CN106324234A - Modified N-acetylneuraminic acid aldolase and preparation method and application thereof - Google Patents
Modified N-acetylneuraminic acid aldolase and preparation method and application thereof Download PDFInfo
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- CN106324234A CN106324234A CN201610643859.3A CN201610643859A CN106324234A CN 106324234 A CN106324234 A CN 106324234A CN 201610643859 A CN201610643859 A CN 201610643859A CN 106324234 A CN106324234 A CN 106324234A
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- G—PHYSICS
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Abstract
The invention provides modified N-acetylneuraminic acid aldolase and a preparation method and application thereof. The amino acid side chain of the modified N-acetylneuraminic acid aldolase is modified through alkylation. The preparation method of the modified N-acetylneuraminic acid aldolase comprises the following steps that N-acetylneuraminic acid aldolase is reacted with an alkylation reagent to remove carboxyl in the amino acid side chain; the N-acetylneuraminic acid aldolase without carboxyl is modified with guanidine hydrochloride, and the modified N-acetylneuraminic acid aldolase is obtained. The modified N-acetylneuraminic acid aldolase can be used for preparing a stable SA kit and is higher in enzymatic activity and better in stability in the reagent; meanwhile, the modified N-acetylneuraminic acid aldolase and a reduced coenzyme do not disturb each other, so that the sensitivity of the reagent is better, a smaller amount of sialic acid can be detected, and accuracy is better in sample detection with a relatively low value.
Description
Technical field
The present invention relates to in-vitro diagnosis field, be specifically related to Neu 5 Ac aldolase and the preparation thereof of a kind of modification
Methods and applications, particularly relate to the method for modifying of a kind of Neu 5 Ac aldolase and the test kit containing this modification enzyme.
Background technology
N-acetyl-neuraminate generates ManNAc and acetone acid under Neu 5 Ac aldolase is catalyzed,
It it is one of enzyme important in sialic acid detection.
Sialic acid is the important component part of glycoprotein, relevant with many biological functions of organism, and with
Malignant change of cell, cancerometastasis, infiltrate, lose inhibition contact, cell adhesion reduce and tumor antigenicity closely related.Measure
Serum sialic acid (SA) concentration, can be as diagnosing tumor Subsidiary Index and parameters for observation on effect.
Disease significantly raised for SA has: acute leukemia, esophageal carcinoma, carcinoma of gastric cardia, gastric cancer, intestinal cancer, hepatocarcinoma, pulmonary carcinoma and ovum
Nest cancers etc., wherein with acute leukemic patient for the highest.Serum of Cancer Patients sialic acid increases and is probably tumor cell ptyalose egg
White synthesis increases the reflection in blood of the change with metabolism.
The change of cancer patient's SA horizontal dynamic is relevant to the state of an illness, may be used for transfer and the recurrence of early discovery tumor.
The principle of sialic acid detection: the sialic acid in sample is acted on by neuraminidase, forms N-acetyl nerve ammonia
Acid, and then under the effect of Neu 5 Ac aldolase (NANA-aldolase), generate acetone acid and ManNAc.
Acetone acid generates lactic acid and NAD+ under being acted on by lactic acid dehydrogenase (LDH) in the presence of NADH.The extinction measuring this NADH declines
Speed can try to achieve sialic acid concentration in sample.
Because Neu 5 Ac aldolase side chain also has carboxyl, carboxyl to have certain oxidisability, reagent 2 contains
Reduced coenzyme, long storage can make reduced coenzyme be aoxidized by Neu 5 Ac aldolase, result in reagent
Stability is the best.
At present, the stability of universal SA detection kit is 6 months to 10 months.Main cause is just N-in reagent
Acetylneuraminate aldolase and reduced coenzyme reciprocal influence.Containing the N-acetyl-neuraminate using the inventive method to modify
The test kit of aldolase, stability reaches 14 months, and stability is greatly improved.
Summary of the invention
For defect of the prior art, it is an object of the invention to provide the modification of a kind of Neu 5 Ac aldolase
Method and the test kit containing this modification enzyme.The present invention provides the method for modifying of a kind of Neu 5 Ac aldolase, simultaneously profit
The Neu 5 Ac aldolase modified by the method, has invented a kind of stable testing agent for sialic acid box.This test kit
Sensitivity is good, good stability, and measurement result is accurately and reliably.
It is an object of the invention to be achieved through the following technical solutions:
First aspect, the invention provides the Neu 5 Ac aldolase of a kind of modification, it is characterised in that described N-
The amino acid side chain of acetylneuraminate aldolase is modified through alkylation modification and enzyme space structure.
Second aspect, the invention provides the preparation method of the Neu 5 Ac aldolase of a kind of modification, including with
Lower step:
A1, Neu 5 Ac aldolase is reacted with alkylating reagent, remove the carboxyl of its amino acid side chain；
A2, the Neu 5 Ac aldolase guanidine hydrochloride after carboxyl will be removed modify,.Use guanidine hydrochloride
Neu 5 Ac aldolase is modified, enzyme molecule can be made again to fold, change its space structure.
Preferably, in step A1, described alkylating reagent includes the one in carbodiimides, iodoacetic acid and iodoacetamide
Or several mixing.
Preferably, in step A1, described reaction is particularly as follows: use triethylamine as solvent, N-acetyl-neuraminate aldehyde
Contracting enzyme mixes with the mass ratio of 1:3～5 with alkylating reagent, is stirred at room temperature overnight.
Preferably, in step A2, described modification method particularly includes: described containing the N-acetyl-neuraminate removing carboxyl
The solution of aldolase adds the guanidine hydrochloride of certain mass, use glacial acetic acid regulation PH to 5.0～6.0 times, stirred overnight at room temperature.
Preferably, the Neu 5 Ac aldolase after described removal carboxyl is 1:5～10 with the mass ratio of guanidine hydrochloride.
The third aspect, the invention provides a kind of test kit containing the Neu 5 Ac aldolase modified, including
Reagent R1 and reagent R2；
Described reagent R1 includes each component of following concentration:
Reagent R2 includes each component of following concentration:
Preferably, described buffer is Good ' s buffer, Tris buffer, glycine-NaOH buffer, HEPES
One or more in buffer, MES buffer；In described R1, the PH of buffer is 5.0-7.0, the PH of buffer in reagent R2
For 8.0-10.0.
Preferably, during described surfactant is polysorbas20, Tween 80, TritonX-100, dodecylbenzene sodium sulfonate
One or more.
Preferably, described stabilizer is bovine serum albumin, trehalose, sucrose, the one or several of chelating agen EDTA apoplexy due to endogenous wind
Kind；Described preservative is sodium azide or proclin series preservative.
Compared with prior art, the present invention has a following beneficial effect:
(1) containing carboxyl in Neu 5 Ac aldolase amino acid side chain.Carboxyl contains certain oxidisability, in examination
Agent can influence each other with reduced coenzyme, make reduced coenzyme content reduce, cause reagent event stability the best.Use this
After dressing agent in bright is modified, eliminate carboxyl, thus improve the stability in the large of reagent.
(2), after Neu 5 Ac aldolase removes carboxyl, the space conformation of enzyme there occurs change, by enzyme space structure
The impact of elephant, the response speed of Neu 5 Ac aldolase has declined.For solving this problem, we use guanidine hydrochloride
Processing enzyme, guanidine hydrochloride can make peptide bond be fully extended, and hydrophobic group intramolecular to enzyme is modified, and makes enzyme again enter
Row folds so that enzyme space conformation changes.Enter the Neu 5 Ac aldolase after persalt guanidine is modified to repair than carboxyl
Before decorations, vigor is higher.
(3) the best stabilized pH value of common Neu 5 Ac aldolase is 7.4-8.0, and reduced coenzyme is stable
PH condition need to reach more than PH9.0.The difference stablizing PH causes under normal circumstances, it is impossible to 2 enzymes are all kept
Good steady-state conditions.And the Neu 5 Ac aldolase after using the inventive method to modify, because of the change of enzyme conformation, enzyme steady
Determine PH and be changed to 8.8-9.2.2 enzymes so can be made all to coexist under optimal steady-state conditions.
(4) enzyme activity after modifying is higher, and in reagent, stability is more preferably, is not concerned with mutually with reduced coenzyme simultaneously
Disturb so that the sensitivity of reagent more preferably, can detect the sialic acid of more trace, in the pattern detection that value is relatively low, accurate
Exactness is more preferably.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 is the calibration curve of test kit of the present invention；
Fig. 2 is the correlation analysis figure of test kit of the present invention.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following example will assist in the technology of this area
Personnel are further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement.These broadly fall into the present invention
Protection domain.
Embodiment 1
The present embodiment relates to Neu 5 Ac aldolase of a kind of modification and preparation method thereof, and described N-acetyl is neural
The amino acid side chain of propylhomoserin aldolase is modified through alkylation.
Described preparation method comprises the steps:
Triethylamine is used to mix with the mass ratio of 1:3 with carbodiimides as solvent, Neu 5 Ac aldolase,
It is stirred at room temperature overnight, removes the carboxyl of its amino acid side chain；
In the above-mentioned solution containing the Neu 5 Ac aldolase removing carboxyl, add guanidine hydrochloride, use glacial acetic acid to adjust
Joint PH to 5.0～6.0 times, stirred overnight at room temperature.Neu 5 Ac aldolase after described removal carboxyl and guanidine hydrochloride
Mass ratio is 1:5.
Embodiment 2
The present embodiment provides the detectable of the Neu 5 Ac aldolase of a kind of modification containing embodiment 1 preparation
Box, its composition is as follows:
The each component of reagent R1 and concentration is:
The each component of reagent R2 and concentration is:
The SA detection kit that the present embodiment describes, it is adaptable to various types of full automatic biochemical apparatus is complete with Hitachi 7170
As a example by automatic biochemical analyzer, its operation is such as table 1.Analysis method: performance rate method, i.e. reagent R1；The consumption of R2 is respectively 240 μ l and 60 μ
L, sample size 8 μ l；240 μ l reagent R1 add 8 μ l samples and add 60 μ lR2 after 37 DEG C of 5min, postpone to start read point in 120 seconds, read
The about 180 seconds time of number；Detection wavelength dominant wavelength 340nm, commplementary wave length 405nm respectively.
Use this reagent and said determination method, the curve of the SA standard substance that employing Hitachi 7170 biochemistry analyzer records
(as shown in Figure 1), wherein X-axis represents SA content (mg/dl)；Y-axis represents absorbance.
Table 1
Embodiment 3: the correlation test of detectable
The purpose of the present embodiment is reagent and the dependency of available reagent of the detection embodiment of the present invention 2 preparation.
The SA using this law invention reagent (concrete formula is with embodiment 2) and contrast agents Japan and Guang Chun medicine company tries
100 parts of human serums (including normal and monstrosity) are measured by each autoregressive parameter, measured value are carried out dependency and divides by agent
Analysis.Measurement result is shown in Fig. 2, X, and Y-axis is measured value (the content mg/dl of SA).
Found out by the result of Fig. 2, the coefficient R of two kinds of reagent2=0.9928, regression equation is y=1.0265x-
0.926.It is good that result shows that this reagent and import reagent measure patients serum's dependency, has good specificity with accurate
Property.
Additionally, above this reagent of testing is that 7170 full automatic biochemical apparatus using Hitachi, Ltd to manufacture are carried out, but this
Bright reagent is not limited to above-mentioned instrument, applies also for other full-automatic or semi-automatic biochemical analyzers.
Embodiment 4 sensitivity test
The purpose of the present embodiment is the detection test kit of the present invention sensitivity when test sample.
Use the reagent of experimental example 2, contrast agents, standard substance.
Operating procedure: measure water sample 20 times, calculates reagent lowest detectable limit.
Table 2 shows, containing the test kit of the Neu 5 Ac aldolase using the inventive method to modify, lowest detectable limit
0.5mg/dl can be arrived.The lowest detection of the test kit of the Neu 5 Ac aldolase containing unmodified is limited to 5mg/dl.Examination
The sensitivity of agent is greatly improved.
Table 2
Embodiment 5 stability test
The purpose of the present embodiment is the stability of detectable.
Use experimental example 1 reagent, standard substance, quality-control product.
Operating procedure:
1., after test kit preserves 7 days under the conditions of heating (37 DEG C), take out each Quality Control of kit measurement 3 times, result such as table 3
Shown in.
2. test kit is under the conditions of cold preservation (2～8 DEG C), within every 2 months, takes out kit measurement Quality Control, and result is as shown in table 4.
Table 3 shows, after reagent preserves 7 days under the conditions of heating (37 DEG C), the accuracy of detection is the most fine.Table 4 shows,
Test kit is under the conditions of cold preservation (2～8 DEG C), and after preserving 14 months, reagent accuracy keeps good.From table, result can be seen that
Reagent stability of the present invention is good, can preserve for a long time.It is suitable for clinical requirement.
37 DEG C of table 3 sample heats the testing result after 7 days
| Low value Quality Control | High level Quality Control | |
| 1 | 59.51 | 102.93 |
| 2 | 58.07 | 102.52 |
| 3 | 59.67 | 98.90 |
| Meansigma methods | 59.083 | 101.450 |
| Target value | 59 | 100 |
| Relative deviation | 0.14% | 1.45% |
The table 4. sample preservation stability of 14 months
Embodiment 6
The present embodiment relates to Neu 5 Ac aldolase of a kind of modification and preparation method thereof, and described N-acetyl is neural
The amino acid side chain of propylhomoserin aldolase is modified through alkylation.
Described preparation method comprises the steps:
Using triethylamine as solvent, Neu 5 Ac aldolase mixes with the mass ratio of 1:5 with iodoacetic acid, in room
It is stirred overnight under temperature, removes the carboxyl of its amino acid side chain；
In the above-mentioned solution containing the Neu 5 Ac aldolase removing carboxyl, add guanidine hydrochloride, use glacial acetic acid to adjust
Joint PH to 5.0～6.0 times, stirred overnight at room temperature.Neu 5 Ac aldolase after described removal carboxyl and guanidine hydrochloride
Mass ratio is 1:10.
The present embodiment also provides for the detection of the Neu 5 Ac aldolase of a kind of modification prepared containing said method
Test kit, its composition is as follows:
The each component of reagent R1 and concentration is:
The each component of reagent R2 and concentration is:
The correlation test result of test kit prepared by the present embodiment is substantially the same manner as Example 3, measures with import reagent
Patients serum's dependency is good, has good specificity and accuracy.Its sensitivity test result be lowest detectable limit up to
0.5mg/dl.Its stability test result is: after preserving 7 days under the conditions of heating (37 DEG C), the accuracy of detection is the most fine；?
Under the conditions of cold preservation (2～8 DEG C), after preserving 14 months, reagent accuracy keeps good.Test kit prepared by the present embodiment can long time
Between preserve, be suitable for clinical requirement.
Embodiment 7
The present embodiment relates to Neu 5 Ac aldolase of a kind of modification and preparation method thereof, and described N-acetyl is neural
The amino acid side chain of propylhomoserin aldolase is modified through alkylation.
Described preparation method comprises the steps:
Triethylamine is used to mix, in room with the mass ratio of 1:4 as solvent, Neu 5 Ac aldolase iodoacetamide
It is stirred overnight under temperature, removes the carboxyl of its amino acid side chain；
In the above-mentioned solution containing the Neu 5 Ac aldolase removing carboxyl, add guanidine hydrochloride, use glacial acetic acid to adjust
Joint PH to 5.0～6.0 times, stirred overnight at room temperature.Neu 5 Ac aldolase after described removal carboxyl and guanidine hydrochloride
Mass ratio is 1:7.
The present embodiment provides the detectable of the Neu 5 Ac aldolase of a kind of modification containing embodiment 1 preparation
Box, its composition is as follows:
The each component of reagent R1 and concentration is:
The each component of reagent R2 and concentration is:
The correlation test result of test kit prepared by the present embodiment is substantially the same manner as Example 3, measures with import reagent
Patients serum's dependency is good, has good specificity and accuracy.Its sensitivity test result be lowest detectable limit up to
0.5mg/dl.Its stability test result is: after preserving 7 days under the conditions of heating (37 DEG C), the accuracy of detection is the most fine；?
Under the conditions of cold preservation (2～8 DEG C), after preserving 14 months, reagent accuracy keeps good.Test kit prepared by the present embodiment can long time
Between preserve, be suitable for clinical requirement.
The concrete application approach of the present invention is a lot, and the above is only the preferred embodiment of the present invention.It should be pointed out that, above
Embodiment is merely to illustrate the present invention, and is not limited to protection scope of the present invention.Common skill for the art
For art personnel, under the premise without departing from the principles of the invention, it is also possible to make some improvement, these improvement also should be regarded as this
Bright protection domain.
Claims (10)
1. the Neu 5 Ac aldolase modified, it is characterised in that the ammonia of described Neu 5 Ac aldolase
Base acid side chain is modified through alkylation modification and enzyme space structure.
2. the preparation method of the Neu 5 Ac aldolase of a modification as claimed in claim 1, it is characterised in that bag
Include following steps:
A1, Neu 5 Ac aldolase is reacted with alkylating reagent, remove the carboxyl of its amino acid side chain；
A2, the Neu 5 Ac aldolase guanidine hydrochloride after carboxyl will be removed modify,.
3. the preparation method of the Neu 5 Ac aldolase modified as claimed in claim 2, it is characterised in that step A1
In, described alkylating reagent includes one or more mixing in carbodiimides, iodoacetic acid and iodoacetamide.
4. the preparation method of the Neu 5 Ac aldolase modified as claimed in claim 2, it is characterised in that step A1
In, described reaction is particularly as follows: use triethylamine as solvent, and Neu 5 Ac aldolase and alkylating reagent are with 1:3
～the mass ratio mixing of 5, it is stirred at room temperature overnight.
5. the preparation method of the Neu 5 Ac aldolase modified as claimed in claim 2, it is characterised in that step A2
In, described modification method particularly includes: in the described solution containing the Neu 5 Ac aldolase removing carboxyl, add salt
Acid guanidine, use glacial acetic acid regulation PH to 5.0～6.0 times, stirred overnight at room temperature.
6. the preparation method of the as claimed in claim 5 Neu 5 Ac aldolase modified, it is characterised in that described in go
Except the mass ratio of the Neu 5 Ac aldolase after carboxyl Yu guanidine hydrochloride is 1:5～10.
7. the test kit containing the Neu 5 Ac aldolase modified as claimed in claim 1, it is characterised in that
Including reagent R1 and reagent R2；
Described reagent R1 includes each component of following concentration:
Reagent R2 includes each component of following concentration:
8. the test kit containing the Neu 5 Ac aldolase modified as claimed in claim 7, it is characterised in that described
Buffer be in Good ' s buffer, Tris buffer, glycine-NaOH buffer, HEPES buffer, MES buffer
One or more；In described R1, the PH of buffer is 5.0-7.0, and in reagent R2, the PH of buffer is 8.0-10.0.
9. the test kit containing the Neu 5 Ac aldolase modified as claimed in claim 7, it is characterised in that described
Surfactant is one or more in polysorbas20, Tween 80, TritonX-100, dodecylbenzene sodium sulfonate.
10. the test kit containing the Neu 5 Ac aldolase modified as claimed in claim 7, it is characterised in that institute
State stabilizer be bovine serum albumin, trehalose, sucrose, chelating agen EDTA apoplexy due to endogenous wind one or more；Described preservative is folded
Nitrogen sodium or proclin series preservative.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866153A (en) * | 2017-05-11 | 2018-11-23 | 广州市伊川生物科技有限公司 | A kind of Application of Sialic Acid Measurement kit and its measuring method |
| CN109097434A (en) * | 2018-08-15 | 2018-12-28 | 山东博科生物产业有限公司 | A kind of testing agent for sialic acid box |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866153A (en) * | 2017-05-11 | 2018-11-23 | 广州市伊川生物科技有限公司 | A kind of Application of Sialic Acid Measurement kit and its measuring method |
| CN109097434A (en) * | 2018-08-15 | 2018-12-28 | 山东博科生物产业有限公司 | A kind of testing agent for sialic acid box |
| CN109097434B (en) * | 2018-08-15 | 2021-08-27 | 山东博科生物产业有限公司 | Sialic acid detection kit |
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| Publication number | Publication date |
|---|---|
| CN106324234B (en) | 2018-05-22 |
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