CN1063484C - Prepn. of selenium-bearing antibody enzyme using monoclonal antibody - Google Patents
Prepn. of selenium-bearing antibody enzyme using monoclonal antibody Download PDFInfo
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- CN1063484C CN1063484C CN94102481A CN94102481A CN1063484C CN 1063484 C CN1063484 C CN 1063484C CN 94102481 A CN94102481 A CN 94102481A CN 94102481 A CN94102481 A CN 94102481A CN 1063484 C CN1063484 C CN 1063484C
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- abzyme
- monoclonal antibody
- gsh
- selenium
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Abstract
The present invention belongs to a technique for preparing a selenium containing abzyme. The method of the combination of a standard single-antibody preparation technique and simple chemical mutation can successfully prepare the selenium containing abzyme with high GPX activity. The abzyme has superior stability, guidance performance and affinity to substrates to those of natural GPX; in addition, the abzyme can expand production and avoid the defect that the source of the natural enzyme is limited. The preparation technology of the present invention is simple and is still capable of preparing the abzyme with high activity under the condition of having no knowledge of reaction mechanism and reaction transition state. The selenium containing abzyme prepared by the present invention has high application potential in medical service.
Description
The present invention is the technology of preparing that belongs to selenium-containing abzyme
The novel method of exploitation preparation abzyme is one of forward position of bio-science research field.In view of the molecule plasticity-of antibody itself, the characteristics such as diversity that molecular recognition specificity and antibody change, the indication abzyme has broad application prospects for fields such as molecular biology, physiotechnology and medical science.Therefore, state such as English, U.S. all prepares the novel method of abzyme at active development.So far, the method for success exploitation has: filter attitude analogue method, entropy trap method and introducing method comprise selective chemical modification and protein engineering method again in the introducing method.The specificity of abzyme generally equals or is better than the specificity of natural enzyme to substrate, yet catalytic efficiency is than low 2-3 the order of magnitude of natural enzyme.Therefore, develop the target that high vigor abzyme technology of preparing has just become the scientist struggle.
Abzyme is medically having huge application potential.The incumbent European free radical president R.Evans of research association pointed out in 1993, and the developing direction of future drugs is, very strong drug effect should be arranged, and the guidance quality of pointing to diseased organ is arranged again.Abzyme is the best candidate that satisfies above-mentioned requirements.Be one of important member of anti-ageing enzyme system in view of Selenoperoxidase (GPX), can remove interior free yl, prevent lipid peroxidation, to preventing and treating various inflammation, cardiovascular and cerebrovascular diseases, tumour etc. have obvious curative effects, and therefore, the abzyme that preparation has the GPX vigor just seems meaningful especially.
The objective of the invention is to: the method that the Monoclonal Antibody technology of application standard combines with chemical mutation successfully prepares the selenium-containing abzyme with very high GPX vigor.
The present invention is, preparation earlier has the monoclonal antibody of substrate binding site, with the chemical mutation method catalytic group is incorporated into the combining site of monoclonal antibody then, and the result produces high vigor abzyme.The preparation process of selenium-containing abzyme is:
Reduced glutathion (GSH) and 2,4-dinitrochlorobenzene (DCNB) reaction, the gsh derivative (GSH-S-DNP) that makes the S-replacement is as haptens; With glutaraldehyde GSH-S-DNP is coupled at bovine serum albumin (BSA), as holoantigen; The synthetic holoantigen is added freund adjuvant, and injection carries out immunity for female mouse, uses standard monoclonal antibody preparation method (seeing document [1]) preparation to have the monoclonal antibody of GSH combining site then.With the hydroxyl on the serine residue (Ser) in phenylmethylsulfonyl fluoride (PMSF) the activation monoclonal antibody variable region, use H again
2Se handles, and then Ser is mutated into seleno-cysteine (SeCys).Because SeCys is the catalytic group of GPX, thereby at the existing substrate binding site in monoclonal antibody variable region, catalytic group is arranged again, the result produces highly active selenium-containing abzyme.
The present invention has following characteristics;
1. the preparation method is simple, only uses standard monoclonal antibody preparation method and simple chemical mutation method.
2. in catalytic mechanism that can not knowing reaction, do not know still can prepare abzyme under the situation of reaction transition state.
3. can enlarge with this law and produce abzyme, overcome the limited shortcoming in natural GPX source.
4. the selenium-containing abzyme vigor height made from the present invention has reached the order of magnitude level of natural GPX.
5. the stability of the selenium-containing abzyme made from the present invention is than natural GPX height.
6. selenium-containing abzyme keeps tiring of original monoclonal antibody, thereby orientable near the pathology target site.
Embodiment
1.02g GSH (3.32 mmol) is dissolved among 10 ml, the 1 mol/L NaOH, lowers the temperature in ice bath.Get DCNB 0.67 g (3.6 mmol) that is dissolved in the 10 ml ethanol again, stir down and slowly join in the GSH solution, mixture keeps 10 min at 0 ℃, transfer about pH to 4 with diluted acid, obtain the glassy yellow crystal, use the hot water recrystallization, 70 ℃ of oven dry, promptly get pure product GSH-S-DNP 1.3 g, productive rate 90%.
10 mg BSA and 5.6 mg GSH-S-DNP are dissolved in 1 ml O.2 in the phosphoric acid buffer of mol/L pH=7.2 (PBS), slowly add O.7ml glutaraldehyde solution, behind the reaction 10h, add 10mg glycine termination reaction, pH=7.5 is used in the back, and (2.0 * 40cm) chromatography purification products are used the level pad wash-out to 0.05 mol/L PBS equilibrated Ultrogel AcA, 54 posts, flow velocity is 1ml/min, and the collection first peak is an antigen protein.
Synthetic antigen is added freund adjuvant, and injection carries out immunity for female mouse (BALB/c).The mice spleen cell of immunity merges with myeloma cell (NS-1) under the PEG effect, and sowing is in the culture plate that adds scavenger cell.Cultivate with the HAT nutrient solution, clone, obtain a positive monoclonal cell strain 4A4, carry out enlarged culturing with the cell strain of the double tests positive of ELISA.The cell culture fluid supernatant is through Biol-gel P200 post (2.0 * 50cm) chromatography purifications.This post pH=7.O, 0.05mol/L PBS damping fluid balance and wash-out, flow velocity are 1ml/min, collect first peak, obtain 4A4 IgG, this monoclonal antibody has substrate (GSH) combining site.
0.5mg O.5ml the freeze-drying monoclonal antibody is dissolved in, pH=7.0, among the 0.05mol/L PBS, adds 10 μ l PMSF solution (21 mg PMSF are dissolved in the 1 ml acetonitrile), room temperature vibration 1h feeds H under high purity nitrogen protection
2Se gas at 40 times insulation 36-40h, finishes reaction to saturated again.With reaction solution to deionized water dialysis 24h after, freeze-drying promptly gets selenium-containing abzyme.
The vigor of selenium-containing abzyme is determined as 1097 μ/μ mol by document [2], has reached the order of magnitude level of natural enzyme vigor (5780 μ/μ mol); Abzyme is decided to be 2.7 * 10 to the Michaelis-Menton constant of GSH by document [3]
-4M is with the Michaelis-Menton constant (3.0 * 10 of natural GPX
-3M) compare, improved 1 order of magnitude; After measured, abzyme tire with suddenly change before monoclonal antibody identical; The selenium content of abzyme is determined as 5Se/mol. by document [4].The stability of abzyme is listed in the table below:
The package stability enzyme state storage requirement half life of selenium-containing abzyme (my god) solution enzyme 15 mg/ml, pH7.2,0.05 mol/L, PBS, 15-20 ℃ of 28 solution enzymes, 15 mg/ml, pH7.2,0.05 mol/L, PBS, vigor is still after 90 days for 4 ℃ of 79 lyophozyme room temperature
Keep more than 90%
Reference: [1] .Koehler, G.And Milstein, C. (1995) Nature, 256:495[2] .Wendel, A. (1981) Methods Enzymol.77:325-333[3] .Lineweaver, H.and Burk, D. (1934) J.Am.chem..soc.56:658[4] .Leibfritz, D. (1972) Angew.chem.Int.Ed.Engl, 11:232-233
Claims (1)
1, a kind of preparation method of selenium-containing abzyme, it comprises Monoclonal Antibody technology and chemical mutation technology, its step is as follows:
Reduced glutathion (GSH) and 2,4-dinitrochlorobenzene (DCNB) reaction, the gsh derivative (GSH-S-DNP) that makes the S-replacement is as haptens; With glutaraldehyde GSH-S-DNP is coupled at bovine serum albumin (BSA), as holoantigen; The synthetic holoantigen is added freund adjuvant, and injection carries out immunity for female mouse, has the monoclonal antibody of GSH combining site then with the preparation of standard monoclonal antibody preparation method; With the hydroxyl on the serine residue (Ser) in phenylmethylsulfonyl fluoride (PMSF) the activation monoclonal antibody variable region, use H again
2Se handles, and then Ser is mutated into seleno-cysteine (SeCys); Because SeCys is the catalytic group of GPX, thereby at the existing substrate binding site in monoclonal antibody variable region, catalytic group is arranged again, the result produces the selenium-containing abzyme of high vigor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94102481A CN1063484C (en) | 1994-03-09 | 1994-03-09 | Prepn. of selenium-bearing antibody enzyme using monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94102481A CN1063484C (en) | 1994-03-09 | 1994-03-09 | Prepn. of selenium-bearing antibody enzyme using monoclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1093405A CN1093405A (en) | 1994-10-12 |
| CN1063484C true CN1063484C (en) | 2001-03-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94102481A Expired - Fee Related CN1063484C (en) | 1994-03-09 | 1994-03-09 | Prepn. of selenium-bearing antibody enzyme using monoclonal antibody |
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| CN (1) | CN1063484C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062600C (en) * | 1996-09-10 | 2001-02-28 | 中国科学院长春应用化学研究所 | Preparation of selenic antibody enzyme with activity higher than that of natural enzyme |
| CN101280015B (en) * | 2008-04-01 | 2012-09-05 | 吉林大学 | Preparation of soluble human selenium-containing single-chain abzyme |
| CN109061172B (en) * | 2018-09-21 | 2021-07-06 | 中国烟草总公司郑州烟草研究院 | Enzyme linked immunosorbent assay kit for detecting butralin and application thereof |
-
1994
- 1994-03-09 CN CN94102481A patent/CN1063484C/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| J.AM.CHEM.SOC 111 1989.1.1 WU,Z.P.ADN HILVERT,D.CONVERSION OF A PROTEASE INTO A ACYL TRANSFERASE SEL * |
| J.AM.CHEM.SOC 111 1989.1.1 WU,Z.P.ADN HILVERT,D.CONVERSION OF A PROTEASE INTO A ACYL TRANSFERASE SEL;METHODS ENZYMOL 11 1967.1.1 GOLD A.M SULFONYLATION WITH SULFONYL HALIDES * |
| METHODS ENZYMOL 11 1967.1.1 GOLD A.M SULFONYLATION WITH SULFONYL HALIDES * |
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| CN1093405A (en) | 1994-10-12 |
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