID NO in protein molecule variable region encoding gene such as sequence table：Shown in 2；The monoclonal neutrality antibody E4-2's SEQ ID NO in light chain protein matter molecule variable region amino acid sequence such as sequence table：Shown in 3, heavy chain protein matter molecule variable region SEQ ID NO in amino acid sequence such as sequence table：Shown in 4；

SEQ ID NO in the amino acid sequence of the complementary determining region CDR1 of the light chain protein matter molecule variable region such as sequence table：5 It is shown, SEQ ID NO in the amino acid sequence of complementary determining region CDR2 such as sequence table：Shown in 6, the amino of complementary determining region CDR3 SEQ ID NO in acid sequence such as sequence table：Shown in 7.

SEQ ID NO in the amino acid sequence of the complementary determining region CDR1 of the heavy chain protein matter molecule variable region such as sequence table：8 It is shown, SEQ ID NO in the amino acid sequence of complementary determining region CDR2 such as sequence table：Shown in 9, the amino of complementary determining region CDR3 SEQ ID NO in acid sequence such as sequence table：Shown in 10.

2. the monoclonal neutrality antibody E4-2 of anti-Staphylococcus aureus eLtaS albumen described in claim 1 is preparing anti-gold Application in the medicine of staphylococcus aureus infection.