CN106831994A - A kind of monoclonal antibody of S adenomethionine synthases and its application - Google Patents

Abstract

The invention discloses a kind of monoclonal antibody of S adenomethionine synthases, the weight chain variabl area sequence of described monoclonal antibody includes the amino acid sequence of the CDR2 shown in the amino acid sequence and SEQ ID NO.4 of the CDR1 shown in SEQ ID NO.2；With amino acid sequence of the light-chain variable sequence comprising the CDR3 shown in SEQ ID NO.6.With the polypeptide of the enzyme as immunogene, immune mouse obtains lymphocyte to the present invention, and fused cell is obtained using hybridoma cell fusion technology, obtains that high-affinity and high specific detection can be produced to set the cell line of the enzyme.The present invention can be used to detect S adenomethionine synthase contents in animal and plant body；The instrument that a targeting proteins are studied is provided with monoclonal antibody of the invention so that the deeper hierarchical research of protein expression change and albumen network collection of illustrative plates to different vegetation types, different lines or different development stage is achieved.

Description

A kind of monoclonal antibody of SAM synzyme and its application

Technical field

The invention belongs to monoclonal antibody biological technical field, more particularly to a kind of Dan Ke of SAM synzyme Grand antibody and its application.

Background technology

SAM (SAM), English entitled S-adenosyl-methionine is in the life entities such as animal, plant Important metabolic intermediate matter, is also a kind of important physiological activator in human body, and (scape is abundant, gland to participate in various biochemical reactions Glycosides methionine, the chemistry of life, 1995,5:49), as participated in transmethylation in organism, turn aminopropyl effect and turn Sulphur effect (LU S C, Regulation of hepatic glutathione synthesis Sem Liv Dis, 1998, 18:331-343).Prior art major part is studied it by starting with from the preparation method of SAM.

SAM be by substrate METHIONINE and ATP through S-adenosylmethionine synzyme enzyme' s catalysis, therefore, S- adenosines Methionine synthetase is the key enzyme for synthesizing SAM, in the content of animals and plants vivo detection S-adenosylmethionine synzyme, can Reflection influence animal and plant body synthesizes the factor of SAM, it can also be used to detect that different animal and plant bodies synthesize containing the S-adenosylmethionine The analysis and research of enzyme.

At present on the market not specifically designed for the targeting antibodies of S-adenosylmethionine synzyme, therefore, in protein groups In aspect, effective antibody instrument is lacked for the research field, for different vegetation types, different lines or different developments Period the expression of enzymes ability level research.

The content of the invention

The present invention provides monoclonal antibody and its application of a kind of SAM synzyme, is deposited with solving prior art Drawbacks described above, the present invention is by providing the instrument that targeting proteins are studied so as to different vegetation types, different lines Or the protein expression change of different development stage and the deeper hierarchical research of albumen network collection of illustrative plates are achieved.

Technical scheme is as follows：

Present invention synthesis SAM synthesizes enzyme polypeptide, i.e., four group ' FTKCPEEIGA ' ' TDETPELMPL ' ' QVTVEYYNDK ' and ' VLISTQHDET ', respectively using it as immunogene, immune mouse obtains lymphocyte, and using hybridization Oncocyte integration technology is obtained fused cell, and through Western blot, limiting dilution assay and ELISA method, obtaining being capable of product parent high With property and the cell line of the monoclonal antibody of high specific, and the monoclonal antibody secreted by the cell line, and through immune enzyme-linked, Protein blot, co-immunoprecipitation adds the checking such as mass spectrum and antibody chip detection, determines most effective antibody.

A kind of monoclonal antibody of SAM synzyme, the weight chain variabl area sequence bag of described monoclonal antibody The amino acid sequence of the CDR2 shown in the amino acid sequence and SEQ ID NO.4 of the CDR1 shown in the NO.2 of ID containing SEQ；And light chain Amino acid sequence of the variable region sequences comprising the CDR3 shown in SEQ ID NO.6.

Preferably, the framework region of described weight chain variabl area sequence is successively comprising the amino acid shown in SEQ ID NO.1 and 3 Sequence, and described light-chain variable sequence framework region successively comprising the amino acid sequence shown in SEQ ID NO.5 and 7.

Monoclonal antibody the invention also discloses above-mentioned SAM synzyme is in detection SAM Application on synzyme.

Preferably, the monoclonal antibody of SAM synzyme SAM synthesis in detection animal and plant body The application of enzyme content.

Compared with prior art, beneficial effects of the present invention are as follows：

A kind of monoclonal antibody of SAM synzyme of the invention, has height to SAM synzyme Affinity and high specific, can be widely used in the detection of SAM synzyme, being particularly useful for detecting animals and plants Internal SAM synzyme content；Meanwhile, enzyme content, should by the expression of SAM in direct reaction body Albumen mainly carries out biochemical reaction in liver, and is widely used in the treatment of the hepatopathys such as alcohol hepatopathy, liver lesion induced by drugs, should Antibody will carry out the Cure of routine observation related liver disease patient as very effective detection means.Additionally, in protein group Learn research field, monoclonal antibody of the invention provides the instrument of targeting proteins research so as to different vegetation types, The protein expression change of different lines or different development stage and the deeper hierarchical research of albumen network collection of illustrative plates are achieved.

Brief description of the drawings

Fig. 1 is the intrinsic protein marking WB the results of cherry holoprotein lysate and clone's 2B7anti-SAM antibody Schematic diagram；

Fig. 2 is the antibody chip detection of cherry holoprotein lysate and clone 2B7anti-SAM antibody and control antibodies Result schematic diagram；

Fig. 3 is that clone 2B7anti-SAM antibody of the invention reacts with various concentrations SAM synzyme ELISA Obtained standard curve.

Specific embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this hair It is bright, rather than limit protection scope of the present invention.Those skilled in the art are according to changing that the present invention makes in actual applications Enter and adjust, still fall within protection scope of the present invention.

The not special explanation of the present invention, technological means used is well known to those skilled in the art in following examples Conventional technical means, and raw material, reagent used is also commercially available commercially available commodity.

A kind of preparation of the monoclonal antibody hybridoma cell of SAM synzyme of embodiment 1 and anti-S- adenosines egg The preparation and purification of the monoclonal antibody of propylhomoserin synzyme

The preparation of 1.1 antigens

Synthesize four groups of SAMs synthesize enzyme polypeptide ' FTKCPEEIGA ' ' TDETPELMPL ' ' QVTVEYYNDK ' and ' VLISTQHDET ', and respectively using it as immunogene, aforementioned polypeptides also even-coupling VLP and tradition KLH systems it is immune Originality enhancer.

1.2 immune mouses

Every group of antigen will be for being immunized 6 Balb/c mouse (8-12 week old), and it is optimal to determine to monitor its serum titer Immune time.Each 6 immunogene polypeptide is mixed into one group.The adjuvant and immunization method for optimizing can be produced for most of anti- The antibody (IgG hypotypes) of the high-affinity of former polypeptide.Mouse blood can will be taken after reinforcement by the reinforcement of 3 to 4 times after initial immunity Clear detection titre (recombinant protein of SAM synzyme is used as anti-antigen coat).The qualified mouse of titre will impact one Secondary and for merging, underproof mouse will continue one to strengthening twice, to titre highest after fusion.

1.3 Virus monitories and screening

Immune mouse eye socket takes blood, and detects that serum titer (make by the recombinant protein of SAM synzyme with ELISA It is antigen coat).Serum titer need to be more than 10K, otherwise continue booster immunization.

1.4 fusions and screening

Full spleen and 1/2 lymph node are taken, is merged with myeloma SP2/0 cell lines.Technique is the PEG fusions for optimizing.Melt Close cell and be taped against on 4 piece of 384 orifice plate (per hole cell 102 to 104), cultivated.The supernatant in all holes is collected, with ELISA pairs Polypeptide detection original is screened, and the positive hole that microscopy has cell goes to 96 orifice plates and continues to cultivate.Growth after a few days, collects all holes Supernatant detected with ELISA and solvable fragment detects former reaction.Further detect the solvable fragment of different dilution factors in positive hole Detection is former to be combined, to carry out affinity sequence.20 Parental clones of each polypeptide immunogen affinity highest enter subclone. 60 Parental clones of each solvable fragment immunogen affinity highest enter subclone.

1.5 subclones and screening

Screened by limiting dilution assay and ELISA and be subcloned, obtain monoclonal hybridoma.Cell spreads 96 holes Plate, and cultivate to the bottom of covering about 1/6.ELISA detects that each hole supernatant is former for the detection of solvable fragment and corresponding polypeptide is examined Former reaction is surveyed, OD values height is taken and two good holes of cell state is subcloned into lower whorl.In steps be repeated alternatively until hole Cell line positive rate 100%.Now we obtain monoclonal cell strain.After last wheel is subcloned, all positive cells Amplification Culture immediately a, part freezes after being provided with and uses, and another part carries out supernatant or prepared by ascites.

1.6 antibody supernatants are prepared and purified

8 plants of monoclonal cell strains are finally obtained, and antibody producing is used for by abdomen injection to F1 mouse.The ascites of generation Purified with Protein A/G, and for subsequent detection.

The checking of the anti-CK18 monoclonal antibodies of embodiment 2

8 plants of cell strain of monoclonal antibody to obtaining carry out immune enzyme-linked, and protein blot, co-immunoprecipitation adds mass spectrum, resists The checking such as body chip, determines most effective antibody.

Elisa (immune enzyme-linked) pair verification of 2.1 antibody and antigen polypeptide

Take ascites antibody to be paired and be coated with 96 hole elisa plates, be incubated, skim milk is overnight closed after washing, PBS washings, 4 DEG C preservation is stand-by.Antigen polypeptide is incubated, PBS washings, while setting control.HRP marks detection antibody, and being added to incubation has foregoing Elisa plate in.TMB chromogenic reactions, ELIASA reading.Screening obtains 8 potency of cell line as shown in Table 1：

Table one

The intrinsic protein marking (WB) checking of 2.2 antibody

Use cherry holoprotein lysate, antibody diluted concentration 1：1000、1：2000 and 1:5000 carry out WB checkings.Experiment Result shows that anti-SAM (clone 2B7) can be consistent with specific identification 40KD bands in WB checkings with expected size, As shown in Figure 1.

Co-immunoprecipitation (IP) plus the mass spectrum checking of 2.3 antibody

Cherry holoprotein 1mg is extracted, immunoprecipitation is carried out using anti-SAM (clone 2B7), it is big that IP products cut 40kd Small band carries out Mass Spectrometer Method.As shown in Table 2, SAM is largely enriched with mass spectral results in IP samples, illustrates that this antibody is known to SAM Other specificity is high, also, this antibody can apply to IP experiments.

Table two

2.4 antibody chip test experiences

Anti-SAM (clone 2B7) antibody and control antibodies point are formed on the glass with NC films as matrix using chip point sample instrument Glass piece, forms the antibody point of a diameter of 100um.Apple holoprotein is carried out into biotin labeling, is incubated in by the concentration of 2ug/ml On antibody chip, half an hour is incubated at room temperature.PBS softly cleaning three times, then be incubated with CY3-SA fluorescence secondary antibodies, PBS Three times, chip is scanned using GenePix Fluorescence chip scanners 523nm.

Experimental result as shown in Fig. 2 Anti-SAM antibody has substantially enrichment to combine effect to target protein, fluorescence intensity compared with By force, and not there is antigen-antibody binding reaction in control antibodies.

A kind of monoclonal antibody of SAM synzyme of the invention of embodiment 3 S- in detection different animals body The application of adenomethionine synthase content

Because antibody of the invention has unique specificity to SAM synzyme, can be as special detection not With the detection antibody of SAM synzyme content in animal and plant body, or this area also can be by according to conventional technical means It is further developed as detection kit.

First, antibody of the present invention is with fixed concentration (1mg/ml) and various concentrations SAM synthetase recombinant protein Standard items carry out ELISA experiments, obtain corresponding OD values, and draw out standard curve, abscissa is that SAM is closed Into the concentration coordinate of enzyme, the OD value (OD values) that ordinate is measured for ELIASA, as shown in Figure 3.After the standard curve is used for The continuous concentration that the OD values that sample to be tested is detected are converted into the enzyme；Wherein standard items are the restructuring egg of SAM synzyme In vain, by BC A protein quantifications and examine dye and run glue and confirm concentration, and by 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25ug/ml Doubling dilution, as shown in Table 3.

Table three

Standard concentration (mg/ml)	10	5	2.5	1.25
					Standard items OD values	2.856	2.154	1.521	0.857

Then, the tissue liquid sample for taking different peach classes carries out ELISA experiments, uses identical AC of the present invention (1mg/ml) is detected, the OD values for obtaining substitute into standard curve and calculate, that is, obtain actual enzyme content in different animal and plant bodies And corresponding expression, as shown in Table 4.

Table four

	Detection OD values	Enzyme concentration conversion mg/ml	Expression
				Honey peach	1.869	1.88	In
Arabidopsis	0.568	0.51	It is low
				Zebra fish	2.058	2.27	In
Serum	3.254	7.52	It is high

Embodiment 4

Clone's 2B7anti-SAM antibody

The hybridoma cell strain of 2B7 clone's anti-SAM antibody is cultivated and extracted total serum IgE, by mRNA reverse transcriptions Into the first chain cDNA；Heavy chain and light chain gene are expanded by PCR and amplification gene is cloned into sequencing vector, carry out multiple positives Cloning and sequencing obtains ultimate sequence result.

Present invention disclosed above preferred embodiment is only intended to help and illustrates the present invention.Preferred embodiment is not detailed All of details is described, it is only described specific embodiment that the invention is not limited yet.Obviously, according to the content of this specification, Can make many modifications and variations.This specification is chosen and specifically describes these embodiments, is to preferably explain the present invention Principle and practical application so that skilled artisan can be best understood by and utilize the present invention.The present invention is only Limited by claims and its four corner and equivalent.

SEQUENCE LISTING

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Claims (4)

1. a kind of monoclonal antibody of SAM synzyme, it is characterised in that the heavy chain of described monoclonal antibody can The amino acid of the CDR2 shown in amino acid sequence of the change region sequence comprising the CDR1 shown in SEQ ID NO.2 and SEQ ID NO.4 Sequence；With amino acid sequence of the light-chain variable sequence comprising the CDR3 shown in SEQ ID NO.6.

2. the monoclonal antibody of SAM synzyme according to claim 1, it is characterised in that described heavy chain The framework region of variable region sequences is successively comprising the amino acid sequence shown in SEQ ID NO.1 and 3, and described light chain variable district sequence The framework region of row is successively comprising the amino acid sequence shown in SEQ ID NO.5 and 7.

3. the monoclonal antibody of the SAM synzyme described in claim 1 is on detection SAM synzyme Application.

4. the monoclonal antibody of SAM synzyme according to claim 3 is detecting S- adenosines in animal and plant body The application of methionine synthase content.