WO2017054371A1 - Cystatin c product capable of serving as standard substance and preparation method and use thereof - Google Patents

Abstract

Provided is a cystatin C product capable of serving as a standard substance, and comprising: a recombinant human cystatin C protein accounting for 95% or more by mass thereof. The protein has an amino acid sequence and physicochemical properties identical to those of a natural cystatin C, and can be used for preparation of a cystatin C standard substance, a cystatin C diagnostic agent, or a cystatin C calibrator or quality control material.

Description

Cystatin C product which can be used as a standard substance, a preparation method thereof and use thereof Technical field

The present application relates to the field of metrology, in particular to the field of reference materials, and more particularly to a cystatin C product useful as a reference material, a process for its preparation and its use.

Background technique

Cystatin C (CysC), also known as cysteine protease inhibitor C, is a member of the cysteine protease inhibitor family. The human CysC has a relative molecular mass of 13 kD and is a secreted low molecular weight protein consisting of 120 amino acids. It is widely distributed in body fluids such as blood, cerebrospinal fluid, saliva and semen. It can be freely filtered by glomeruli, and its serum concentration is negatively correlated with glomerular filtration rate (GFR), regardless of gender or age. Interference with factors such as diet, inflammation, blood lipids, and liver disease. CysC is an ideal marker for reflecting impaired renal function.

In recent years, the clinical application of human CysC and the literature reports of detection methodologies have confirmed its important value. In view of the increasing incidence of chronic kidney disease worldwide, the diagnostic value of serum CysC as a clinical examination has attracted more and more attention.

CysC assays typically use PENIA, PETIA or ELISA methods. Test kits from a variety of different manufacturers are available on the market and can be run on different test platforms. However, it has been reported in the literature that there are large differences in the magnitude of the values between different laboratories, which leads to the inconsistency of measurements in different laboratories and even the same sample (Christine A. White et al., 2013). . This inter-room variation is, to a large extent, derived from the differences between calibrators (or standards) provided by different manufacturers.

Therefore, the development of CysC reference materials and/or reference methods will help to harmonize the differences between manufacturers. After years of hard work, finally in 2008, the International Federation of Clinical Chemistry (IFCC) announced the successful development of CysC certified reference materials (certificate number: ERM-DA471/IFCC). However, it is too expensive, and most manufacturers and medical institutions, especially those in developing and underdeveloped countries, cannot afford it; and the ordering cycle is extremely long and it is difficult to meet clinical needs. Therefore, it has not been widely used in the clinical community so far.

Conventional human CysC is extracted from a patient's blood, urine, etc., and the preparation method has various disadvantages. For example, the collection of samples is affected by ethical factors, large-scale sample collection is not possible, and it is difficult to produce on a large scale. This is especially disadvantageous for secondary (ie, working-grade) reference materials because it is directly used for on-site analytical measurements. The demand is very large; due to the difference in the source of the samples collected in different batches, it is easy to cause a large difference between the batches of the finished products; the concentration of CysC in the sample is low, which leads to more impurities in the finished product, which is difficult to purify, so the purity is poor; the production cost is also relatively Higher. Take ERM-DA471/IFCC as an example, prepared from the serum of volunteers in two centers (see ERM-DA471 certification report, EUR 24408EN–2010).

In recent years, some studies have reported the use of genetic engineering methods for the production of recombinant human CysC, but most of their amino acid sequences are not completely faithful to natural CysC.

Zhang et al., 2007, used pET-28a(+) expression vector to achieve soluble expression of CysC in E. coli expression system. However, it was repeatedly found in our experiments that most of the CysC expressed in pET-28a(+) existed in the form of inclusion bodies, and the soluble form was extremely small, making it impossible to carry out large-scale production of CysC.

Sichuan Mike Technology Co., Ltd. (Chinese Patent Application No.: 200810147715.4) applied the pET-32a(+) expression vector to achieve soluble expression of CysC in E. coli. However, the expression product is a fusion protein with a Trx tag. Even with the use of enterokinase for excision of the tagged protein, there are still additional amino acid residues that do not result in a protein whose amino acid sequence is completely faithful to native CysC.

Chen et al., 2012, constructed the prokaryotic expression plasmid pCold TF-CysC of human CysC gene and obtained soluble expression of CysC, but the expression product still has extra TF tag residue after excision by 3C protease.

Wuhan Youzhi Bio-Pharmaceutical Co., Ltd. (a method for preparing recombinant cystatin C in Pichia pastoris, Chinese Patent Application No.: 201210104241.1) uses the pPIC9K expression vector to achieve soluble expression of CysC in Pichia pastoris, but The expression product has a histidine tag at the C-terminus and is therefore not fully faithful to the native CysC in the amino acid sequence.

According to the requirements of the reference material, it should have at least the same composition and characteristics as the measured substance. When using the reference substance to determine the amount of the substance to be tested, in order to eliminate the difference between the reference material and the measurement range due to the difference between the standard substance and the substance to be tested For systemic influences, it should be selected as the candidate for the reference material, which is preferably the same as the nature and composition of the substance to be tested. This is the most basic principle to be followed in the development and use of reference materials.

In view of this, in the preparation of standard materials, producers often consciously choose certain materials or artificially synthesize some materials, such as: collecting leaves to simulate the substrate of plants in biochemistry and environmental analysis; artificially synthesizing glass containing trace elements as minerals The matrix of the components; the simulated seawater, river water, acid rain as the matrix of the water quality standard substances, etc., all of which are designed to eliminate the influence of the matrix difference when measuring with the standard substance.

In this respect, the above-mentioned principles have not been met by the CysC reference materials or preparation methods provided by Zhang et al., Chen Te et al., Sichuan Mike et al. It can be seen that such a standard substance cannot ideally impart a true amount to the substance to be tested when it is actually applied.

Summary of the invention

According to an aspect of the present application, the present application provides a CysC product useful as a standard substance containing ≥95% by mass of recombinant human CysC. In other words, the purity of the CysC product is ≥95%. In some embodiments, the purity is > 96%, > 97%, > 98%, > 99%, or > 99.5%.

Purity characterizes the extent to which a substance contains impurities. The less impurities, the higher the purity. Determination of protein purity allows for the use of any method known to those skilled in the art, including current and future assays, including but not limited to electrophoresis, chromatography. In some embodiments, the purity of the CysC product of the present application is the purity determined by electrophoresis. Specifically, in the electrophoresis method, the CysC product of the present application is applied to a gel for electrophoresis, and then the gel is dyed to represent the staining intensity of the band of the recombinant human CysC and the staining intensity of all the bands. For comparison, the content (i.e., purity) of the recombinant human CysC protein in the CysC product was determined by the ratio obtained.

In other embodiments, the purity of the CysC product of the present application is the purity determined by chromatography. For example, the CysC product is loaded onto the column, and when the component flows out of the column, the UV detection signal is used to compare the chromatographic peak area representing the recombinant human CysC protein with the total peak area, and the recombination is determined by the obtained ratio. The content of human CysC protein in CysC products. In some embodiments, the amino acid sequence of recombinant human CysC is SEQ ID No. 2. SEQ ID No. 2 is the amino acid sequence of native human CysC, total 120 The amino acid, sequence of the CysC precursor protein is available from the NCBI public database under accession number NP_000090.1.

According to another aspect of the present application, the present application provides a CysC solution that can be used as a standard substance, which is produced from the above CysC product. This CysC solution itself can be used as a standard solution for the solution. In view of the different analytical methods and different reagent systems used in the analytical instrument, the CysC solution can be prepared in a suitable reagent system according to actual needs.

In some embodiments, the reagent system in the CysC solution is selected from one or more of the group consisting of water, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, Tris-HCl buffer, and acetic acid-sodium acetate buffer. . The skilled person also allows the CysC product of the present application to be formulated in a biological fluid matrix; the biological fluid matrix is selected from the group consisting of: serum matrix, plasma matrix, and urine matrix. In a preferred embodiment, the CysC solution is a CysC solution obtained by dissolving the CysC product of the present application in a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system.

According to another aspect of the present application, the present application provides a method of preparing a CysC product useful as a standard substance, comprising:

Providing a construct comprising the nucleotide of SEQ ID NO: 1;

The construct is loaded into an expression vector to obtain a recombinant expression vector;

Converting the obtained recombinant expression vector into a host cell;

The host cell transformed with the recombinant expression vector is cultured, and the host cell is expressed to express the labeled recombinant human CysC;

Labeled recombinant human CysC isolated from culture;

Removing the tag of recombinant human CysC, preferably removing the tag by enterokinase cleavage;

Purification of recombinant human CysC;

Purified recombinant human CysC was fixed to obtain a CysC product that can be used as a standard substance.

Optionally, after the value of the purified recombinant human CysC is determined, the step of packaging the recombinant human CysC after the setting is also included.

SEQ ID No. 1 is the nucleotide sequence of native human CysC, a total of 360 nucleotides, which is available from the NCBI public database under accession number NM_000099.

In some embodiments, the nucleoside represented by SEQ ID NO: 1 is used for ease of handling. The acid is provided in the form of a construct, and thus an operation in the construct that allows introduction of an enzyme cleavage site, a stop codon, and the like to facilitate recombinant expression. In some embodiments, the construct comprises an entire human CysC coding sequence, a restriction site, and a nucleotide encoding an enterokinase recognition site. In a specific embodiment, the nucleotide sequence of the construct is SEQ ID NO:3. In a specific embodiment, the enterokinase recognition site is located at the 5' or 3' end of the human CysC coding sequence; preferably the 5' end.

In some embodiments, the tag is a histidine tag. The histidine tag is also known in the art as a His-tag. A plurality of histidine-stringed peptides are fused at the end of the recombinant protein, and the chelation of the histidine peptide with the divalent metal ions (nickel, zinc, etc.) facilitates purification of the protein by metal chelate affinity chromatography.

In some embodiments, the expression vector is a prokaryotic expression vector. The prokaryotic expression vector is selected from the group consisting of: pET-39b (+), pET-41a (+), and pET-32a (+). Preferably, the prokaryotic expression vector is pET-39b (+). pET-39b(+) is commercially available.

In some embodiments, the construct is loaded into an expression vector to obtain a recombinant expression vector, particularly the construct is loaded into an expression vector according to the reading frame. In the process of constructing a recombinant expression vector, the loading is obtained by this method: the expression vector and the construct are respectively cleaved by restriction endonucleases to produce a matching end (which may be a sticky end or a blunt end), and then DNA ligase The cleavage construct is ligated to the cleaved expression vector to obtain a recombinant expression vector.

In a specific embodiment, the KpnI enzyme and the XhoI enzyme are used for enzymatic cleavage. Additional restriction sites are also provided on the expression vector.

In some embodiments, the host cell is E. coli. The E. coli is selected from the group consisting of Escherichia coli BL21 (DE3) pLysS, C43 (DE3), BL21 (DE3) and Rosetta (DE3). Preferably, the Escherichia coli is Escherichia coli BL21(DE3)pLysS. BL21(DE3)pLysS is commercially available.

In some embodiments, the recombinant expression vector is transformed into E. coli using methods known in the art. Such methods include, but are not limited to, electroporation or preparation of competent cells.

In some embodiments, a host cell transformed with a recombinant expression vector is cultured such that the host cell expresses a tagged recombinant human CysC. E. coli BL21(DE3)pLysS can be cultured by methods well known in the art. For example: BL21(DE3)pLysS can be used The recommended culture method, or the method taught in the Guide to Molecular Cloning. In a specific embodiment, E. coli is cultured in an LB medium containing antibiotics; the culture temperature is 25-38 °C.

In a specific embodiment, the host cell is expressed to express a tagged recombinant human CysC by induction. Methods of inducing expression include, but are not limited to, IPTG induction, lactose induction. The specific manner of induction may vary depending on the expression vector. In some embodiments, the method of induction is IPTG induction. The induction temperature was 22-28 ° C; the induction time was 15-20 h; the final concentration of IPTG was 0.4-1.0 mM.

In some embodiments, the separating is performed by affinity. Preferably, the affinity mode is nickel affinity chromatography.

In a specific embodiment, the nickel affinity chromatography is performed using a nickel chromatography column. Those skilled in the art can select available commercially available nickel chromatography columns according to production scale and environmental conditions, or self-fill nickel chromatography columns according to known methods.

In some embodiments, the recombinant human CysC of the present application is expressed in fusion with a histidine tag. In some embodiments, the histidine kinase is used to remove a histidine tag on recombinant human CysC. In some embodiments, the concentration of enterokinase is 10 U enterokinase/mg of labeled recombinant human CysC. After digestion, the digested product obtained by enterokinase digestion is obtained by affinity-binding binding of the excised tag, and the recombinant human CysC which does not bind to the affinity column flows through the affinity column, thereby obtaining the purified recombinant body of the present application. CysC. In some embodiments, the affinity mode is nickel affinity chromatography.

In a specific embodiment, the nickel affinity chromatography is performed using a nickel chromatography column. Those skilled in the art can select available commercially available nickel chromatography columns according to production scale and environmental conditions, or self-fill nickel chromatography columns according to known methods.

In some embodiments, the purified recombinant human CysC is assayed. The fixed value refers to the method of traceability of recombinant human CysC to international standard substances or traceability to international standards. By this setting, the recombinant human CysC is assigned, and this value has a clear uncertainty. The valued CysC product (or in the form of a solution) can be used as a standard material. Optionally, purified recombinant human CysC can be diluted prior to setting for subsequent settings and applications.

According to another aspect of the present application, the present application provides usable by the above method. A standard material for CysC products.

According to another aspect of the present application, the present application provides the use of the above-described CysC product usable as a standard substance as a standard substance.

In some embodiments, a CysC product useful as a standard material according to the present application is used as a standard material for calibrating an instrument.

In other embodiments, a CysC product useful as a standard material according to the present application is used as a standard substance for quantitative analysis of CysC content in a sample to be tested.

According to another aspect of the present application, the present application provides the use of the above-described CysC solution usable as a standard substance as a standard substance.

In some embodiments, a CysC solution that can be used as a standard substance according to the present application, as a standard substance, is used to calibrate an instrument.

In other embodiments, a CysC solution that can be used as a standard substance according to the present application is used as a standard substance for quantitative analysis of CysC content in a sample to be tested.

DRAWINGS

Figure 1 is a purified electropherogram of recombinant human CysC protein induction.

Among them, M: pre-stained protein molecular weight marker (Marker); 1: IPTG did not induce lysate supernatant; 2: IPTG induced lysis supernatant after 18 h; 3: Ni-purified fusion protein; 4: Ni-column again Purified recombinant human CysC protein.

Figure 2 is a diagram showing the Western hybridization analysis of recombinant human CysC protein.

Among them, M: pre-stained protein molecular weight marker; 1: recombinant human CysC protein, primary antibody is rabbit anti-human CysC polyclonal antibody; 2: recombinant human CysC protein, primary antibody is rabbit anti-human serum albumin polyclonal antibody.

Figure 3 is an electropherogram of the CysC product of the present application and CysC International Standard Material DA471.

Among them, M: pre-stained protein molecular weight marker; 1: CysC product of the present application, 2: CysC international standard substance DA471.

detailed description

the term:

Reference material (also called reference material): according to ISO A material or substance having one or more characteristic values that are sufficiently uniform and well defined to calibrate a measuring device, evaluate a measuring method, or assign a value to a material. The reference material can be a pure or mixed gas, liquid or solid.

Certified reference material: A standard substance with a certificate whose one or more characteristic quantities are determined by a process established by traceability to traceable to accurately reproducible measurements indicating the value of the characteristic. Units, each of the identified characteristic magnitudes are accompanied by an uncertainty of the given confidence level.

Fusion protein: A fusion expression product obtained by recombinant techniques. In the context of the present application, a fusion protein is used to mean a tagged recombinant human CysC; in particular, a fusion protein refers to a recombinant human CysC with a histidine tag.

Example

Unless otherwise indicated, the following examples were carried out according to conventional methods known to those skilled in the art using reagents of the type commonly used in the corresponding assays and preparations in the art. % stated herein is mass/volume percentage unless otherwise indicated.

The plasmid vector used in the present application was preserved in the laboratory, pET-39b (+) was purchased from Novagen; Escherichia coli BL21 (DE3) pLysS was purchased from Novagen, and restriction endonuclease and T4 DNA ligase were purchased from NEB Corporation.

Example 1: Synthesis of a polynucleotide encoding human CysC

The coding sequence of human CysC SEQ ID No. 1 was obtained according to the sequence of the human CysC precursor protein gene NP_000090.1 published by NCBI. The construct of SEQ ID No. 3 was synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd., and the enterokinase recognition site, complete SEQ ID No. 1, stop codon and double enzyme were included in SEQ ID No. 3. Cut the spot.

Example 2: Construction of recombinant expression vector

The construct obtained in Example 1 was cleaved with KpnI and XhoI restriction enzymes, and ligated with the same double-cut pET-39b (+) to construct a prokaryotic expression plasmid. After double-digesting the plasmid with KpnI and XhoI restriction enzymes, DNA sequencing was performed, and the sequencing result was selected to show that the plasmid including SEQ ID No. 3 was a positive plasmid.

Example 3: Transformation of host cells

The positive plasmid was transformed into competent E. coli BL21(DE3)pLysS by competent cells to obtain a positive expression strain.

Example 4: Culture of host cells

The above positive strains were inoculated in 10 ml LB+Kan liquid medium, cultured at 37 ° C, 220 rpm for 16 to 20 hours; then inoculated in fresh LB + Kan medium according to 1% inoculum, cultured at 37 ° C, 220 rpm 3 4h; Take 1ml of bacterial solution as a control under uninduced conditions, add the remaining bacterial solution to IPTG with a final concentration of 1 mM, induce culture for 16-20 hours at 25 ° C, 220 rpm, and take 1 ml of bacterial solution as the sample after induction.

The bacterial bacterial liquid before and after induction was centrifuged to remove the supernatant, and the cells were resuspended in 100 μl of PBS buffer, boiled for 5 min, and detected by SDS-PAGE electrophoresis. Coomassie blue staining showed that there was a concentrated band at the predicted molecular weight in the supernatant of the cell lysate after induction (see Figure 1, lane 2), and the cells before induction had no such protein at the same molecular weight. Strip (see Figure 1, lane 1). Analysis by BandScan software revealed that the target protein accounted for about 15% by mass of the total protein. Positive expression strains can be used in the practice of the present application, and it is preferred to select strains having a higher expression level.

The above results demonstrate that the CysC fusion protein is present in a soluble form in the supernatant after induction, while no corresponding characteristic protein bands are present in the corresponding positions in the supernatant.

Example 5: Pilot scale host cell culture

The culture amount was amplified according to the conditions of the small-scale culture, and the positive expression bacteria preserved in 1% of glycerol were inoculated into 50 ml of LB+Kan medium, and cultured at 37 ° C, 220 rpm for 16-20 hours.

Thereafter, the cells were inoculated in 1 L of LB+Kan medium at 37 ° C according to a 2% inoculation amount, cultured at 220 rpm for 4-5 hours to an OD ₆₀₀ of 0.6-1.0, and IPTG was added to a final concentration of 1 mM, and cultured at 25 ° C, 220 rpm for 18 hours.

The cells were collected by centrifugation and added to the binding buffer according to the ratio of 1 g of the cells to 10 ml of binding buffer (20 mM PB, 500 mM NaCl, 20 mM imidazole, pH 7.4), sonicated, and centrifuged again to obtain the supernatant, which was filtered with 0.22 μm. The membrane was filtered and purified for purification.

Example 6: Isolation of labeled recombinant human CysC from culture

The HisTrap-FF (GE) column was equilibrated with 5 column volumes of binding buffer.

The supernatant after filtration in Example 5 was directly loaded onto the column. After loading, the column was washed with binding buffer to the baseline, and then washed with an elution buffer (20 mM PB, 500 mM NaCl, 200 mM imidazole, pH 7.4). The elution peak was taken off and the fusion protein (labeled recombinant human CysC) was obtained.

The fusion protein was analyzed for purity by SDS-PAGE electrophoresis, and the purity of the fusion protein was over 80% (see Figure 1, lane 3). The concentration of the fusion protein was determined by the BCA method to be 4-5 mg/ml. The 8L culture solution can obtain 320-350 mg of the fusion protein.

Example 7: Removal of the tag of the fusion protein

The desalting column was equilibrated with 3 column volumes of enterokinase digestion buffer (25 mM Tris-Cl, 200 mM NaCl, 2 mM CaCl ₂ , pH 7.4).

The fusion protein obtained by the above separation was directly applied to the column, washed with enterokinase digestion buffer, and the protein elution peak was collected.

Enterokinase was then added to the eluted solution to a final concentration of 10 U of enterokinase per mg of fusion protein, placed at 23 ° C, and digested for 16-20 hours in a 120 rpm shaker to remove the His-tag.

Example 8: Purification of recombinant human CysC

Finally, the digested product was loaded onto a HisTrap-FF column equilibrated with 5 column volumes of enterokinase digestion buffer to collect a breakthrough peak, ie, recombinant human CysC according to the present application (see Figure 3, lane 1). .

Example 9: Analysis of the properties of recombinant human CysC

1. Molecular weight identification:

The molecular weight of CysC was analyzed by SDS-PAGE electrophoresis of recombinant human CysC harvested in Example 8 and native human CysC, respectively.

The experimental results show that the recombinant human CysC (see Figure 3, Dao 1) and natural human CysC (see Figure 3, Dao 2) have a molecular weight of about 13 kDa.

2. Isoelectric point analysis:

The isoelectric point of recombinant human CysC was determined by isoelectric focusing electrophoresis (IEF) technique of recombinant human CysC harvested in Example 8.

The experimental results show that the recombinant human CysC is between pH 9.0-9.5 and has its characteristic protein band, and its isoelectric point is about pI=9.3, which is basically the same as the isoelectric point of natural human CysC reported in the literature.

3. Amino acid sequence identification:

The sequence of the recombinant human CysC protein of the present application was determined using the American ABI Procise 491 protein sequence sequencing system (based on Edman degradation).

The results of the assay showed that the sequence of the recombinant human CysC protein of the present application was identical to that of native human CysC. For example, the mass spectrometer captures the N-terminal sequence SSPGKPPRLVGGPMDASVEEEGV of the recombinant human CysC protein, which is identical to the N-terminal sequence of native human CysC.

It is indicated that the N-terminal fusion protein tag of the recombinant human CysC of the present application has been completely removed and does not contain any residual His.

4. Purity test:

The recombinant human CysC harvested in Example 8 was analyzed by SDS-PAGE electrophoresis. The method has a protein purity detection sensitivity of 0.5% (relative mass concentration), and no other recombinant cells were observed on the electrophoresis map of recombinant CysC (see Figure 3, lane 1) and CysC international standard material DA471 (see Figure 3, lane 2). Protein impurities. The calculation was repeated 3 times, and the average purity of the recombinant human CysC protein was 97.5% by mass.

5. Pilot production:

The recombinant human CysC harvested in Example 8 was subjected to BCA assay, and the final protein concentration was 2-3 mg/ml, and 8 L of the culture solution finally obtained 70-80 mg of recombinant human CysC protein having a purity of 95% or more by mass.

6. Antigenicity analysis:

The antigenicity of the recombinant CysC protein was identified by Elisa and Western blotting, respectively. After diluting the recombinant human CysC harvested in Example 8 to 10 μg/ml, the plate was coated according to the Elisa standard protocol, and a polyclonal antibody against human CysC and a polyclonal antibody against human serum albumin were used. Anti-HRP-labeled goat anti-rabbit IgG was used as the secondary antibody. The results showed that the CysC polyclonal antibody was positive for the platelet of the primary antibody, while the anti-human serum albumin polyclonal antibody was negative for the platelet of the primary antibody. Western hybridization results showed significant hybridization bands at a molecular weight of 13 kDa (see Figure 2).

The test results indicate that the recombinant human CysC of the present application has high immunogenicity. The recombinant human CysC of the present application was demonstrated to have the same immunogenicity as native CysC.

7. Comparison of recombinant human CysC and natural human CysC in detection system

The concentration of recombinant human CysC and natural human CysC protein (purchased from Danish DAKO) was determined on a fully automatic biochemical analyzer using different manufacturers of Cystatin C assay kits;

Recombinant human CysC and natural CysC (DAKO) were diluted to a concentration of 5 mg/L with PBS buffer; respectively, using the CysC assay kit (latex enhanced immunoturbidimetry) of Gcell, Roche and DAKO, respectively, in Hitachi 7180 The mass concentration of CysC was determined on a biochemical analyzer.

The results of the assay are shown in Table 1. The recombinant human CysC has a very close quantitative measurement with the natural human CysC, and the standard error is much less than 5%, which can be applied to the development of high quality CysC kit.

Table 1: Comparison of CysC mass concentration comparison results

summary:

The results of Example 9 fully demonstrate that the recombinant human CysC of the present application is highly consistent with natural CysC in terms of physicochemical properties, biological properties and metrological properties.

Example 10: Preparation of reference materials

The CysC recombinant protein prepared by the present application is firstly diluted with physiological saline containing a final concentration of 0.1% NaN ₃ and diluted to a final concentration of 5-10 mg/L;

Subsequent application of CysC International Standard Substance

-DA471/IFCC (5.48 mg/L) as a calibration substance, and the concentration of recombinant CysC after dilution was measured;

And continue to dilute the recombinant CysC to a final concentration of 4.9-5.1 mg / L;

Finally, it was dispensed into a brown glass bottle with a pipette, 1 ml per bottle.

Example 11: Setting of recombinant human CysC

1. Uniformity check:

Five samples were taken from different bottles of the recombinant human CysC standard material prepared in Example 10, and each measurement was repeated 4 times, and the measurement results were used as the results of the uniformity test between the bottles. Take any of the 5 samples and repeat the measurement 4 times. The measurement results are taken as the result of the in-bottle uniformity test.

The test results between the bottle and the bottle were statistically calculated by statistical method (F test) to judge the uniformity. The test results are shown in Table 2.

As a result of the test, it was found that the recombinant human CysC standard material prepared in Example 10 had good uniformity.

Table 2. Uniformity test results of recombinant human CysC

2. Stability check:

The recombinant human CysC solution prepared above was optionally taken and placed at room temperature (25 ± 2 ° C). The measurement was carried out seven days later, and the measurement was repeated three times at each point, and the measurement results were taken as the stability test results at room temperature, and the test results are shown in Table 3.

From the results, it was found that the recombinant human CysC solution of the present application was stable at room temperature for one week.

Table 3. Results of room temperature stability test of recombinant human CysC

3. Traceability:

The standard substance of the present application (recombinant human CysC solution prepared in Example 10) was subjected to constant value by electrospray-quadrupole-time of flight mass spectrometry.

Before setting the value, first adopt CysC international standard substance

-DA471/IFCC (5.48 mg/L, whose own uncertainty is U _CRM = 0.15 mg/L) Calibrate or verify the mass spectral mass axis. Then, the standard substance of this application is assigned by mass spectrometry, so that it is traceable to the international standard substance.

-DA471/IFCC, and algorithms known in the art calculate uncertainty.

Assignment and uncertainty calculation methods can refer to international standards, national standards or industry standards, and can also be carried out according to the methods in the textbook. For example, China Science Press's "Using Measurement Uncertainty Evaluation", Science Press's "Experimental Error Principles and Data Processing", Shanghai Metrology and Testing Technology Research Institute Press, "Common Measurement Uncertainty Evaluation Methods and Application Examples The algorithm is given in detail in the Measurement Uncertainty Evaluation and Representation Guide of China Metrology Publishing House. Therefore, it will not be listed in detail in this application. Of course, it can also be carried out in accordance with the method in Section 7 of ERM-DA471/IFCC. The factors to be considered in determining the uncertainty will vary with laboratory conditions, including but not limited to: the uncertainty of the DA471 itself, the factors of the mass spectrometer itself, the effects of personnel operations, and the solution preparation process. The uncertainty introduced.

Example 12: Calibration of a biochemical analyzer

The sequenced recombinant human CysC obtained in Example 11 was sequentially diluted with physiological saline containing a final concentration of 0.1% NaN ₃ so that the final concentrations were 5.0 mg/L, 2.5 mg/L, and 1.25 mg, respectively. /L, and 0.625 mg / L; the above physiological saline was further taken as 0 mg / L. A total of 5 points were used as calibrators for the measurement of the magnitude.

Then use the above 5-point calibrator to determine the CysC international standard substance after the calibration of Gell's CysC latex-enhanced immunoturbidimetric reagent on the automatic biochemical analyzer Hitachi 7180.

The ratio of -DA471/IFCC (5.48 mg/L) was 2.74 mg/L, 1.37 mg/L, and 0.685 mg/L, and 0 mg/L. The measurement results are shown in Table 4.

Table 4. Calibration of the biochemical analyzer by the CysC reference material of the present application

Example 13: Recombinant human CysC of the present application for detection of a substance to be tested

Serum samples were assayed using the assigned recombinant human CysC multiple dilutions obtained in Example 11 as kit calibrators, while CysC International Standards was used as a calibrator control.

The Gys CysC assay kit was calibrated and determined in 20 cases using the concentration solutions of the standard substances of this application at 5.0 mg/L, 2.5 mg/L, 1.25 mg/L, and 0.625 mg/L, 0 mg/L. Serum sample

Simultaneously with CysC International Standard Substance

-DA471/IFCC (5.48mg/L) of the concentration of 5.48 mg / L, 2.74mg / L, 1.37 mg / L, and 0.685mg / L, and 0mg / L, as a calibrator for the Gcell CysC kit Calibration, as a control to determine the serum samples of the above 20 cases;

The measurement results show that the standard substance of the present application is almost the same as the serum sample sample and the international standard substance calibration serum sample value as the calibrator, and there is no difference; the measurement results are shown in Table 5.

Table 5. Determination of Serum Samples as a Calibrator for the CysC Reference Material of the Present Application

Sample number	Standard substance of the application		DA471
1	1.01	0.99
2	1.46	1.45
3	1.03	1.00
4	0.81	0.80
5	0.64	0.64
6	0.85	0.84
7	0.90	0.88
8	1.10	1.09
9	1.04	1.03
10	0.72	0.71
11	1.18	1.17
12	0.84	0.82
13	4.45	4.65
14	5.24	5.28

15	3.21	3.25
16	6.44	6.38
17	0.70	0.70
18	0.68	0.69
19	2.36	2.41
20	3.59	3.58

references

Christine A. White et al. The Impact of Interlaboratory Differences in Cystatin C Assay Measurement on Glomerular Filtration Rate Estimation. CJASN. 2013, vol 8(1);

ERM-DA471 certification report, EUR 24408 EN–2010;

Zhang et al., Expression and purification of recombinant human cystatin C in Escherichia coli, International Journal of Laboratory Medicine, 2007, 28(5): 39;

Chen Te et al., Preparation of Recombinant Proteins Without Fusion Labeled Human Cystatin C, Basic Medicine and Clinic, 2012, 32(6): 697-701.

Claims (9)

1. A cystatin C product useful as a standard substance containing ≥95% by mass of recombinant human cystatin C.
2. The cystatin C product usable as a standard substance according to claim 1, wherein the amino acid sequence of the recombinant human cystatin C is SEQ ID No. 2.
3. A cystatin C solution usable as a standard substance prepared by formulating the cystatin C product of claim 1 in a reagent system selected from the group consisting of water, sodium dihydrogen phosphate-hydrogen phosphate One or more of sodium buffer, Tris-HCl buffer, and acetic acid-sodium acetate buffer; sodium dihydrogen phosphate-disodium hydrogen phosphate buffer is preferred.
4. A method for preparing a cystatin C product useful as a standard substance, comprising:

   Providing a construct comprising the nucleotide set forth in SEQ ID NO: 1;

   The construct is loaded into an expression vector to obtain a recombinant expression vector;

   Converting the obtained recombinant expression vector into a host cell;

   The host cell transformed with the recombinant expression vector is cultured, and the host cell expresses the labeled recombinant human cystatin C;

   The labeled recombinant human cystatin C is isolated from the culture;

   Removing the tag of recombinant human cystatin C;

   Purification of recombinant human cystatin C;

   Purification of recombinant recombinant human Cystatin C to obtain a Cystatin C product that can be used as a standard substance;

   Optionally, packaging the recombinant human cystatin C after the setting,

   Wherein the label is preferably a histidine tag,

   Preferably, the construct comprises the nucleotide, cleavage site, enterokinase recognition site and stop codon represented by SEQ ID NO: 1; more preferably, the nucleotide sequence of the construct is SEQ ID NO :3.
5. Preparation of a Cystatin C Product As a Standard Substance According to Claim 4 Method, where:

   The expression vector is a prokaryotic expression vector;

   The host cell is Escherichia coli;

   The separation is carried out by affinity;

   The purification is carried out by affinity.
6. A method of producing a cystatin C product useful as a standard substance according to claim 5, wherein:

   The prokaryotic expression vector is selected from the group consisting of: pET-39b (+), pET-41a (+), and pET-32a (+);

   The Escherichia coli is selected from the group consisting of Escherichia coli BL21 (DE3) pLysS, C43 (DE3), BL21 (DE3) and Rosetta (DE3);

   The affinity is nickel affinity chromatography.
7. A cystatin C product useful as a standard material prepared by the method of any one of claims 4-6.
8. Use of a cystatin C product usable as a standard substance according to any one of claims 1-2 and 7 as a standard substance.
9. Use of a cystatin C solution usable as a standard substance according to claim 3 as a standard substance.

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