Recombinant nt-probnp protein for preparation of nt-probnp quality control and candidate reference material and preparation method thereof

Abstract

The application discloses a kind of recombinant NT-proBNP protein for NT-proBNP quality control product and candidate reference substance preparation and a preparation method thereof, belong to biological quality control product preparation technical field.The recombinant protein is according to the optimization NT-proBNP coding sequence of e. coli codon bias, constructs 6His-NT-proBNP-pET recombinant plasmid, is induced expression in e. coli prokaryotic expression system, and is obtained after purification.The recombinant protein can be accurately recognized by multiple clinical mainstream immune analysis system, and show good detection linearity and stability and uniformity.The application lays a foundation for the development of NT-proBNP quality control product and candidate reference substance.

Description

Technical Field

[0001] This invention relates to the field of biomass control product preparation technology, and in particular to a recombinant NT-proBNP protein for the preparation of NT-proBNP quality control products and candidate reference materials, and a method for preparing the same. Background Technology

[0002] Heart failure (HF) is a clinical syndrome that represents the end-stage progression of various cardiovascular diseases, characterized by high incidence, high recurrent hospitalization rates, and poor prognosis. N-terminal pro-B-type natriuretic peptide (NT-proBNP), an inactive linear polypeptide cleaved during the secretion of B-type natriuretic peptide (BNP), is the preferred biomarker for diagnosing and differentially diagnosing HF, assessing disease severity, and predicting prognosis. It has been adopted as a Level I recommendation for HF diagnosis by multiple clinical practice guidelines.

[0003] As the clinical value of NT-proBNP becomes increasingly prominent, ensuring the accuracy and comparability of its test results is receiving more and more attention. One of the important ways to achieve accurate and comparable clinical testing is to use reference materials (in a broad sense, reference materials include quality control materials, quality assessment materials, calibrators, certified reference materials, etc.) (ISO 17511:2020). However, high-concentration NT-proBNP reference materials are extremely difficult to obtain, and there is no internationally recognized reference method or primary reference material for NT-proBNP. Therefore, the need to develop NT-proBNP quality control materials and candidate reference materials is very urgent.

[0004] In layman's terms, quality control materials are divided into external quality assessment materials and internal quality control materials. External quality assessment materials are used for external quality evaluation, assessing the consistency and comparability of test results between different laboratories. Their concentrations cover both normal and abnormal ranges, focusing on external comparison and standardization. Internal quality control materials are selected by the laboratory itself and are used to continuously monitor the precision and stability of the testing system, taking into account the medical decision level within the normal range. They focus on internal monitoring and real-time error detection. The two complement each other in terms of source, purpose, and frequency, together forming a complete testing quality assurance system. Currently, the raw materials for the preparation of traditional quality control materials and reference materials are mostly derived from human serum. However, for the NT-proBNP test, the activity, degradation rate, and fragment composition of endogenous proteases in human serum from different individuals vary. In fact, NT-proBNP, proBNP, BNP, and related peptides formed to varying degrees of degradation are all present in serum. In addition, due to the special nature of the NT-proBNP index, it is impossible to collect large quantities of high-value serum (patients in severe heart failure or critical condition). Therefore, natural human serum of this type suffers from problems such as limited sources, long collection cycles, difficulty in obtaining large quantities, and significant individual variability. It is also easily affected by factors such as disease status, storage conditions, and the number of freeze-thaw cycles, thus impacting the stability of the target component in quality control materials or candidate reference materials. In contrast, using recombinant NT-proBNP protein as the source of the target component and adding it exogenously to a low-background mixed human serum matrix can preserve the characteristics of the human serum matrix while making the source of the target component more clearly defined and reducing the influence of disease status, endogenous NT-proBNP, protease activity, and its complex degradation fragments on naturally high-value patient serum.

[0005] Compared to eukaryotic expression, prokaryotic expression offers advantages such as faster generation speed, lower cost, simpler operation, and less impact on proteins like NT-proBNP, which have relatively simple modifications. However, the human NT-proBNP coding sequence was not designed for the *E. coli* expression system. Directly using the human coding sequence for prokaryotic expression may affect the recombinant protein expression efficiency and yield. Therefore, it is necessary to optimize the NT-proBNP coding sequence based on *E. coli* codon preferences to obtain recombinant NT-proBNP protein suitable for the *E. coli* expression system. Furthermore, the preservation method of serum matrix samples containing recombinant NT-proBNP protein also affects their stability and subsequent applications. Frozen liquid samples are susceptible to temperature fluctuations and repeated freeze-thaw cycles during storage, transportation, and use, while lyophilization is generally more conducive to stable sample preservation and transportation.

[0006] For raw materials used in the development of NT-proBNP quality control products and candidate reference materials, obtaining recombinant protein alone is insufficient. Further confirmation is needed regarding its recognition by mainstream clinical immunoassay systems, the linear relationship between detection values ​​and dilution factors, its homogeneity and stability after preparation as a candidate sample, and its ability to obtain quantitative detection results on different detection platforms. Therefore, developing recombinant NT-proBNP protein that is stable in origin, low in cost, and easily prepared on a large scale is of great significance for the subsequent development of NT-proBNP quality control products and candidate reference materials. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant NT-proBNP protein and its preparation method for NT-proBNP quality control materials and candidate reference materials, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing recombinant NT-proBNP protein for the preparation of NT-proBNP quality control materials and candidate reference materials, comprising the following steps: (1) The E. coli codons were optimized to obtain the gene fragment with the nucleotide sequence shown in SEQ ID NO.1; (2) After adding the 6His tag sequence to the gene fragment, it was cloned into the pET-28a(+) prokaryotic expression vector to obtain the recombinant expression vector; (3) The recombinant expression vector was transformed into E. coli BL21 Codon Plus competent cells, and the recombinant NT-proBNP protein was obtained by IPTG induction and protein purification.

[0009] Furthermore, the conditions for IPTG-induced expression are as follows: 0.4 mmol / L IPTG is used for induction at 30°C for 20 h.

[0010] Furthermore, the protein purification method is as follows: collect the bacterial cells after induced expression, perform ultrasonic lysis, and collect the supernatant by centrifugation; after filtering the supernatant through a filter membrane, nickel ion affinity chromatography, ultrafiltration concentration and molecular sieve chromatography, the purified recombinant NT-proBNP protein is obtained.

[0011] Optionally, the pore size of the filter membrane is 0.22 μm; The nickel ion affinity chromatography was performed under the following conditions: sequential elution with 20 mmol / L imidazole and 250 mmol / L imidazole. The ultrafiltration concentration method involves centrifugation using a 30KD ultrafiltration tube and a 10KD ultrafiltration tube in sequence. The molecular sieve is Superdex 75 increase 10 / 300 GL molecular sieve.

[0012] The present invention also provides a recombinant NT-proBNP protein prepared by the above preparation method.

[0013] The present invention also provides the application of the above-mentioned recombinant NT-proBNP protein in the preparation of NT-proBNP quality control products.

[0014] Optionally, the NT-proBNP quality control materials include external quality assessment materials and internal quality control materials.

[0015] Preferably, the NT-proBNP quality control material is an interlaboratory quality assessment material.

[0016] The present invention also provides the application of the above-mentioned recombinant NT-proBNP protein in the preparation of NT-proBNP reference material.

[0017] The present invention also provides the application of the above-mentioned recombinant NT-proBNP protein as a quality control in the preparation of a kit for detecting NT-proBNP.

[0018] The present invention also provides an NT-proBNP quality control product, which is prepared by adding the above-mentioned recombinant NT-proBNP protein to a serum matrix.

[0019] The present invention also provides a kit for detecting NT-proBNP, wherein the kit contains the above-mentioned NT-proBNP quality control sample.

[0020] The present invention discloses the following technical effects: This invention provides a recombinant NT-proBNP protein for the preparation of NT-proBNP quality control materials and candidate reference materials, and a method for preparing the same, which has the following advantages: (1) Stable source: The present invention prepares recombinant NT-proBNP protein through genetic engineering technology, avoiding the problems of limited source of natural high-value human serum samples, poor stability and strict ethical review; (2) Convenient preparation: The present invention can rapidly obtain the target protein using the Escherichia coli prokaryotic expression system, and has the characteristics of relatively simple operation, short preparation cycle, low cost and easy large-scale preparation; (3) Easy to purify. The recombinant NT-proBNP protein contains an N-terminal 6×His tag, and the protein can be further purified by nickel ion affinity chromatography, ultrafiltration concentration and molecular sieve chromatography. (4) Good detectability: The purified recombinant NT-proBNP protein can be accurately identified and detected by mainstream clinical immunoassay systems. Moreover, the detection value after serial dilution has a good linear relationship with the dilution factor, indicating that it has the basis for subsequent preparation of NT-proBNP quality control products and candidate reference materials. (5) The preliminary applicability is good. After the purified recombinant NT-proBNP protein is prepared into frozen liquid samples and lyophilized samples with mixed human serum as matrix, the homogeneity and short-term stability at 4℃ are evaluated. The results show that the two samples have good homogeneity. The lyophilized sample shows better short-term stability at 4℃ than the frozen liquid sample, and is more suitable as a preservation form for NT-proBNP quality control and candidate reference material. (6) It has a certain basis for multi-platform detection applicability. The recombinant NT-proBNP sample prepared by the present invention can obtain quantitative detection results on multiple clinically commonly used NT-proBNP detection platforms. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results of agarose gel electrophoresis analysis of the 6His-NT-proBNP-pET recombinant plasmid after double digestion with restriction endonucleases are shown. Lane 1 contains plasmid DNA, and lane 2 contains the product of double digestion of the recombinant plasmid with Mlu I and Xho I. M is the DNA marker. Figure 2 SDS-PAGE analysis results of expression and purification products of recombinant bacteria transformed with 6His-NT-proBNP-pET plasmid; where M is the marker, "+" indicates that recombinant bacteria transformed with 6His-NT-proBNP-pET plasmid were induced with IPTG, and "-" indicates that recombinant bacteria transformed with 6His-NT-proBNP-pET plasmid were not induced with IPTG. Figure 3 The results of SDS-PAGE gel Coomassie Brilliant Blue staining of four different batches of purified protein after affinity chromatography, ultrafiltration concentration, and molecular sieve chromatography are shown; where 1-4 are four different batches of purified protein. Figure 4The results of SDS-PAGE analysis of supernatant of bacterial cell lysis products induced by different IPTG concentrations (A), SDS-PAGE analysis of purified products (B), and grayscale analysis of protein band quantification (C) are shown. Figure 5 SDS-PAGE analysis of supernatant of bacterial cell lysis products induced by different temperatures (A), SDS-PAGE analysis of purified products (B), and grayscale analysis of protein band quantification (C) results; Figure 6 SDS-PAGE analysis of supernatant of bacterial cell lysis products induced at different time points (A), SDS-PAGE analysis of purified products (B), and grayscale analysis of protein band quantification (C) results; Figure 7 The graph shows the linear relationship between the dilution factor and the concentration measurement value after different dilutions of the purified recombinant NT-proBNP protein. Figure 8 The results show the short-term stability trends of recombinant NT-proBNP frozen liquid and lyophilized samples with mixed human serum as the matrix after storage at 4°C for 0 to 9 days. Figure 9 The results show the detection results of the current samples and recombinant NT-proBNP samples using different detection systems; where A is a linear relationship graph of the target values ​​of the current samples and recombinant NT-proBNP samples using different detection systems, and B is a box plot of the normalized target values ​​of the current samples and recombinant NT-proBNP samples using different detection systems. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe the methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the contents of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0029] The research approach of this invention is as follows: A nucleotide fragment encoding recombinant NT-proBNP was designed and synthesized based on the human NT-proBNP gene sequence. The synthesized NT-proBNP gene and the pET-28a(+) prokaryotic expression vector were digested using Nco I and Xho I restriction endonucleases, respectively. A recombinant expression plasmid was constructed using T4 DNA ligase. This plasmid was transformed into *E. coli* clone host cells. After successful construction was confirmed by PCR amplification, double enzyme digestion verification, and sequencing analysis, the plasmid was further transformed into *E. coli* expression strains for induced expression. Following nickel ion affinity chromatography, ultrafiltration concentration, molecular sieve chromatography protein purification, and optimization of induction expression conditions, recombinant NT-proBNP protein recognizable by clinical immunoassay systems was obtained. The protein was further prepared into frozen liquid samples and lyophilized samples for homogeneity evaluation, short-term stability evaluation at 4°C, and comparison of results from multiple platforms.

[0030] Using genetic engineering and molecular biology techniques, the nucleotide fragment encoding recombinant NT-proBNP was cloned into the pET-28a(+) prokaryotic expression vector to construct the 6His-NT-proBNP-pET recombinant expression plasmid. This recombinant expression plasmid was then transformed into... E. coliAfter BL21 Codon Plus competent cells were selected, single clones were inoculated into LB medium to induce expression of recombinant NT-proBNP protein. The expressed cells were lysed by sonication, and the supernatant was collected by centrifugation. The protein was purified using nickel affinity chromatography, ultrafiltration concentration, and molecular sieve chromatography to obtain the recombinant NT-proBNP protein. SDS-PAGE analysis showed a target band around 10 kDa, and the obtained protein was detectable by a clinical immunoassay system. The purified recombinant NT-proBNP protein was exogenously added to a low-background mixed human serum matrix to prepare frozen liquid and lyophilized samples. Homogeneity and short-term stability at 4°C were evaluated for these samples. The detection results of the recombinant NT-proBNP samples on different detection platforms were compared and analyzed based on interlaboratory quality assessment (EQA) data. The results showed that both the frozen liquid sample and the lyophilized sample prepared from recombinant NT-proBNP had good homogeneity. The lyophilized sample showed good short-term stability at 4℃. In addition, the recombinant NT-proBNP sample could be quantitatively detected on multiple NT-proBNP detection platforms.

[0031] Example 1 (a) Construction of the 6His-NT-proBNP-pET recombinant expression plasmid 1. Search for the human NT-proBNP gene sequence from NCBI, and based on... E. coli BL21 Codon Plus codon-biased gene sequence optimization was used to synthesize a nucleotide fragment encoding recombinant NT-proBNP, as shown in SEQ ID NO.1. A 6His tag sequence was added to this gene fragment, and Nco I and Xho I restriction sites were designed at both ends, resulting in a nucleotide fragment with the sequence shown in SEQ ID NO.2. The amino acid sequence of the encoded recombinant 6His-NT-proBNP protein is shown in SEQ ID NO.3.

[0032] SEQ ID NO.1: CACCCGCTGGGTTCTCCGGGTTCTGCTTCTGACCTGGAAACCTCTGGTCTGCAGGAACAGCGTAACCACCTGCAGGGTAAACTGTCTGAACTGCAGGTTGAACAGACCTCTCTGGAACCGCTGCAGGAATCTCCGCGTCCGACCGGTGTTTGGAAATCTCGTGAAGTTGCTACCGAAGGTATCCGTGGTCACCGTAAAATGGTTCTGTACACCCTGCGTGCTCCGCGT.

[0033] SEQ ID NO.2: CCATGGGCAGCAGCCATCATCATCATCACCACCCGCTGGGTTCTCCGGGTTCTGCTTCTGACCTGGAAACCTCTGGTCTGCAGGAACAGCGTAACCACCTGCAGGGTAAACTGTCTGAACTGCAGGTTGAAC AGACCTCTCTGGAACCGCTGCAGGAATCTCCGCGTCCGACCGGTGTTTGGAAATCTCGTGAAGTTGCTACCGAAGGTATCCGTGGTCACCGTAAAATGGTTCTGTACACCCTGCGTGCTCCGCGTTAACTCGAG.

[0034] SEQ ID NO.3: MGSSHHHHHHHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR.

[0035] 2. The NT-proBNP gene fragment (SEQ ID NO.2) and the pET-28a(+) prokaryotic expression vector were cut using Nco I and Xho I restriction endonucleases, respectively, and ligated using T4 DNA ligase to obtain the 6His-NT-proBNP-pET recombinant expression plasmid.

[0036] 3. Transform the constructed product into E. coli DH5α competent cells were used, and single colonies were selected for PCR amplification and double restriction enzyme (Mlu I and Xho I) digestion verification. The digestion products were analyzed by 1% agarose gel electrophoresis, which showed fragments containing the target gene and vector fragments. Figure 1 The successfully identified positive recombinant plasmids were sent for sequencing analysis to verify the recombinant plasmid construction results. The sequencing results are shown in SEQ ID NO.4, which show that the NT-proBNP target gene sequence in the recombinant plasmid is consistent with the designed sequence, confirming the successful construction of the prokaryotic expression plasmid.

[0037] SEQ ID NO.4:

[0038] (II) Verification of the preliminary expression of the recombinant expression plasmid in Escherichia coli 1. Transform the recombinant expression plasmid confirmed by sequencing into... E. coli BL21 Codon Plus competent cells were selected, and single clones were inoculated into LB medium for induced expression. The OD of the bacterial culture was then calculated. 600 The value reached approximately 0.6 when IPTG was added to induce expression.

[0039] 2. After inducing culture with 0.5 mmol / L IPTG at 20℃ and 160 r / min for 20 h, the bacterial cells were collected. The cells were lysed by sonication, and the supernatant and precipitate were collected separately after centrifugation. The protein expression was detected by SDS-PAGE analysis.

[0040] SDS-PAGE analysis of protein expression showed that the bacterial culture induced by IPTG exhibited a significant differential band around 10 kDa, and the target protein was mainly present in the supernatant, with almost no detectable amount in the precipitate. This indicates that the recombinant NT-proBNP protein was successfully expressed and mainly exists in a soluble form. Figure 2 ).

[0041] (III) Purification of recombinant NT-proBNP protein 1. Collect recombinant bacterial cells induced by IPTG expression, lyse the cells by sonication, collect the cell lysis supernatant after centrifugation, filter the cell lysis supernatant through a 0.22 μm filter membrane and mix it thoroughly with Ni-NTA affinity resin at 4℃.

[0042] 2. Under the conditions of equilibration buffer at pH 8.0, NaCl concentration of 300 mmol / L, and phosphate concentration of 50 mmol / L, wash with 20 mmol / L imidazole to remove non-specific conjugates, and then elute recombinant protein with 250 mmol / L imidazole.

[0043] 3. After elution, the crude purified protein is first centrifuged using a 30KD ultrafiltration tube, and the eluent is collected to remove impurities with a molecular weight >30KD. Then, it is concentrated by centrifugation using a 10KD ultrafiltration tube, and the retentate is collected to remove impurities with a molecular weight <10KD.

[0044] 4. The protein sample after ultrafiltration concentration was purified using Superdex 75 increase 10 / 300 GL molecular sieve to finally obtain pure protein.

[0045] The purified protein was subjected to SDS-PAGE electrophoresis and then stained with Coomassie Brilliant Blue. The results showed that the target band appeared at approximately 10 kDa in different batches of purified protein, indicating that high-purity and stable recombinant NT-proBNP protein could be obtained after nickel affinity chromatography, ultrafiltration concentration, and molecular sieve chromatography. Figure 3 ).

[0046] (iv) Optimization of recombinant NT-proBNP protein induction expression conditions After confirming that the recombinant protein could be induced to express and that the purified protein could be obtained through the purification procedure described in (III), the induction conditions were further optimized. Samples obtained under different induction conditions were all processed using the same purification procedure in (III), and the expression levels of the target protein were compared by SDS-PAGE Coomassie Brilliant Blue staining and grayscale analysis.

[0047] (1) Select the optimal IPTG concentration Using IPTG concentration gradients of 0.2, 0.3, 0.4, and 0.5 mmol / L, recombinant NT-proBNP protein was induced for 20 h at 20℃ and 160 r / min. The expression of recombinant NT-proBNP protein under different IPTG concentrations was compared.

[0048] Purified recombinant NT-proBNP was stained with Coomassie Brilliant Blue by SDS-PAGE, and the protein expression level was analyzed by grayscale. The results showed that the protein expression level was high when the final IPTG concentration was 0.4 mmol / L, so 0.4 mmol / L was selected as the optimal IPTG induction concentration. Figure 4 ).

[0049] (2) Select the optimal induction temperature After selecting the optimal IPTG concentration, 16, 20, 30, and 37 °C were set as induction temperature gradients. The recombinant protein was induced for 20 h at 0.4 mmol / L IPTG and 160 r / min, and the expression of the recombinant protein at different temperatures was compared.

[0050] The purified recombinant NT-proBNP was stained with Coomassie Brilliant Blue by SDS-PAGE, and the protein expression level was analyzed by grayscale. The results showed that the protein expression level was higher when the induction temperature was 30℃, so 30℃ was selected as the optimal induction temperature. Figure 5 ).

[0051] (3) Select the optimal induction time After selecting the optimal IPTG concentration and optimal induction temperature, 8, 12, 16, and 20 h were set as induction time gradients. The recombinant protein was induced at 0.4 mmol / L IPTG, 30℃, and 160 r / min, and the expression of the recombinant protein under different induction times was compared.

[0052] The purified recombinant NT-proBNP was stained with Coomassie Brilliant Blue by SDS-PAGE, and the protein expression level was analyzed by grayscale. The results showed that the protein expression level was high when the induction time was 20 h, so 20 h was selected as the optimal induction time. Figure 6 ).

[0053] Example 2 (I) Detection and dilution linearity analysis of recombinant NT-proBNP protein 1. The concentration of purified recombinant NT-proBNP protein was determined using the BCA method.

[0054] 2. The purified protein was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining for protein verification and purity analysis.

[0055] 3. The purified NT-proBNP protein was diluted 5000, 10000, 20000, 40000, and 80000 times with ddH2O. The content of recombinant NT-proBNP was quantitatively detected using a Roche Cobas 801 fully automated chemiluminescence immunoassay analyzer and matching detection kit. Each dilution was measured in triplicate.

[0056] 4. Relative quantification of protein bands was performed using ImageJ for grayscale value calculation and analysis, and GraphPad Prism9 was used to generate grayscale analysis graphs.

[0057] Analysis of the linear relationship between dilution factor and detection concentration showed that pure NT-proBNP protein could not only be detected in clinical laboratories, but also exhibited a high degree of fit to the standard curve (R0). 2 >0.99 indicates that the recombinant protein prepared by this invention maintains a good linear relationship between the dilution factor and the concentration measurement value over a wide dilution range, which can serve as an effective basis for subsequent methodological evaluation, quality control, and candidate reference material research. Figure 7 ).

[0058] (II) Evaluation of the uniformity of recombinant NT-proBNP protein 1. The purified recombinant NT-proBNP protein was prepared into frozen liquid samples and lyophilized samples using mixed human serum (collected from the Department of Laboratory Medicine of Beijing Hospital, NT-proBNP concentration <35 pg / mL) as the matrix. The recombinant NT-proBNP protein concentrations in the samples were 2075 pg / mL and 1900 pg / mL, respectively (measured by Roche platform).

[0059] 2. Ten samples were randomly selected from the same batch of frozen liquid samples and lyophilized samples, and each sample was tested twice in parallel. One-way ANOVA was used to calculate the population mean, standard deviation, coefficient of variation, between-bottle mean square, within-bottle mean square, F value and P value (P < 0.05 indicates that the difference or trend is statistically significant) based on the test mean of the 10 samples.

[0060] The results (see Tables 1 and 2) showed that the mean values ​​of the 10 frozen liquid samples ranged from 2045.0 to 2099.5 pg / mL, with a total mean of 2074.95 pg / mL, a standard deviation between the means of the two samples of lyophilized product, and a coefficient of variation of 0.87%. The mean values ​​of the 10 lyophilized product samples ranged from 1858.5 to 1933.0 pg / mL, with a total mean of 1899.55 pg / mL, a standard deviation between the means of the two samples of lyophilized product, and a coefficient of variation of 1.38%. For the frozen liquid samples, F=1.752, P=0.197; for the lyophilized product samples, F=2.686, P=0.070. There were no statistically significant differences between the two samples (P>0.05). Therefore, both the frozen liquid and lyophilized product samples from the same batch containing a mixed human serum matrix showed good homogeneity.

[0061] Table 1. Homogeneity test results of recombinant NT-proBNP frozen liquid and lyophilized samples Table 2. Results of one-way ANOVA on homogeneity of recombinant NT-proBNP frozen liquid and lyophilized samples. (III) Evaluation of the short-term stability of recombinant NT-proBNP protein at 4°C 1. The purified recombinant NT-proBNP protein was prepared into frozen liquid samples and lyophilized samples with mixed human serum as the matrix.

[0062] 2. Frozen liquid samples and lyophilized samples were stored at 4℃ and sampled and tested from 0 to 9 days. Two parallel repeated tests were performed at each time point. Linear regression analysis was used to compare the trend of the test values ​​with storage time under different storage methods, and the slope b1, slope standard error S_b1, t value and p value were calculated.

[0063] The results (see Tables 3 and 4) showed that the detection values ​​of frozen liquid samples decreased with prolonged storage time. The average value of parallel detections decreased from 2028 pg / mL at 0 days to 1721 pg / mL at 9 days, a decrease of approximately 15.1%, with a slope b1 of -33.600 pg·mL. -1 ·d -1The standard error of the slope, S_b1, was 3.426, t = -9.808, and P < 0.001, indicating a significant decreasing trend in frozen liquid samples at 4℃. The mean values ​​of lyophilized samples remained between 1793 and 1850 pg / mL from 0 to 9 days, with a slope b1 of 0.806 pg·mL. -1 ·d -1 The slope standard error S_b1 was 2.649, t=0.304, P=0.769, showing no significant downward trend. Therefore, compared with frozen liquid samples, lyophilized samples have better short-term stability at 4℃ and are more suitable as the preferred preservation form for subsequent studies of recombinant NT-proBNP quality control materials and candidate reference materials. Figure 8 ).

[0064] Table 3. Short-term stability test results of recombinant NT-proBNP frozen liquid and lyophilized samples at 4℃. Table 4. Linear regression analysis results of recombinant NT-proBNP frozen liquid and lyophilized samples at 4℃. (iv) Comparison of detection results of recombinant NT-proBNP samples in different detection systems During the formal EQA activity, a trial survey of recombinant NT-proBNP samples was conducted. Lyophilized recombinant samples, along with the currently used EQA quality control materials (purchased from China Resources Pharmaceutical Commercial Group Medical Devices Co., Ltd., with a concentration of 1782 pg / mL measured on the Roche platform), were distributed to laboratories nationwide. Data from 24 mainstream detection systems were collected. The test results of the currently used EQA quality control samples and recombinant NT-proBNP samples from these 24 detection systems were statistically analyzed according to ISO 13528, and robust means were calculated (Note: robust means are not affected by outliers or extreme values, nor by sample distribution; they are an iterative convergent calculation method and can be used as the target value for each detection system), as shown in Table 5.

[0065] Table 5. Robust mean test results of 24 mainstream testing systems Pearson correlation analysis, Spearman rank correlation analysis, and Pearson correlation analysis after natural logarithmic transformation were used to evaluate the correlation between the currently used sample and the recombinant NT-proBNP sample across different detection systems. First, the original target values ​​of each detection system were used to calculate the Pearson correlation coefficient, Spearman rank correlation coefficient, and Pearson correlation coefficient after natural logarithmic transformation. Then, the target values ​​were normalized, resulting in a mean target value of 2293.6 pg / mL for the currently used sample and 2387.8 pg / mL for the recombinant NT-proBNP sample. The ratio of the target value of each detection system to the corresponding mean target value was calculated, and then Pearson correlation analysis, Spearman rank correlation analysis, and Pearson correlation analysis after natural logarithmic transformation were performed on these ratios.

[0066] The normalized CVs of the target values ​​of the currently used sample and the recombinant NT-proBNP sample were calculated separately to evaluate the dispersion of the detection values ​​of the currently used sample and the recombinant NT-proBNP sample across different detection systems.

[0067] The results show (see Table 6 and...) Figure 9 The Pearson correlation coefficient between the target values ​​of the currently used sample and the recombinant NT-proBNP sample (A) is 0.729, and the normalized Pearson correlation coefficient is 0.731. The Spearman rank correlation coefficient of the target values ​​is 0.560, and the normalized Spearman rank correlation coefficient is 0.560. The Pearson correlation coefficient of the target values ​​after ln transformation is 0.683, and the normalized Pearson correlation coefficient after ln transformation is 0.686. This indicates that the detection results of the two types of samples have a certain correlation in different detection systems, and the correlation is very stable. Further comparison of the normalized numerical distribution of the target values ​​of the currently used sample and the recombinant NT-proBNP sample shows the results (see...). Figure 9 The normalized distribution trends of the detection values ​​of the currently used sample and the recombinant NT-proBNP sample (B) are similar. The medians and interquartile ranges of the two groups are 0.9215 (0.7900, 1.1315) and 0.917 (0.8035, 1.0800), respectively, indicating that the detection levels of the recombinant sample and the currently used sample are well consistent across different detection systems. Furthermore, the detection values ​​of the recombinant sample are more concentrated across systems, with lower dispersion, demonstrating better inter-system consistency. Meanwhile, the normalized CV of the target value for the currently used sample across 24 detection systems is 33.61%, while that for the recombinant sample is 29.26%, further indicating that the dispersion of the detection values ​​of the recombinant NT-proBNP sample across systems is lower than that of the currently used sample.

[0068] Table 6. Target values ​​and their correlation after normalization between the current sample and the recombinant NT-proBNP sample. The above results show that the recombinant NT-proBNP sample prepared by this invention can be identified and quantitatively detected by multiple mainstream NT-proBNP detection systems, and has a certain basis for multi-platform detection applicability. It has a stable correlation with the detection result change trend of the currently used EQA sample in different detection systems, and the dispersion of the target value of the detection value in different platforms is lower than that of the EQA sample, indicating that the recombinant sample has better consistency between systems and is more advantageous.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing recombinant NT-proBNP protein for the preparation of NT-proBNP quality control materials and candidate reference materials, characterized in that, Includes the following steps: (1) The E. coli codons were optimized to obtain the gene fragment with the nucleotide sequence shown in SEQ ID NO.1; (2) After adding the 6His tag sequence to the gene fragment, it was cloned into the pET-28a(+) prokaryotic expression vector to obtain the recombinant expression vector; (3) Transform the recombinant expression vector into E. coli Recombinant NT-proBNP protein was obtained from BL21 Codon Plus competent cells after IPTG-induced expression and protein purification.

2. The preparation method according to claim 1, characterized in that, The conditions for IPTG-induced expression were as follows: 0.4 mmol / L IPTG was used for induction at 30°C for 20 h.

3. The preparation method according to claim 1, characterized in that, The protein purification method is as follows: collect the bacterial cells after induced expression, perform ultrasonic lysis, and collect the supernatant by centrifugation; after filtering the supernatant through a filter membrane, nickel ion affinity chromatography, ultrafiltration concentration and molecular sieve chromatography, the purified recombinant NT-proBNP protein is obtained.

4. The preparation method according to claim 3, characterized in that, The filter membrane has a pore size of 0.22 μm; The nickel ion affinity chromatography was performed under the following conditions: sequential elution with 20 mmol / L imidazole and 250 mmol / L imidazole. The ultrafiltration concentration method involves centrifugation using a 30KD ultrafiltration tube and a 10KD ultrafiltration tube in sequence. The molecular sieve is Superdex 75 increase 10 / 300 GL molecular sieve.

5. A recombinant NT-proBNP protein prepared by the preparation method according to any one of claims 1-4.

6. The use of the recombinant NT-proBNP protein according to claim 5 in the preparation of NT-proBNP quality control products.

7. The use of the recombinant NT-proBNP protein according to claim 5 in the preparation of NT-proBNP reference material.

8. The use of the recombinant NT-proBNP protein according to claim 5 as a quality control in the preparation of a kit for detecting NT-proBNP.

9. An NT-proBNP quality control product, characterized in that, The NT-proBNP quality control product was prepared by adding the recombinant NT-proBNP protein of claim 5 to a serum matrix.

10. A kit for detecting NT-proBNP, characterized in that, The kit contains the NT-proBNP quality control material as described in claim 9.

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