CN106811443A - The preparation and application of Apolipoprotein C-III monoclonal antibody - Google Patents

Abstract

The invention discloses a kind of preparation and application of mouse anti human apoC-III monoclonal antibodies.Specifically, the present invention provides the amino acid sequence of apoC-III antigen polypeptides, and preserving number is the mouse hybridoma cell strain WLC001 of CCTCC No.C2015191, and the thus apoC-III monoclonal antibodies of hybridoma cell strain generation.The invention further relates to application of the apoC-III monoclonal antibodies in apoC-III Western blottings are detected.

Description

The preparation and application of apoC-III monoclonal antibodies

Technical field

The present invention relates to technical field of bioengineering.Specifically, the present invention relates to a kind of monoclonal antibody and the monoclonal is secreted The hybridoma cell strain of antibody, and application of the monoclonal antibody in apoC-III Western blottings are detected.

Background technology

The micro-molecule glucoprotein that apoC-III is made up of 79 amino acid, is that content is most abundant in apoC race One class, mainly synthesizes in liver, and only fraction synthesizes in small intestine, and apoC-III is distributed mainly on chylomicron, extremely low In density lipoprotein and HDL.ApoC-III genes are located at No. 11 chromosome long arm q23 areas, are carrying fat egg In vain between A-I and Apolipoprotein A-IV, three collectively constitutes a gene family.Insulin is received in the transcription of apoC-III And the regulation and control of many factors such as peroxisome proliferation-activated receptors.

ApoC-III has the function of suppressing lipoprotein lipase and hepatic lipase activity, can suppress liver intake rich in triglycerides Lipoprotein and lipidolysis, be the main regulatory factors of plasma triglyceride level.ApoC-III is in plasma high density fat Mutually shifted between albumen and VLDL, in the serum of blood fat normal individual, apoC-III is most of and high Density lipoprotein is combined, and in primary hypertriglyceridemiapatients patients serum, then major part is combined with VLDL.It is high sweet Oily three ester mass formed by blood stasis are the most common abnormalities of sugar/lipid metabolism diseases of China, are one of the independent hazard factors for causing coronary heart disease.With me The increase of state's obese people, the quantity of primary hypertriglyceridemiapatients patients is also increasing year by year, and its morbidity is mainly due to rich in glycerine The dyslipoproteinemia of three esters causes, and apoC-III contents are significantly higher than normal population, apolipoprotein in patients blood plasma The promising tumor marker that the hypertriglyceridemia that C-III turned into develops, prepares apoC-III monoclonal antibodies and can be used for out The diagnostic reagent of hypertriglyceridemia risk is sent out, the pathogenesis to inquiring into hypertriglyceridemia is significant.

ApoC-III monoclonal antibodies are had at present, but it is higher to still need dilution factor, and more preferably, expression quantity is more for compatibility Monoclonal antibody.Activity apoC-III monoclonal antibodies high are obtained to grind the clinical diagnosis of hypertriglyceridemia and science Study carefully significant.

The content of the invention

It is an object of the present invention to provide one plant can stabilization, efficient secretion apoC-III monoclonal antibodies hybridoma Strain and preparation method thereof；

It is a further object to provide apoC-III monoclonal antibodies；

A further object of the present invention is that described apoC-III monoclonal antibodies are applied into apoC-III Diagnosis of Sghistosomiasis In mark detection.

Above-mentioned purpose of the invention is realized by following technical approach：

A kind of apoC-III antigen polypeptides, amino acid sequence such as SEQ ID NO：Shown in 1.Synthesize the polypeptide, and with load Body protein is coupled, coupled product immune mouse, draws myeloma cell's suspension and the spleen B from coupled peptide immune mouse Lymphocyte suspension carries out cell fusion, and limiting dilution assay obtains monoclonal, and ELISA screening positive hybridoma cells obtain institute State hybridoma cell strain.Its microbial preservation is numbered：CCTCC N0.2015191；Depositary institution：Chinese Typical Representative culture Collection.

Mouse ascites are prepared, Protein G column chromatography apoC-III monoclonal antibodies, ELISA method determines antibody purification Potency, Western blot verify the sensitivity and specificity of the monoclonal antibody.

Brief description of the drawings

Fig. 1 is apoC-III immunogenicities, hydrophilic and hydrophobic and surface accessibility analysis result, is shown from synthesis in square frame Polypeptide sequence fragment.

Fig. 2 is the apolipoprotein in immune-blotting method human plasma and liver cell HepG2 with apoC-III monoclonal antibodies as primary antibody C-III protein expression situations.Swimming lane 1：Human normal plasma；Swimming lane 2：HepG2 cells.

Fig. 3 is the invention flow of this experiment.

Specific embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for explaining the present invention, and It is non-for limiting the present invention.On the premise of technical scheme is not departed from, ordinary skill made for the present invention Any change that personnel easily realize is fallen within scope of the presently claimed invention.

Embodiment 1：ApoC-III antigen polypeptides are designed

1.1 according to apoC-III GenBank accession number GI：4557323, obtain human apolipoprotein C-III protein sequences (UniProt：P02656), 79 amino acid are contained.

1.2 use ProtParam (http：//web.expasy.org/protparam/) on-line analysis apoC-III protein characteristics, point Analysis the results are shown in Table 1, and the molecular weight of the albumen is 8.7646KD, and isoelectric point is 4.72, is acidic protein.

ApoC-III basic physical and chemical the analysis results of table 1

1.3 use DNAstar software analysis apoC-III protein immunogenics, hydrophilic and hydrophobic and surface accessibility, as a result table The bright nearly C-terminals of apoC-III have 16 amino acid antigenicities, hydrophily and surface accessibility relatively strong (see Fig. 1).

1.4 apoC-III antigen polypeptides are screened

16 amino acid sequences that will be filtered out are analyzed through Blastp, and final screening polypeptide sequence is： KFSEFWDLDPEVRPTS (60aa-75aa, SEQ ID NO：1).

Embodiment 2：ApoC-III antigen polypeptides synthesize and are coupled

2.1 apoC-III Peptide systhesis

Polypeptide is synthesized by Shanghai gill biochemical corp, and purity is 93.85%.

2.2 polypeptides and carrier protein couplet

Take 5mg polypeptides and be dissolved in 50 μ l DMSO, add coupling buffer to be diluted to final volume for 1ml, take out 200 μ l and use In ELISA detections, after adding the OVA solution that 400 μ l concentration are 10mg/ml to mix in remaining 800 μ l polypeptide solutions, The EDC that 200 μ l concentration are 10mg/ml is added, is mixed immediately, room temperature reaction 2h.

The ultrafiltration of 2.3 coupled products and packing

Coupled product is diluted to 4ml with PBS, ultrafiltration is carried out to coupled product with 10KD Milipore ultra-filtration centrifuge tubes, often Filtrate in down tube is discarded after secondary ultrafiltration, when remaining liq about 1ml in upper pipe, continues to carry out ultrafiltration centrifugation after being diluted with PBS, After loop ultrafiltration 4 times, remaining liq 1ml adds PBS to be diluted to 3ml mixings in taking pipe, using NanoDrop light splitting light Degree meter determine coupling after protein concentration be 1.272mg/ml, with 0.22 μm of filter filtration sterilization after, often pipe packing coupling after albumen 500 μ l, -80 DEG C of preservations.

Embodiment 3：The preparation of the hybridoma of anti-apoC-III

3.1 immune animals

6 6-8 week old cleaning grade BALB/c mouses are chosen, the coupling protein after packing is taken with isometric corresponding adjuvant emulsion Immune mouse after mixing, immune programme for children is as shown in table 2.

The animal immune program of table 2

3.2ELISA methods detect serum antibody titer

Blood is taken within 7 days after third time is immune to immune mouse afterbody, ELISA detection serum antibody titers are comprised the following steps that：

With the apoC-III polypeptides (2 μ g/ml) of synthesis for antigen coat ELISA Plate, per the μ l of hole 100,4 DEG C of overnight incubations, Abandon coating buffer, add the skimmed milk powers of 200 μ l 15%, 37 DEG C of closing 1h to wash per hole 3 times, add with 1: 1000,1: 2000, The blood serum sample of 1: 4000,1: 8000,1: 16000,1: 32000 dilution proportion, per the μ l of hole 100, is made with non-immune serum It is negative control, used as blank, 37 DEG C of incubation 1h are not washed 3 times increase serum, add the sheep anti mouse two of HRP marks Anti- (1: 5000), 37 DEG C of incubation 1h, is washed 3 times, and the μ l of substrate developer 50 are added per hole, is stored at room temperature 5min, is added per hole Enter the μ l of terminate liquid 50, the OD values at ELIASA detection 450nm wavelength.Treat that gaging hole OD values are more than or equal to negative control hole 2.1 times be judged as the positive, meet the maximum dilution multiple as serum antibody titer of the testing sample of this condition, the results are shown in Table 3.

The serum antibody titer of table 3

3.3 cell fusions

Choosing antibody titer highest mouse carries out cell fusion, and eyeball excise depletion method is put to death mouse, spleen is taken out under aseptic condition Dirty, polishing prepares mouse boosting cell suspension, the SP2/0 that myeloma cell is originated using BALB/c mouse, by mouse boosting cell Mix with 10: 1 with myeloma cell, and add 1ml50% polyethylene glycol to promote cell fusion.Using HAT Selective agar mediums Carry out the selectivity culture and screening of hybridoma.ELISA detects Hybridoma Cell Culture supernatant, is entered using limiting dilution assay The clone of row positive hybridoma cell, obtains the hybridoma cell strain of stably excreting apoC-III.Hybridoma is expanded Culture, is stored in China typical culture collection center (abbreviation CCTCC, address：Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University's collection, postcode 430072), preserving number is CCTCC No.2015191, and Classification And Nomenclature is：Human apolipoprotein C- The strain of III monoclonal antibody hybridoma cells.

Embodiment 4：A large amount of preparation and purifications of monoclonal antibody

It is prepared by 4.1 ascites

Take 8 week old BALB/c mouses, the pretreatment of Intraperitoneal injection norphytane, 1 week pneumoretroperitoneum injection hybridoma, 7-10 days After there is ascites, ascites 10ml is gathered under aseptic condition.

4.2 ascites are purified

By ascites under the conditions of 4 DEG C, 10000rpm centrifugation 10min remove lipid material, collect supernatant and are ascites Dan Ke Grand antibody.With PBS by ascites with 1: 2 dilution proportion, 10000rpm is centrifuged 25min, and the ascites after centrifugation is with 0.22 μm The filtering with microporous membrane in aperture, crosses Protein G affinity columns, and eluent (Gly-HCl, pH 2.7) is eluted, ultrafiltration AC after concentration after NanoDrop spectrophotometric determinations affinity purification is 3.7mg/ml to determine AC.

Embodiment 5：ELISA method detection antibody titer

With the apoC-III polypeptides (2 μ g/ml) of synthesis for antigen coat ELISA Plate carries out ELISA, specific method is with implementation Example 3.2, testing result is as shown in table 4.

Anti- apoC-III antibody titers ELISA the testing results of table 4

Embodiment 6：ApoC-III monoclonal antibody Western blot application detections

Western blot detect the expression of apoC-III in human serum and HepG2 cells, comprise the following steps that：

Human plasma and HepG2 cells are taken respectively, after being cracked, extraction sample are measured with BCA determination of protein concentration kits Protein concentration, take after 20 μ g loadings carry out SDS-PAGE electrophoresis, 200mA constant currents 1h goes to pvdf membrane, uses TTBS Buffer 5% (M/V) skimmed milk power closes pvdf membrane, room temperature closing 1h.The pvdf membrane that will have been closed is put into miscellaneous Hand in bag, the anti-apoC-III monoclonals for obtaining are purified in the embodiment 4 for adding the dilution of 5% (M/V) skimmed milk power and is resisted Body (1: 500), is sealed, 4 DEG C of overnight incubations with film laminator.Pvdf membrane is taken out, and is put into TBST, and 4 are washed on shaking table It is secondary, each 5min.Add the sheep anti mouse secondary antibody (1: 5000) of the HRP marks of 5% skim milk dilution, 37 DEG C of incubations 45min, adds ECL colour developings to detect the expression of apoC-III.Result is shown in Fig. 2.Anti- load fat egg in the present invention White C-III monoclonal antibodies can detect the single band of molecular weight 11KD or so in human plasma and HepG2 cell pyrolysis liquids, Relative molecular weight is expected with human apolipoprotein C-III to be consistent.

Claims (4)

1. a kind of hybridoma cell strain, it is characterised in that its microbial preservation number is：CCTCC NO.C2015191.

2. a kind of monoclonal antibody for apoC-III, is produced as hybridoma cell strain secretion described in claim 1.

3. application of the monoclonal antibody described in claim 2 in apoC-III Western blottings are detected.

4. the preparation method of the hybridoma cell strain described in a kind of claim 1, it is characterised in that comprising step：

1. according to human apolipoprotein C-III (UniProt：P02656) protein characteristic, filters out polypeptide sequence： KFSEFWDLDPEVRPTS(60aa-75aa)；

2. according to the apoC-III antigen protein synthesis polypeptide SEQ ID NO of expression：1, and and carrier protein couplet；

3. by the polypeptide immune mouse after coupling, ELISA detection serum antibody titers；

4. antibody titer highest mouse spleen bone-marrow-derived lymphocyte suspension is chosen thin with the SP2/0 myeloma that BALB/c mouse is originated Born of the same parents are merged；

5. selectivity culture hybridoma, ELISA detection hybridoma potency, screening positive cell strain, obtain described hybridization Tumor cell strain.