CN106399294B - A kind of preparation of the monoclonal antibody 7H8 of anti-human Procalcitonin protein N terminal epitope - Google Patents

Abstract

The invention discloses the hybridomas of monoclonal antibody 7H8 for secreting anti-human PCT protein N terminal amino acid sequence a kind of, by people's PCT complete genome sequence after the prokaryotic expression codon optimization in Escherichia coli, BALB/c mouse is immunized using the people PCT albumen of purifying as antigen.By cell fusion and screening, obtain can stably excreting be directed to people PCT protein N terminal epitope monoclonal antibody hybridoma, be named as 7H8.7H8 monoclonal antibody can be applied to the detection of blood-serum P CT concentration, and the sensitivity and specificity with height.7H8 antibody identifies people PCT protein N terminal amino acid sequence, it avoids and cross reaction occurs with internal calcitonin, improve the specificity of identification PCT albumen, a kind of good new material is provided for the exploitation corresponding diagnostic reagent of PCT, it can be widely used in cell biology and immunological testing, there is good application value.

Description

A kind of monoclonal antibody 7H8's of anti-human Procalcitonin protein N terminal epitope Preparation

Technical field

The invention belongs to field of biotechnology, are related to a kind of monoclonal antibody, and in particular to a kind of anti-human PCT albumen of secretion The preparation of the hybridoma and its monoclonal antibody specific of the monoclonal antibody of N-terminal epitope.

Background technique

Calcitonin I gene of the Procalcitonin (procalcitonin, PCT) on No. 11 chromosome of people, the base After the accurate translation Preprocalcitonin (proprocalcitonin) in parafollicular cells of thyroid gland, sheared by endogenous polypeptide enzyme The signal peptide for falling 25 amino acid of N-terminal becomes peptide material before the calcitonin without hormonal activity containing 116 amino acid, as Procalcitonin (PCT).PCT is made of N-terminal (1-57aa)-calcitonin (60-91aa)-katacalcin (96-116aa) three parts, It is connected respectively by 2 peptides (- Lys-Arg-) and 4 peptides (- Gly-Lys-Lys-Arg-), relative molecular mass is about 13 KDa, and PCT is again The polypeptide with hormonal activity comprising 32 amino acid, as calcitonin are hydrolyzed by a series of processing of intracellular proteases (calcitonin, CT).So far, seldom about the basic research of Procalcitonin, source and pathophysiological role are not yet Clear, biological function in vivo is also unknown.But according to changes of contents of the PCT in serum and inflammatory reaction Correlation, PCT are applied in various clinical detection and observation.

Under normal physiological conditions, the calcitonin I gene expression outside thyroid gland is suppressed, and is only limitted in thyroid gland and lung It is expressed in cell, therefore, PCT content is few in Healthy People blood, usually less than 0.10 ng/mL.Serious fungal, bacterium, parasitism Calcitonin I gene table in the meeting inducing systemic Various Tissues such as insect infection and pyemia and multiple organ failure and cell It reaches, the content of PCT in serum is caused to will rise rapidly, PCT concentration can even rise quickly to hundreds of to ten thousand times of normal level, but CT Content then remain unchanged or slightly increase.However, allergy and virus infection and autoimmune disease will not cause blood Middle PCT raising, localized bacterial infection and chronic inflammation not will lead to the significant change of PCT level equally.Therefore, PCT is important Marker of inflammation, inflammatory reaction caused by bacterium infection and virus infection, autoimmune disease can be distinguished.Also, with it His inflammation indicator is compared, and PCT has better sensitivity and specificity.PCT can be detected for 2 hours after infection It arrives, reaches peak value within 6-12 hours after infection, 12-24h is plateau, for 24 hours rear concentration decline, and half-life period is longer, is that 20-24 is small When, high level can maintained for a period of time.PCT level and gradient of infection are positively correlated simultaneously, and PCT concentration is higher to be meaned to be felt Dye degree is more serious, therefore PCT level all has important meaning to the diagnosis and treatment prognosis etc. of the occurrence and development of inflammation Justice.Other inflammatory factors such as IL-6, IL-10, TNF-α reach peak value for 2 hours after infection, and decline rapidly, are not easy to be detected It arrives；And c reactive protein (CRP) just reaches peak value for 24-48 hours after infection, and concentration is still kept compared with Gao Shui after inflammation control It is flat, it is unfavorable for early diagnosis and therapy evaluation.In clinical conditions, traditional detection index such as body temperature, white blood cell count(WBC) (WBC) sensitivity and specificity be not high, and PCT is compared with past biological marker, with ideal standard True property and specificity are an ideal infection detection marker, facilitate clinical early diagnosis and formulate reasonable treatment Scheme.

The method of PCT detection inflammation has obtained the license of Food and Drug Adminstration of the US (FDA).In early days, detection PCT is main Using gel chromatography analytic approach and high performance liquid chromatography, both methods is not only time-consuming, and high operation requirements, is not easy automatic Change.In recent years, the detection of PCT mostly uses immunological method to carry out, and has high specificity, high sensitivity and easy to operate etc. excellent Point, including double antibodies sandwich immunochemiluminescence method, colloidal gold method and radioimmunology etc..The premise of all these immunological methods All be the monoclonal antibody specific prepared for PCT, PCT content is extremely low in human normal plasma, 0.2 ng/mL with Under, it is therefore, very high to parameter requests such as the affinity of prepared antibody.And antibody used in detection architecture is all from foreign countries at present Major company, it is expensive.Therefore, PCT monoclonal antibody specific is prepared, provides critical materials for the immune detection of PCT, For expanding its clinical application, medical treatment cost is reduced, is of great significance.

Summary of the invention

Currently, the monoclonal antibody for PCT albumen of domestic independent research, mainly using the prediction epitope peptide of synthesis Duan Zuowei mice immunized with antigen is obtained by hybridoma technology, and the antigen peptide fragment used is 34-60aa and 75- respectively 88aa sequence exists with calcitonin sequence (60-91aa) and intersects, therefore can not distinguish PCT and CT.The purpose of the present invention is The monoclonal antibody of the amino acid sequence of specific recognition PCT protein N terminal is filtered out, cross reaction is reduced, is allowed to wide In the general clinical detection and basic research applied to PCT.

In view of the deficiencies of the prior art, the present invention passes through repetition test, using prokaryotic expression technology, expresses simultaneously Purification of Human PCT full-length proteins, people's PCT albumen after purification have very high purity, and Western blot experiment shows that the albumen has very Strong immunoreactivity.People's PCT protein immunization BALB/c mouse after purification screens 13 plants of secretions by hybridoma technology The hybridoma of anti-human PCT protein monoclonal antibody, it is identified, wherein the site of 1 plant of identification is located at PCT protein N terminal 4- 18aa sequence.The present invention has carried out Identification of Biological Characteristics to this strain of hybridoma (hybridoma cell strain 7H8).

Present invention provide the technical scheme that it is anti-to screen a kind of monoclonal for secreting anti-human PCT protein N terminal epitope The hybridoma of body, the hybridoma (hybridoma cell strain 7H8) are Chinese Typical Representative training on September 15th, 2015 Supporting the preservation of object collection (Wuhan) CCTCC institute, (preservation address is: Wuhan University, Wuhan, China city, postcode: 430072), protecting Hiding number is CCTCC NO:C2015155, is survived through detection.The cell is that circular half attached cell (can be by cell with bend pipe Blown down from bottle wall, collect cell and do not need pancreatin digestion, be also normal if there is cell suspension exists), cell division times The time of increasing is 16-18 hours.

Meanwhile the present invention also provides a kind of hybridomas of the monoclonal antibody of anti-human PCT albumen n end amino acid sequence Preparation method, this method comprises the following steps:

(1) people PCT albumen and Freund's complete adjuvant is fully emulsified, using subcutaneous inoculation method, with every mouse immune Protein concentration is respectively the dosage of 30 μ g, 50 μ g, 100 μ g and 150 tetra- gradients of μ g, and 36 ~ 8 week old are immunized in each gradient BALB/c female mice；

(2) the 2nd immune, people PCT albumen and incomplete Freund's adjuvant is fully emulsified, use and step are carried out after two weeks (1) same dosage and method booster immunization BALB/c mouse in；

It carries out the 3rd time after (3) 2 weeks to be immunized, immunizing dose and method are immune with the 2nd time；

(4) preceding 3 d people's PCT antigen that adjuvant is not added is merged, using booster immunization under spleen, every dosage is 100 μ g；

(5) preceding 1 d people's PCT antigen booster immunization that adjuvant is not added, every 50 μ g of dosage are merged；

(6) mouse docking carries out tail point blood sampling, and 4 DEG C, 4000 g are centrifuged 5 min, takes supernatant, and indirect elisa method detects blood Clear potency chooses mouse of the serum titer greater than 1,000,000 and is ready for cell fusion assay；

(7) spleen cell of the selection good SP2/0 myeloma cell of growth conditions and immune mouse is mixed with 1:5 ~ 1:10 It closes, 1500 g are centrifuged 5 min, abandon supernatant；

(8) jog centrifuge tube keeps cell evenly dispersed, is slowly added into the PEG 1500 of 37 DEG C of 1 mL preheatings and gently mixes Then 20 mL, 1640 culture medium is added in even 1.5 min；

(9) 800 g abandon supernatant after being centrifuged 5 min, and the HAT-1640 culture medium containing 20% serum is added in cell and mixes It is even；

(10) cell of mixing is uniformly laid in 96 orifice plates, at 37 DEG C, 5% CO₂Cell incubator in cultivate 5 ~ 7 Syncretizing effect is observed after d and changes liquid；

(11) it changes after liquid the 4th d and detects hybridoma supernatant with indirect ELISA method, select positive value high and cell The few hole of colony number, is subcloned by limiting dilution assay, and every hole cell number theoretical value is 1 ~ 2；

(12) after 3 ~ 4 subclones, that is, the hybridoma cell strain for capableing of stably excreting antibody, the sun filtered out are obtained Property hybridoma can be used for preparing anti-human PCT protein monoclonal antibody.

(13) epitope that further screening and identification can secrete identification is located at the N-terminal amino acid of PCT albumen, without Identify the hybridoma cell strain of the monoclonal antibody of the amino acid sequence of calcitonin (CT).

The present invention also provides the monoclonal antibodies of hybridoma secretion.

The present invention also provides application of the monoclonal antibody in the kit for preparing PCT Protein Detection.

The application of institute's art, wherein the method for detection PCT albumen uses double crush syndrome method.

The present invention provides a kind of preparation method of monoclonal antibody for Procalcitonin PCT protein N terminal epitope. Present invention monoclonal antibody obtained be use overall length PCT albumen as after mice immunized with antigen by hybridoma technology screening It obtains, the epitope of its identified identification is located at the N-terminal amino acid of PCT albumen, the amino acid sequence of nonrecognition calcitonin (CT) Column, therefore the cross reactivity between calcitonin can be effectively avoided, specific recognition PCT albumen is immune for double antibodies sandwich Chemoluminescence method detection PCT albumen provides a kind of new pairing monoclonal antibody, false positive rate, the raising to PCT detection is reduced The specificity of detection has great importance.Compared with prior art, the invention has the following advantages:

(1) the present inventor makes it by people's PCT complete genome sequence after the prokaryotic expression codon optimization in Escherichia coli It is expressed in endochylema supernatant, by technologies such as the specific affinity purifications in this laboratory, can simplify people and recombinate the pure of PCT albumen Change process, purifying descendant PCT albumen keep its natural activity, ensure that the integrality of protein steric conformation to greatest extent, by it BALB/c mouse is immunized as antigen, the antibody for screening acquisition can preferably identify natural PCT albumen in clinical serum.

(2) by cell fusion and subclone screening, 7H8 monoclonal antibody is obtained, by using PCT all amino acid sequence Column truncate the peptide fragment of synthesis, carry out Epitope Identification using monoclonal antibody of the ELISA method to acquisition, the antibody has accurately been determined The sequence of identification is people PCT protein N terminal (4-18aa) epitope sequences, and Western blot and ELISA testing result is shown, 7H8 monoclonal antibody has specificity, stability and sensitivity well.

(3) HRP enzyme label is carried out to 7H8 antibody, establishes double crush syndrome method, clinical serum sample is detected, The natural PCT albumen in blood serum sample can be captured, shows that there is feasibility for clinical detection with the antibody of independent research, to open Hair PCT diagnostic kit provides a kind of good new material.

Detailed description of the invention

People's PCT fusion protein mass spectrum of Fig. 1 prokaryotic expression identifies that software is analyzed the results show that Protein score is 282, it is very high with PCT protein matches degree, as a result reliably.

It is identified after Fig. 2 PCT protein purification, lane M: protein relative molecular mass standard items；Lane 1:30% sulphur Sour ammonium deposit sample；The albumen of 2 ~ 5:Ni-IDA of lane resin after purification；Lane 6: the egg after molecular sieve chromatography purification It is white.

It is identified after Fig. 3 PCT protein purification, Fig. 3 (A): SDS-PAGE qualification result；Fig. 3 (B): Western blot mirror Determine as a result, lane M: protein relative molecular mass standard items；Lane 1: the albumen after molecular sieve chromatography purification.

Fig. 4 7H8 monoclonal antibody subtype identification, 7H8 monoclonal antibody hypotype are IgG1 subclass.

Identification after Fig. 5 is monoclonal antibody-purified, Fig. 5 (A): SDS-PAGE qualification figure, lane M: opposite point of protein Protonatomic mass standard；Lane 1: the ascites before purification that do not boil；Lane 2: the ascites before purification boiled；Lane 3: it does not boil Monoclonal antibody after purification；Lane 4: the monoclonal antibody after purification boiled；Fig. 5 (B): immunologic specificity identification, lane M: Protein relative molecular mass standard；Lane 1: recombination PCT albumen.

The detection of Fig. 6 7H8 Antibody stability, is respectively placed in -80 DEG C, -20 DEG C, 4 DEG C for monoclonal antibody 7H8 after purification It in 37 DEG C of environment, was sampled in 1,2,3,9,27,60 day, indirect ELISA detects the variation of its potency, as the result is shown: being put in Potency is just begun to decline 37 DEG C of 7H8 antibody after one month, under the conditions of 4 DEG C 60 days it is still stable.

Specific embodiment

Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but is not to limit of the invention System, only illustrates.

The present embodiment material therefor is as follows:

(1) strain, sequence and cell strain: Escherichia coliE.coliTop 10, ER2566 bacterial strain are saved by this laboratory, PCT complete genome sequence, 2/0 murine myeloma cell of SP of codon optimization are taught by Xiamen University's School of Public Health summer peaceful Shao It awards and give.

(2) main agents: HRP- goat-anti rabbit is given by Xiamen University professor Xia Ningshao；Standard PCT polyclonal antibody is purchased from Proteintech company；Protein relative molecular mass standard is purchased from Thermo company；Freund's adjuvant is public purchased from U.S. sigma Department；HT, HAT culture medium additive are purchased from U.S. sigma company；1640 culture mediums are purchased from Gibco company.

(3) it clinical serum sample: is collected by clinical laboratory, People's Hospital, Hunan Prov..

The present invention is prepared by the following method to obtain the hybridoma for secreting anti-human PCT protein monoclonal antibody:

(1) gene order

The people's PCT complete genome sequence being given by Xiamen University, be gene codon is replaced into Escherichia coli preference it is close Numeral makes it be more easier the nucleotides sequence after expression in escherichia coli, optimization and is classified as shown in SEQ ID NO:1；

The 4-18 aa polypeptide sequence of epitope mapping is as shown in SEQ ID NO:2.

(2) PCT albumen bacterial strain inducing expression is recombinated

Picking screening monoclonal bacterial strain, be inoculated into 5 mL LB liquid mediums (containing 100 μ g/mL sulfuric acid cards that Mycin (Kan⁺) 37 DEG C of cultures are to OD₆₀₀Value reaches 0.6, and the IPTG that final concentration of 0.4 mmol/L is added carries out inducing expression, Induction is carried out as negative control to convert the bacterial strain of empty plasmid simultaneously, and other condition of culture are consistent with test group.37 DEG C of inductions 4 h are cultivated, 12000 g of room temperature is centrifuged 1min, collects thallus.Protein sample is prepared respectively, analyzes recombination with 12% SDS-PAGE The expression of albumen.As the result is shown: it can be observed that a relatively thick protein band, size and reason in test group By almost the same, and do not occur this band in control group, illustrates that recombined human PCT albumen may obtain in recombinant bacterial strain High efficient expression.

(3) mass spectrography detects recombinant protein property

Recombinant protein carries out the matching analysis after spectrometer analysis, by obtained data input base, as the result is shown Detection albumen is consistent with PCT albumen in database.Software analysis the results show that Protein score be 282, illustrate matching degree It is very high, as a result reliably；Mass spectral results also show that the relative molecular mass of PCT recombinant protein is about 13kD, with natural PCT albumen Relative molecular mass size meets, this also further improves the confidence level (Fig. 1) of PCT recombinant protein.

(4) destination protein great expression and protein purification

It picks from the plate single colonie and is inoculated into 50 mL LB(Kan⁺) 12 h are cultivated in culture medium, then it is inoculated with 1% Recombinant bacterium is to 2 L LB culture medium (Kan⁺) in culture.To bacterium solution OD₆₀₀After value reaches 0.6, IPTG is added and carries out inducing expression (final concentration of 0.4 mmol/L).After 37 DEG C of 4 h of culture, 12000 g are centrifuged 10 min and collect thallus.100 mL lysates are added (50 mmol/L TrisHCl, 1 mmol/L EDTA, 100 mmol/L NaCl, pH 8.0), 10 mm, 200 W function of probe Rate ultrasonication thallus on ice.The supernatant being collected by centrifugation is put into beaker, by formula V3=V* (100-Q1)/(Q1-Q2) (V generation Table supernatant volume；Q1 represents this ammonium sulfate concentrations；Q2 represents previous sulphoxylic acid ammonium concentration；V3 represents the saturation sulphur being added Sour ammonium volume) it is slowly dropped into saturated ammonium sulfate (pH 7.4), 30 min, 4 DEG C of 2 h of standing are stirred on ice.4 DEG C, 12 000 G is centrifuged 10 min, is dissolved precipitating with PBS(pH 7.4).Take turns doing 20% to 80% seven gradient preliminary purification recombination PCT egg It is white, collect sample SDS-PAGE identification.

With buffer 0(50 the mM NaH2PO4,300 mM NaCl, pH 8.0 of 5 to 10 times of column volumes) with 1 mL/ The velocity balance His-taq column of min, then the recombination PCT albumen of PBS dissolution crosses column with identical speed, then molten with buffer 0 20,50,100,250 mM imidazole gradient wash-out recombinant proteins of solution, eluent are dialysed with PBS(pH 7.4) in 4 DEG C and are removed Imidazoles collects sample SDS-PAGE identification.

With the flow velocity of 0.5 mL/min, TSK-GEL G3000 is balanced with the mobile phase PBS(pH 7.0 of 10 times of bed volumes) SW chromatographic column, without significant change, will test device absorption value zero, 2 ml of sample injected in loading ring to 280 nm absorption values, with The flow velocity of 0.5 mL/min crosses column, collects the protein peak that absorption value at 280 nm is greater than 0.5 AU.SDS-PAGE analysis is collected Protein peak (Fig. 2).

Descendant PCT protein sample is purified after electrophoresis, SDS-PAGE testing result shows that destination protein is dissolved mainly in In supernatant, it is deposited in 30% saturated ammonium sulfate.People's PCT albumen passes through His-taq column and TSK-GEL G3000 SW chromatographic column After purification, a protein band (Fig. 3 A) is only observed in SDS-PAGE electrophoresis, illustrates that purity is greatly improved, and is used The purity that Bandscan software analyzes albumen has been more than 99%.

Western blot analysis is carried out to albumen after purification.After purifying protein transferring film, with the TN of the defatted milk containing 5% 2 h of (50 mmol/L TrisHCl, 150 mmol/L NaCl, pH7.5) buffer blind.The ratio in 1:2000 is added PCT polyclonal antibody, 37 DEG C of 2 h of reaction are diluted with defatted milk.With TNT buffer (50 mmol/L TrisHCl, 150 Mmol/L NaCl, 0.05%Tween-20, pH7.5) wash film three times, 10 min, is added HRP enzyme mark goat-anti rabbit every time, and room temperature is anti- Answer 2 h.Film is washed three times with TNT, every time 10 min, developed the color with HRP-DAB colour reagent box.Western blot the results show that Specific reaction (Fig. 3 B) occurs for the recombinant protein of expression and the PCT rabbit source polyclonal antibody of Proteintech company, further Illustrate that people's PCT gene obtains high efficient expression in Escherichia coli, and there is good antigenicity.

(5) animal immune and serum titer detect

After PCT albumen after purification is dialysed with PBS buffer solution, being diluted to every mouse immune protein concentration is respectively 30 3 mouse are immunized in tetra- μ g, 50 μ g, 100 μ g and 150 μ g gradients, each gradient, and it is sufficiently newborn that Freund's complete adjuvant is added in 1:1 Change.The method being immunized using subcutaneous multiple spot carries out initial immunity respectively to every mouse with the dosage of every 500 μ l；Two weeks Afterwards, protein solution and Fu Shi Freund's incomplete adjuvant is added in 1:1 when be immunized again, small with same dose immunization after fully emulsified Mouse, altogether booster immunization 2 times, every minor tick 2 weeks.Mice serum, which is collected, in the previous day immune every time carries out indirect ELISA, with Detect the immunogenicity and serum antibody titer of PCT recombinant protein.

After 8 d are immunized in third time, mouse tail vein blood sampling, 4 DEG C, 4 000 g are centrifuged 5 min, take supernatant, indirect ELISA Method detects serum titer, chooses the mouse that serum titer is higher than 1,000,000, and 4 d reinforce primary immunization again before fusion, Spleen injects 30 μ g PCT albumen, 20 μ g albumen of tail vein injection after 2 d.

(6) cell fusion, positive hybridoma cell screening and subclone

The selection good SP2/0 myeloma cell of growth conditions is mixed with the spleen cell of immune mouse with 1:5 ~ 1:10, 1500 g are centrifuged 5 min, abandon supernatant.Jog centrifuge tube keeps cell evenly dispersed, is slowly added into the PEG of 37 DEG C of 1 mL preheatings It 1500 and mixes gently, the termination of 20 mL, 1640 culture medium is added after merging 90s.800 g abandon supernatant after being centrifuged 5 min, thin The HAT-1640 culture medium of 20% serum is added in born of the same parents' precipitating and mixes.

Cell is mixed uniformly to be laid in 96 orifice plates, at 37 DEG C, 5% CO₂Observation fusion is imitated after cultivating 5 ~ 7 d in incubator Fruit simultaneously changes liquid.It changes after liquid the 4th d and detects hybridoma supernatant with indirect ELISA method, select positive value high and cell collection The few hole of number is fallen, is subcloned by limiting dilution assay, every hole cell number theoretical value is 1 ~ 2.By 3 ~ 4 Asias gram After grand, that is, the hybridoma cell strain for capableing of stably excreting antibody is obtained, this cell strain part is used for the production of monoclonal antibody, Part in liquid nitrogen container in freezing.

Passing through observation after merging 7 d and the fusion rate of this cell is calculated is 80.1%, is passed through indirectly after merging 10 d It is 31.2% that ELISA method, which measures positive clone rate,.Positive hybridoma cell is screened using indirect ELISA and limiting dilution assay, is passed through After 3 ~ 4 subclones, 13 plants of cell strains for capableing of the anti-human PCT protein monoclonal antibody of stably excreting are obtained.

(7) generation and purifying of monoclonal antibody

8-10 week old BALB/c mouse is taken, sterile paraffin oil, every 0.5 mL is first injected intraperitoneally.To abdomen after 7 d Intracavitary injection hybridoma, adjustment cell density are 1 × 10⁶ - 2×10⁶/ mL, every mouse about inject 0.5mL. Start to acquire ascites after 8-10 d.The ascites 1:1(V/V that will be collected) it is slowly added to saturated ammonium sulfate, it stirs on ice 30 min, 4 DEG C, 12000 g abandon supernatant after being centrifuged 10 min.The PBS of precipitating certain volume dissolves, and moves to bag filter Middle dialysis equilibrium is provided into the Binding buffer(Protein A Sefinose Kit of 20 times of volumes) in remove The ion of high concentration is removed, albumin A column purification is then used.By before purification with ascites antibody after purification through SDS-PAGE electrophoresis, There are three bands: the band of the heavy chain of 50kDa, the light chain of 25kDa and about 100kDa size in antibody after purification, but The predominantly band of 100 kDa when not boiling, boils rear heavy chain band and obviously becomes by force, and the band of 100 kDa dies down, and big Small is twice of heavy chain, boils front and back light chain bands power and is not much different (Fig. 5), therefore, the band of 100 kDa be heavy chain with The condensate of heavy chain, the reason is that the disulfide bond between two heavy chains does not destroy completely.It can't see other foreign protein items from gel Band, for this explanation after affinity column chromatography, monoclonal antibody has reached quite high purity, can be used for subsequent experimental.

(8) analysis of monoclonal antibody epitope

Truncation synthesis carried out to the end N- of the PCT full amino acid sequence of prokaryotic expression, every 15 amino acid, one peptide chain, Every peptide chain repeats 5 amino acid with previous peptide chain, and overall length synthesizes 12 peptide chains altogether, truncates synthetic proteins and is respectively designated as 1R, 2R, 3R, 4R, 5R, 6R, 7R, 8R, 9R, 10R, 11R, 12R are carried out using epitope of the indirect ELISA to monoclonal antibody Positioning.

The 12 polypeptide chains CB liquid (15mmol/L Na2CO3,35mmol/L NaHCO3) for truncating synthesis is diluted to Concentration is 100ug/mL, coating ELISA plate, 37 DEG C of 4h, then 4 DEG C are stayed overnight.Board-washing is primary, is added 37 DEG C of ED confining liquid and incubates After educating 2h, board-washing 5 times, the monoclonal antibody of 100 ug/ml is added, after 37 DEG C of incubations 60min, board-washing 5 times, addition HRP- sheep anti mouse secondary antibody, 37 DEG C of 30 min of incubation, board-washing 5 times, is eventually adding tmb substrate color developing agent, 37 DEG C of reactions 10 Terminate liquid, 450nm/630nm double UV check is added in min.Pass through screening, 7H8 and the 2nd article of peptide chain significant reaction, with other Peptide chain all fails specificity occur, between the epitope behaviour PCT albumen n end 4-18aa for illustrating 7H8 identification, that is, successfully obtains needle To the monoclonal antibody of people's PCT protein N terminal epitope.Other strain antibodies of screening are primarily directed to 43-69aa and 75- 88aa epitope.

(9) monoclonal antibody subtype identification

The PCT albumen of purifying is diluted with 1 × CB buffer (15mmol/L Na2CO3,35mmol/L NaHCO3) It is 1ug/mL to concentration, is coated with ELISA plate, 4 DEG C overnight.After board-washing is primary, ED confining liquid is added, after 37 DEG C of 2h, Board-washing 5 times, hybridoma supernatant is added, after 37 DEG C of incubations 30min, board-washing 5 times, addition HRP- sheep anti mouse secondary antibody (IgG, IgG1, IgG2a, IgG2b, IgG3, IgM), 37 DEG C of incubation 30min, board-washing 5 times, is eventually adding TMB Terminate liquid, 450/630nm double UV check is added in substrate color developing agent, 37 DEG C of reaction 10min.As the result is shown: 7H8 monoclonal Antibody is IgG1 subclass (Fig. 4).

(10) identification of hybridoma cell line and monoclonal antibody stability

Carry out passing processing of being recovered behind 20 generations and liquid nitrogen cryopreservation 3 months again, indirect ELISA detection culture respectively to 7H8 in vitro The potency of anti-human PCT McAb in supernatant.Ascites monoclonal antibodies are respectively placed in 37 DEG C, 4 DEG C, -20 DEG C and -80 DEG C of environment.In 1, 2, it samples within 3,9,27,60 days, indirect ELISA detects the variation of its potency.The sample saved using -20 DEG C is small as positive control Mouse serum is as negative control.

After the hybridoma that the present invention screens acquisition passed for 20 generations in vitro, using single in indirect ELISA detection cell conditioned medium The potency of clonal antibody, potency are held essentially constant, and still can achieve 1 × 10⁴.By freezing, the hybridoma in liquid nitrogen is thin Born of the same parents recover culture to produce monoclonal antibody, as a result, it was also found that its potency is also essentially unchanged after freezing 3 months, it can achieve 1 × 10⁴, illustrate that the hybridoma cell line stability obtained is good.Ascites monoclonal antibodies are respectively placed in 37 DEG C, 4 DEG C, -20 DEG C and -80 DEG C In environment, potency is just begun to decline 37 DEG C of 7H8 antibody after one month, 60 days still stable (Fig. 6) under the conditions of 4 DEG C.Explanation The stability of monoclonal antibody is good, complies fully with the shelf life stability requirement of commercially available antibody, has a good application prospect.

(11) identification of the potency of monoclonal antibody, specificity and the scope of application

96 hole elisa Plates are coated with people's PCT albumen of 1 μ g/mL, detect antibody purification potency with indirect ELISA method.With Maximum antibody extension rate of the ratio greater than 2.0 of positive value and negative value is determined as antibody titer.The result shows that hybridoma is thin The antibody titer of born of the same parents' culture supernatant is 1 × 10⁴, the antibody titer of ascites is 1 × 10⁶, titer of ascites be apparently higher than supernatant effect Valence.

In order to study the scope of application of 7H8, with recombination PCT albumen, recombinant C ys albumen, recombination NGAL albumen, P27 albumen, GAPDH albumen, pp65 albumen and recombination HFABP albumen are recombinated as antigen, PBS buffer solution carries out ELISA as negative control Detection, the ultimate density of primary antibody 7H8 antibody incubation are 100 ng/mL.7H8 monoclonal antibody only occurs with PCT albumen special as the result is shown Property reaction, and response value is particularly evident, illustrates that 7H8 has the specificity of height.

(12) monoclonal antibody relative affinity measures

With anti-PCT monoclonal antibody in PCT antigenic competition combination 7H8 antibody determination affinity constant (1) 1. reaction system The initial concentration of 7H8 is 1 ug/mL, and the initial concentration of PCT antigen forms 8 reactions from 1 ug/mL doubling dilution down System, in 37 DEG C of 1 h of reaction；2. reaction solution is added to diplopore with every 100 uL of hole and has been coated with the immune of PCT antigen Reaction plate, board-washing 5 times after 37 DEG C of 1 h of incubation；3. 100 uL sheep anti mouse secondary antibody enzyme conjugates, 37 DEG C of solution 1 h of reaction are added, Board-washing 5 times, terminate liquid is added after 10 min of substrate colour developing are added, the measurement nm/630 of OD=450 nm calculates each reaction system The antigen binding rate of system calculates affinity constant.(2) change condition and repeat (1) measuring affinity constant.

Antibody initial concentration in experimental method is changed to 2 ug/mL, the initial concentration of antigen from 10 ug/ml down Doubling dilution establishes reaction system, according to the step in (1) 2., 3. detect antigen binding rate, and calculate affinity constant.From Experimental result shows that body formation Competitive assays could excessively be fought by needing antigen much, can directly use i0 pairs of antigen initial concentration It is k=1.43 (r=0.8702) that B/ (1-B) mapping, which acquires slope (affinity constant), has and is used as immune diagnostic reagent The potentiality of raw material.Change condition according to formed reaction series measure as a result, it is direct with antigen initial concentration i0 to B/ (1-B) Mapping acquires slope (affinity constant), show that Kd value is 0.4461, reduction when experimental result does not improve than antigen, antibody concentration Nearly 4 times.

(13) HRP enzyme labelled antibody and double crush syndrome method detect people PCT albumen

1. the HRP enzyme mark of 7H8: monoclonal antibody dialysis after purification, 4 DEG C of stirrings dialysis 2h are changed liquid 2 times；HRP is put down It weighs to room temperature, after taking HRP and NaIO4 to be dissolved in water respectively, then NaIO4 solution is slowly added in HRP solution, solution is by palm fibre Color is transformed into green, is placed in 4 DEG C and is protected from light 30 min of activation；Ethylene glycol is added in HRP and NaIO4 mixed liquor, room temperature is protected from light end Only 30 min, until solution becomes brown from green；The HRP of activation is added in the antibody of dialysis, 4 DEG C are protected from light dialysis 2h, change liquid 2 It is secondary；The boric acid hydrogen sodium solution of preparation 20mg/ml, the NaBH4 solution of addition HRP enzyme volume 1/10,4 DEG C of placement 2h, every 30min mixes primary；It is drained after being centrifuged 10min with isometric saturated ammonium sulphate HRP labelled antibody；50% glycerol is molten Solve protein precipitation, -20 DEG C of preservations.Indirect ELISA method measures HRP labelled antibody activity.As the result is shown: the enzyme mark of 7H8 antibody Effect is fine, and colour developing is obvious.

2. double crush syndrome method detect people PCT albumen: using the PCT polyclonal antibody of Proteintech company as Antibody coating is captured, HRP marks 7H8 antibody as secondary antibody, detects people and recombinate PCT albumen.It is polyclonal that 100 ul are added in every hole Capture antibody is coated on ELISA Plate surface by antibody-solutions (10ug/ml is dissolved in PBS).37 DEG C are incubated for 2h or 4 DEG C overnight.Board-washing 1 time；It is added 200 μ L ED confining liquids (0.25% Casein), 37 DEG C of placement 2 h, board-washing 5 times；The people PCT of purifying is added Albumen and clinical serum sample, it is 100 μ g/mL that people PCT recombinant antigen, which is diluted to concentration with 1 × PBS(pH 7.45), then presses 1: 10,1:100,1:1000,1:10000,1:100000 gradient dilution, PBS buffer solution are negative control, and setting diplopore compares, often Hole is added 100 μ L of sample, 37 DEG C of placement 2 h, and board-washing 5 times；7H8 monoclonal antibody is marked by 1:5000 dilution HRP with ED confining liquid, Every hole is added the HRP-7H8 after 100 μ L dilution, 37 DEG C of incubation 30 min, and board-washing 5 times；It is aobvious that 100 μ L TMB substrates are added Color liquid, 37 DEG C are protected from light 10 min of colour developing；50 μ L terminate liquids are added and terminate reaction；It is bis- using 450 nm/630 nm in microplate reader The OD value in each hole of wavelength detecting.The results show that the PCT positive and hospital's detection are tied in double crush syndrome method detection blood serum sample Fruit is consistent, and detects that 0.1 μ g/mL, negative control OD450 are lower than 0.1, show 7H8 antibody to the people PCT albumen of purifying is reachable There are good recognition reaction, high sensitivity to PCT, the research for subsequent PCT diagnostic kit provides good material.

Since nineteen eighty-two Fukushi H is prepared for AIV hypotype monoclonal antibody for the first time, monoclonal antibody technique is with its spy Some high sensitivities and specificity and be widely used.In monoclonal antibody technique influence cell fusion because being known as very More, immune effect is particularly important.By the immune system of subcutaneous multidigit point immunostimulation mouse of people PCT albumen after purification.Three After secondary subcutaneous inoculation, mice serum dilute millionfold potency still can obvious test positive, then carry out spleen injecting immune It is immune with tail vein injection, the state that the bone-marrow-derived lymphocyte in Mice Body keeps a kind of high activity is allowed in this way.The present invention is logical The assurance for crossing this committed step of animal immune ensure that preferable positive clone rate.Hole floor space is grown into after cell fusion 1/2 ~ 1/3 when the occurrence of being subcloned, avoiding positive cell missing inspection and chromosome diminution.Using easy to operate Limiting dilution assay, it is ensured that the high feature of the stability and antibody-secreting potency of hybridoma cell line.After 4 time clonings, The hybridoma positive rate of acquisition is 100%.It adds and raises preferably in HT-1640 culture medium when carrying out cell subclone Cell or Porcine HGF are supported, to prevent hybridoma death due to out of order during the cultivation process.

Currently, the purification process of monoclonal antibody has very much, such as caprylic acid-ammonium, affinity chromatography, ion exchange layer Analysis, gel filtration and high performance liquid chromatography (HPLC) etc..The present invention is used again using first saturated ammonium sulfate fractional precipitation preliminary purification Protein A column purification antibody, odd contradictive hydroperitoneum antibody titer after purification is up to 1 × 10⁶.And the supernatant warp of Hybridoma Cell Culture Very high purity can be reached after ammonium sulfate precipitation, can satisfy laboratory requirement and commercial requirements completely, because The mainly monoclonal antibody of cell secretion.Since cell culture serum price is relatively high, the antibody titer of generation is again relatively low, Therefore during producing antibody, it can take and first cultivate a certain amount of hybridoma, then induce ascites using mouse Antibody is produced, not only potency is high for production antibody in this way, and production cost can substantially reduce.The 7H8 that this experiment screening obtains Monoclonal antibody energy specific recognition people's PCT protein N terminal 4-18aa sequence, and natural PCT albumen in blood serum sample can be captured, This provides a new material for the detection of PCT in follow-up clinical inflammation infection.Hybridoma cell line and the inspection of monoclonal antibody stability Result explanation is surveyed, the hybridoma cell line of monoclonal antibody and the stability of monoclonal antibody are good, and long-term preservation will not be such that antibody titer reduces.It is multinomial Statistics indicate that the monoclonal antibody of 7H8 secretion can be widely used in cell biology and the immunology examination of a variety of biologies and tissue It tests, there is applications well prospect and commercial value.

<110>Hunan Normal University

<120>a kind of preparation of the monoclonal antibody 7H8 of anti-human Procalcitonin protein N terminal epitope

<160> 2

<210> 1

<211> 378

<212> DNA

<400> 1

ATGCACCACCACCCCCACCACGGATCTGCACCATTCCGTTCTGCCCTGGAGAGCTCTCCAGCAGATCCG GCAACACTGAGTGAAGACGAAGCGCGCCTTCTGCTGGCTGCACTGGTGCAGGACTATGTGCAGATGAAGGCCAGTGA GCTGGAGCAGGAGCAAGAGCGTGAGGGTTCCAGCCTGGATAGCCCACGTTCTAAGCGTTGCGGTAATCTGAGTACCT GCATGCTGGGCACCTACACGCAGGACTTCAACAAGTTCCACACGTTCCCACAAACTGCAATCGGTGTTGGTGCACCT GGTAAGAAGCGTGACATGTCCAGCGACCTGGAGCGTGACCATCGCCCTCATGTTAGCATGCCACAGAATGCCAACTA A

<210> 2

<211> 15

<212>polypeptide

<400> 2

PFRSA LESSP ADPAT

Claims (4)

1. a kind of hybridoma for the monoclonal antibody for secreting anti-human PCT albumen n end amino acid sequence, is that deposit number is The hybridoma of CCTCC NO:C2015155, is preserved in China typical culture collection center.

2. the monoclonal antibody of hybridoma secretion as described in claim 1.

3. application of the monoclonal antibody as claimed in claim 2 in the kit for preparing PCT Protein Detection.

4. application as claimed in claim 3, wherein the method for detection PCT albumen uses double crush syndrome method.