CN107383199A

CN107383199A - A kind of monoclonal antibody of S adenomethionine synthases and its application

Info

Publication number: CN107383199A
Application number: CN201710509182.9A
Authority: CN
Inventors: 翁炜宁; 孟逊
Original assignee: Abby Mart (shanghai) Co Ltd Pharmaceutical Technology
Current assignee: Abby Mart (shanghai) Co Ltd Pharmaceutical Technology
Priority date: 2017-03-15
Filing date: 2017-06-28
Publication date: 2017-11-24
Anticipated expiration: 2037-06-28
Also published as: CN106831994A; CN107383199B

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 CDR shown in SEQ ID NO.2,4 and 6；The amino acid sequence of the CDR shown in SEQ ID NO.9,11 and 13 is included with light-chain variable sequence.For the present invention using the polypeptide of the enzyme as immunogene, immune mouse obtains lymphocyte, and fused cell is made using hybridoma cell fusion technology, obtains that high-affinity can be produced and high specific detection sets the cell line of the enzyme.The present invention can be used for S adenomethionine synthase contents in detection animal and plant body；The instrument of a targeting proteins research is provided with the monoclonal antibody of the present 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), the entitled S-adenosyl-methionine of English, is in the life entities such as animal, plant Important metabolic intermediate matter, and a kind of important physiological activator in human body, participating in a variety of biochemical reactions, (scape is abundant, gland 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 is most of by starting with from SAM preparation method to study it.

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

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

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 passes through the instrument that targeting proteins are studied that provides 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 synthesis enzyme polypeptide, i.e. four groups ' FTKCPEEIGA ' ' TDETPELMPL ' ' QVTVEYYNDK ' and ' VLISTQHDET ', respectively using it as immunogene, immune mouse obtains lymphocyte, and utilizes hybridization Fused cell is made in oncocyte integration technology, and through Western blot, limiting dilution assay and ELISA method, obtaining being capable of the high parent of product With the cell line of property and the monoclonal antibody of high specific, and the monoclonal antibody secreted by the cell line, and through immune enzyme-linked, Protein blot, co-immunoprecipitation add the checking such as mass spectrum and antibody chip detection, determine 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 and SEQ ID of CDR2 shown in the amino acid sequence of CDR1 shown in the NO.2 of ID containing SEQ, SEQ ID NO.4 The amino acid sequence of CDR3 shown in NO.6；The amino acid of the CDR1 shown in SEQ ID NO.9 is included with light-chain variable sequence The amino acid sequence of CDR3 shown in the amino acid sequence and SEQ ID NO.13 of CDR2 shown in sequence, SEQ ID NO.11.

Preferably, the framework region of described weight chain variabl area sequence is successively comprising the ammonia shown in SEQ ID NO.1,3,5 and 7 Base acid sequence, and described light-chain variable sequence framework region successively comprising the ammonia shown in SEQ ID NO.8,10,12 and 14 Base acid sequence.

The invention also discloses the monoclonal antibody of above-mentioned SAM synzyme 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 present invention, there is height to SAM synzyme Affinity and high specific, it can be widely used in the detection of SAM synzyme, being particularly useful for detecting animals and plants Internal SAM synzyme content；, should meanwhile enzyme content is by the expression of SAM in direct reactant 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.In addition, 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 of different lines or different development stage changes and the deeper hierarchical research of albumen network collection of illustrative plates is achieved.

Brief description of the drawings

Fig. 1 is the intrinsic protein marking WB the results of cherry holoprotein lysate and clone's 2B7 anti-SAM antibody Schematic diagram；

Fig. 2 is that cherry holoprotein lysate detects with clone's 2B7 anti-SAM antibody and the antibody chip of control antibodies Result schematic diagram；

Fig. 3 is that clone's 2B7 anti-SAM antibody and various concentrations SAM synzyme ELISA of the invention are anti- Should obtained standard curve.

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 change according to what the present invention made in actual applications Enter and adjust, still fall within protection scope of the present invention.

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

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 synthesis enzyme polypeptides ' FTKCPEEIGA ' ' TDETPELMPL ' ' QVTVEYYNDK ' and ' VLISTQHDET ', and respectively using it as immunogene, aforementioned polypeptides also even-coupling VLP and traditional KLH systems it is immune Originality enhancer.

1.2 immune mouse

Every group of antigen will be used for that 6 Balb/c mouse (8-12 week old) are immunized, 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 optimized can be produced for most of anti- The antibody (IgG hypotypes) of the high-affinity of former polypeptide.The reinforcement of 3 to 4 times can be passed through after initial immunity, mouse blood will be taken after reinforcement Clear detection titre (recombinant protein of SAM synzyme is 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 merge.

1.3 Virus monitories and screening

Immune mouse orbit takes blood, and detects serum titer (the recombinant protein work of SAM synzyme with ELISA For antigen coat).Serum titer need to be more than 10K, otherwise continue booster immunization.

1.4 fusion and screening

Full spleen and 1/2 lymph node are taken, is merged with myeloma SP2/0 cell lines.Technique is the PEG fusions optimized.Melt Close cell and be taped against on 4 piece of 384 orifice plate (per hole cell 102 to 104), cultivated.The porose supernatant of institute 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 detect former reaction with solvable fragment with ELISA detections.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. Each solvable 60 Parental clones of fragment immunogen affinity highest enter subclone.

1.5 subclones and screening

It is subcloned by limiting dilution assay and ELISA screenings, obtains monoclonal hybridoma.Cell spreads 96 holes Plate, and cultivate to the bottom of covering about 1/6.ELISA detects each hole supernatant for solvable fragment detection original and corresponding polypeptide inspection Former reaction is surveyed, takes two holes that OD values are high and cell state is good to be subcloned into lower whorl.It steps be repeated alternatively until in hole Cell line positive rate 100%.Now we obtain monoclonal cell strain.After last wheel subclone, all positive cells Expand culture immediately, a part freezes be provided with after use, another part carries out supernatant or ascites and prepared.

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.Caused ascites Purified with Protein A/G, and be used for subsequent detection.

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

8 plants of cell strain of monoclonal antibody of acquisition are carried out immune enzyme-linked, protein blot, co-immunoprecipitation adds mass spectrum, resists Body chip etc. is verified, determines most effective antibody.

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

Taking ascites antibody to be paired to be coated with 96 hole elisa plates, be incubated, skim milk is closed overnight after washing, PBS washings, and 4 DEG C preservation is stand-by.Antigen polypeptide is incubated, PBS washings, while sets control.HRP mark detection antibody, be added to incubation have it is foregoing Elisa plate in.TMB chromogenic reactions, ELIASA reading.Screening obtains the potency of 8 cell lines 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 As a result showing, anti-SAM (clone 2B7) can be consistent in WB checkings with specific identification 40KD bands 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, carries out immunoprecipitation 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 to the glass using NC films as matrix using chip point sample instrument Glass piece, form a diameter of 100um antibody point.Apple holoprotein is subjected to biotin labeling, is incubated in by 2ug/ml concentration 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 obvious enrichment to combine effect to target protein, fluorescence intensity compared with By force, and antigen-antibody binding reaction does not occur for control antibodies.

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

Because the antibody of the present invention has unique specificity to SAM synzyme, special detection can be used as not It can also be incited somebody to action according to conventional technical means with the detection antibody of SAM synzyme content in animal and plant body, or this area 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 SAM conjunction Into the concentration coordinate of enzyme, ordinate is the OD value (OD values) that ELIASA measures, as shown in Figure 3.After the standard curve is used for The continuous OD values by sample to be tested detection are converted into the concentration of 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 press 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, take the tissue liquid sample of different peach classes to carry out ELISA experiments, use identical antibody concentration of the present invention (1mg/ml) is detected, and obtained OD values substitute into standard curve and calculated, that is, obtains actual enzyme content in different animal and plant bodies And corresponding expression, as shown in Table 4.

Table four

	Detect 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 2B7 anti-SAM antibody

The hybridoma cell strain of 2B7 clone's anti-SAM antibody is cultivated and extracts 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, carries 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 details are described, it is only described embodiment also not limit the invention.Obviously, according to the content of this specification, It 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

1. a kind of monoclonal antibody of SAM synzyme, it is characterised in that the heavy chain of described monoclonal antibody can Become the amino acid sequence that region sequence includes the CDR1 amino acid sequence shown in SEQ ID NO.2, the CDR2 shown in SEQ ID NO.4 The amino acid sequence of row and the CDR3 shown in SEQ ID NO.6；Included with light-chain variable sequence shown in SEQ ID NO.9 CDR3's shown in the amino acid sequence and SEQ ID NO.13 of CDR2 shown in CDR1 amino acid sequence, SEQ ID NO.11 Amino acid sequence.

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.1,3,5 and 7, and described light chain variable The framework region of region sequence is successively comprising the amino acid sequence shown in SEQ ID NO.8,10,12 and 14.

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 S- adenosines in detection animal and plant body The application of methionine synthase content.