CN111909948A - A kind of stable troponin I standard substance and preparation method thereof - Google Patents

Abstract

The invention discloses a stable troponin I standard substance and a preparation method thereof. On the basis of the amino acid sequence of the cTnI stable fragment, transforming BL21 (DE3) competent cells, ultrasonically disrupting the inducible expressed engineering bacteria to obtain cTnI inclusion bodies, and obtaining cTnI inclusion bodies through 1 % Triton X-100, washed with 2M urea, dissolved in 8M urea, and then renatured on a CM-FF column and purified by dilution and renaturation to obtain the standard substance of troponin I. Wherein, the amino acid sequence of the cTnI stable fragment is selected from the 30th-110th amino acid sequence fragment in the cTnI protein sequence, and the cTnI protein sequence is shown in SEQ No: 1. The invention adopts a new method to realize the preparation of troponin I standard substance, and solves the problem of troponin instability. The standard material can maintain long-term stability under low temperature conditions, meeting the requirements of the national first-class standard material. The standard material is a stable fragment of the troponin sequence, which can cover the epitope requirements of most kits on the market.

Description

A kind of stable troponin I standard substance and preparation method thereof

technical field

The invention relates to the technical field of biochemistry, in particular to a method for preparing a stable troponin I standard substance.

Background technique

Cardiac Troponin (cTn) is a contractile regulatory protein of cardiomyocytes, composed of Ca ²⁺ -binding subunit Troponin C (cTnC), tropomyosin-binding subunit Troponin T (cTnT), inhibitory The three subunits of basal troponin I (cTnI) form a complex that regulates the relative sliding between thick and thin filaments, thereby regulating muscle contraction. When the myocardium is damaged, the three subunits are released outside the cells and can be detected in peripheral blood. Cardiac troponin is a specific marker of myocardial injury and a gold indicator of myocardial infarction.

The kit for cardiac troponin T has been monopolized by Roche, and there is no problem of uniformity. However, according to reports in the literature, the US FDA alone has approved at least 15 manufacturers to produce troponin I detection kits, and the differences in the detection results of the same sample can even reach 100 times between the kits of different manufacturers. The American Institute of Standardization NIST and the American Council of Clinical Chemistry (AACC) established the Troponin I Standardization Committee as early as 2001 to specialize in the standardization of Troponin I. In 2005, NIST in the United States developed the human cardiac muscle-derived troponin standard substance SRM2921, which made a great contribution to the standardization of troponin I at that time. However, with the use of users, more and more articles report that the standard material will be unstable with the change of storage time and temperature.

Regarding the instability of troponin I, the Chinese Academy of Metrology has reorganized the stable fragment sequence of troponin I, and purified it to obtain the standard material candidate of troponin I. Due to the lack of reference materials for troponin I in China and the lack of relevant detection reference methods, the standardization of troponin I detection cannot be achieved, resulting in many research results that are completely different and even contradictory. Therefore, accurate and quantitative determination of the content of troponin I in body fluids and establishment of its quantitative value system play an important role in the clinical examination of troponin I and the diagnosis of diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a stable troponin I standard substance and a preparation method thereof, which have good accuracy, reliability and traceability. The invention clarifies the substance to be tested and realizes the delineation of the range of the substance to be tested; the protein pretreatment steps are simplified, and other variables introduced in the process and the resulting uncertainty are eliminated; the detection method of the characteristic peptide segment entering the mass spectrometer is not selected. , which eliminates the difference in results caused by the change of mass-to-charge ratio due to heterogeneity; the isotope-labeled recombinant cTnI, which is difficult to characterize in the preparation process, is not used as the internal standard, which simplifies the experimental scheme. The invention utilizes isotope dilution mass spectrometry to accurately quantify recombinant troponin I, establishes its standard determination system, and makes the detection result traceable to SI.

To achieve the above object, the technical scheme adopted in the present invention is as follows:

In view of the instability of troponin I, based on the amino acid sequence of the cTnI stable fragment, BL21 (DE3) competent cells were transformed, and the engineered bacteria induced to express were obtained by ultrasonication to obtain cTnI inclusion bodies. After washing, it was dissolved in 8M urea, and cTnI was purified by renaturation and dilution on a CM-FF column to obtain the standard substance of troponin I; wherein, the amino acid sequence of the cTnI stable fragment was selected from the first cTnI protein sequence in the cTnI protein sequence. 30-110th amino acid sequence fragment, the cTnI protein sequence is shown in SEQ No: 1. The amino acid sequence of the cTnI stable fragment is shown in SEQ No: 2. Specifically include the following steps:

(1) Preparation of human serum samples;

(2) Preparation of stable cardiac troponin I standard material: 1) Transformation of recombinant plasmid into BL21(DE3) competent cells and screening; 2) IPTG-induced expression of recombinant human cTnI; 3) cleavage of recombinant human cTnI inclusion body protein, Washing; 4) Purification of recombinant human cTnI.

Wherein, the step (1) is specifically as follows: the collected human serum samples are subjected to human immunodeficiency virus antibody (HIV), syphilis, hepatitis B surface antigen (HBV), hepatitis C antibody (HCV) and a full set of conventional biochemical tests; The cTnI content was detected by the method, and the serum samples were divided into three concentration levels: low, medium and high, which were recorded as 1-2-1, 1-2-2, and 1-2-3 respectively. Filter into sterile filter bottles; divide the serum samples into aliquots and store them in a -80°C freezer.

Wherein, the transformation of the recombinant plasmid into BL21(DE3) competent cells and the screening steps are as follows:

1) Take out a tube of competent cells (BL21, 0.5ml) from the refrigerator and quickly place them on ice, and thaw the cells on ice for 20 minutes;

2) Add 1 ul of recombinant PET-32a plasmid DNA directly to the cells, then reset the tube to ice for 30 min;

3) 42 ℃ water bath for 60 seconds, do not shake, quickly put on ice for 5 minutes;

4) Add 900ul blank LB medium, 37°C, 160rpm, 1h, take 300ul LB plate coated with kanamycin;

5) Put the plate on the workbench for a few minutes to absorb the coated liquid, invert, and incubate at 37°C overnight;

The next day, use a sterile inoculating loop to pick a single colony in good condition from the solid plate, inoculate it into 5ml kanamycin-resistant (K ⁺ ) LB liquid medium, 37°C, 230rpm, 12-18h, until about OD600. is 0.6-0.8, one tube is added with inducer IPTG (final concentration is 1mM), the other tube is not added as a control, 37 ° C, 230rpm, 5h, 5000rpm, centrifuge for 15min to collect bacteria, discard the supernatant, add 5ml of bacterial lysis buffer Liquid lysed bacteria, 5000rpm, 15min, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on the supernatant and precipitate to identify the expression of the target protein; screen out single colonies expressing the target protein; and analyze the solubility of the target protein, - The strain containing the recombinant plasmid pET-32a-cTnI (800ul bacterial solution + 200ul 80% glycerol) was stored at 20°C as a seed solution for future use.

The specific operation method of the SDS-PAGE is:

1) Fix the glass tank, use deionized water to check whether there is leakage, prepare 12% separation glue, mix it and inject it into the gap between the glass plates, and slowly add a few milliliters of deionized water to cover it;

2) The gel polymerization is completed in about 30 minutes at room temperature, pour out the covering water, prepare a 5% stacking gel, mix well and inject it into the upper end of the separating gel, and carefully insert a comb to avoid air bubbles;

3) Polymerization of concentrated gelatin at room temperature for about 30min, remove the comb for use;

4) Add 1x electrode buffer to both the inner and outer tanks to check for leakage. At the same time, mix the sample with the loading buffer, boil at 100 °C for 5 min, 3000 rpm, 1 min, take the supernatant and load the sample;

5) The voltage at the beginning of electrophoresis is 8V/cm gel. After the dye enters the separating gel, the voltage is increased to 15V/cm gel. When the dye reaches the bottom of the separating gel, disconnect the power and remove the gel;

6) Soak the gel with staining solution and shake it at room temperature for about 1h;

7) Soak the gel with decolorizing solution, shake slowly to decolorize until clear, take pictures, or seal in 20% glycerol.

Wherein, the IPTG induction expression step of described recombinant human cTnI is specifically:

1) Inoculate 50ul of glycerol inoculum bacteria into 5ml of K ⁺ resistant LB liquid medium, 37°C, 160rpm, 12-16h, so that the OD600 value reaches 0.6-0.8;

2) Add above-mentioned 5ml bacterial liquid to 500ml K ⁺ resistant LB liquid medium, 37°C, 230rpm, when the OD600 value reaches 0.6-0.8, add IPTG storage solution (final concentration 1mM), and continue to cultivate;

3) Induction for 5-8h, 5000rpm, centrifugation for 15 minutes, and discard the supernatant;

4) After resuspension in deionized water, centrifuge at 5000 rpm for 15 minutes, discard the supernatant, and freeze at -20°C.

Wherein, the cleavage and washing steps of the recombinant human cTnI inclusion body protein are specifically,

1) Preparation of crude inclusion bodies: Take out 5 g of the stored induced bacteria, add 250 ml of bacterial lysis buffer to resuspend after thawing, sonicate in ice bath, ultrasonicate for 3 s, interval for 5 s, power 400 W, 20 min, then centrifuge at 6000 rpm for 20 min, discard the clear, and sampled to obtain crude cTnI inclusion bodies;

2) Washing of inclusion bodies: The crude inclusion bodies were resuspended in 300ml of 1% Triton X-100 washing solution and 2MUre washing solution in turn, washed with magnetic stirring for 1h and 0.5h at 4°C, and finally resuspended in Tris buffer. Centrifuge at 6000rpm for 20min, discard the supernatant and sample;

3) Dissolution of inclusion bodies: Add 0.1 g of wet weight inclusion bodies after washing into 50 ml of inclusion body dissolution buffer, overnight at 4°C, and the precipitation solution becomes transparent and clear, ready for use;

4) Perform denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on the samples in the washing process respectively to analyze the washing and dissolution of the target protein.

Wherein, the purification steps of the recombinant human cTnI include dilution and renaturation of inclusion bodies and cationic column renaturation of inclusion bodies.

Among them, the dilution and renaturation of inclusion bodies is as follows: 50ml of 0.1g wet weight inclusion body dissolution buffer is added into 1L of renaturation buffer by pulse, the whole process lasts for 2-3h, and the ice bath is magnetically stirred for renaturation for 18-24h. After passing through a 0.22um filter membrane, concentrated by a 5KD ultrafiltration membrane package, dialyzed in 4°C dialysate (Tris: 20mM pH=7.4), and finally loaded on a cation column;

The specific operation steps are as follows:

1) Equilibrate the cation column (5ml, CM-FF) with 5CVTris buffer, and adjust the UV value to zero;

2) The sample is loaded at 0.5-1ml/min, and the UV value is washed back to the baseline with Tris buffer after the end;

3) Elute with 30% and 100% 1M NaCl elution buffer respectively, flow rate 5ml/min, pay attention to the collection and sampling of samples.

Wherein, the specific operation steps of renaturation on the cation column of the inclusion body are as follows:

1) Equilibrate the cation column (5ml, CM-FF) with 5CV inclusion body dissolution buffer, and adjust the UV value to zero;

2) Pass 45ml of 0.1g inclusion body denaturation solution through a 0.22um microporous membrane, load 0.5-1ml/min, and wash the UV value back to the baseline with inclusion body dissolution buffer after completion;

3) Gradually increase the ratio of Tris buffer/inclusion body dissolution buffer from 0 to 100%, flow rate 0.5ml/min, and complete within 2h;

4) Elute with 8%, 30%, and 80% 1M NaCl elution buffer, respectively, at a flow rate of 5 ml/min, and collect the second protein peak;

5) Finally, use 8M Ure elution buffer to elute the protein that failed to renature on the column, and pay attention to sampling during the whole process.

Compared with the prior art, the outstanding effect of the present invention is:

1) The present invention adopts a new method to realize the preparation of troponin I standard substance, and solves the problem of instability of troponin.

2) The reference material can maintain long-term stability under low temperature conditions, meeting the requirements of the national first-class reference material.

3) The standard substance is a stable fragment of the troponin sequence, which can cover the antigenic epitope requirements of most kits on the market.

4) For the first time, it is proposed to replace the complete protein with a specific protein sequence as a standard material.

The stable troponin I standard substance of the present invention and its preparation method will be further described below with reference to the accompanying drawings and specific examples.

Description of drawings

Fig. 1 is SDS-PAGE analysis of the renaturation of cTnI inclusion bodies;

Fig. 2 is the high performance liquid chromatogram of recombinant troponin, the inset shows that the purity of recombinant troponin I obtained by the normalization method is 97.3%;

Figure 3 is a troponin stable fragment stability data.

Detailed ways

(1) Main reagents

Trifluoroacetic acid (chromatographic grade), formic acid (analytical grade), perfluoroheptanoic acid (analytical grade): purchased from Sigma-Aldrich Corporation; Tris base (guaranteed reagent): from Angus Chemical Company;

Acetonitrile (chromatographically pure): from J.T Baker Chemical Company;

Urine, iodoacetamide: Beijing Baidi Biotechnology Co., Ltd.;

Tris (ultra pure): from Amresco, Inc;

Ammonium bicarbonate (analytical grade): from China National Pharmaceutical Group Co., Ltd.;

DTT: from Inalco;

Trypsin: from Promega Corporation;

Hydrochloric acid (guaranteed reagent): Beijing Chemical Reagent Co., Ltd.;

Phenylalanine (purity 99.0%): National Reference Substance, GBW(E)100061;

Valine (purity 99.0%): national reference substance, GBW(E)100055;

Proline (purity 99.0%): national reference substance, GBW(E)100084;

Leucine (purity 99.0%): national reference material, GBW(E)100058;

13D8-L-phenylalanine (purity 98.0%), 13C5-L-valine (purity 98.0%), 13C5-L-proline (purity 98.0%), 13D10-L-leucine (purity 98.0 %): from Cambridge Isotope Laboratories, Inc.

(2) Instruments and equipment

Vortex mixer: product of SCILOGEX;

Centrifuge: product of Sigma-Aldrich Corporation;

KS260 desktop thickener: IKA product;

1200 high performance liquid chromatograph: Agilent product;

Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS): Waters Corporation;

XP26 analytical balance: Mettler-Toledo product, with a maximum measuring range of 22g and an accuracy of 0.001mg;

ME235S Analytical Balance: Sartorius product with a maximum measuring range of 230 g and an accuracy of 0.01 mg;

MALDI-TOF-MS: Bruker product;

Pipettes (20, 200 and 1000 μL): products of Thermo Fisher Scientific (Germany);

Model 6410 High Performance Liquid Chromatography Triple Quadrupole Mass Spectrometer (HPLC-MS/MS): a product of Agilent.

1. Preparation of human serum samples

The collected human serum samples were tested for human immunodeficiency virus antibody (HIV), syphilis, hepatitis B surface antigen (HBV), hepatitis C antibody (HCV) and routine biochemical panel.

According to the cTnI content detected by conventional methods, serum samples were divided into three concentration levels: low, medium and high, which were recorded as 1-2-1, 1-2-2 and 1-2-3 respectively. Filter to a sterile filter bottle after homogenization. The serum samples were aliquoted and stored in a -80°C freezer.

2. Preparation of stable cardiac troponin I standard material

2.1 Transformation of BL21(DE3) competent cells with recombinant plasmid and screening

1) Take out a tube of competent cells (BL21, 0.5 ml) from the refrigerator and quickly put them on ice, and the cells are thawed on ice for 20 minutes.

2) 1 ul of recombinant PET-32a plasmid DNA was directly added to the cells, and then the tube was reset to ice for 30 min.

3) 42 ℃ water bath for 60 seconds, do not shake, quickly placed on ice for 5 minutes.

4) Add 900ul blank LB medium, 37°C, 160rpm, 1h, take 300ul LB plate coated with kanamycin.

5) Place the plate on the bench for a few minutes to allow the coating liquid to be absorbed, invert, and incubate at 37°C overnight.

The next day, use a sterile inoculating loop to pick a single colony in good condition from the solid plate, inoculate it into 5ml kanamycin-resistant (K ⁺ ) LB liquid medium, 37°C, 230rpm, 12-18h, until about OD600. is 0.6-0.8, one tube is added with inducer IPTG (final concentration is 1mM), the other tube is not added as a control, 37 ° C, 230rpm, 5h, 5000rpm, centrifuge for 15min to collect bacteria, discard the supernatant, add 5ml of bacterial lysis buffer The bacterial cells were lysed in liquid, 5000 rpm, 15 min, and the supernatant and precipitate were subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) to identify the expression of the target protein. A single colony expressing the target protein was screened out. And the solubility of the target protein was analyzed, and the strain containing the recombinant plasmid pET-32a-cTnI (800ul bacterial liquid + 200ul 80% glycerol) was stored at -20°C as a seed solution for later use.

The specific operation method of SDS-PAGE is as follows:

1) Fix the glass tank, use deionized water to check whether there is leakage, prepare 12% separation glue, mix it and inject it into the gap between the glass plates, and slowly add a few milliliters of deionized water to cover it.

2) After about 30 minutes at room temperature, the gel polymerization is completed, pour out the covering water, prepare a 5% stacking gel, mix well and inject it into the upper end of the separating gel, and carefully insert a comb to avoid air bubbles.

3) The stacking gel is polymerized for about 30 minutes at room temperature, and the comb is removed for use.

4) Add lx electrode buffer to both the inner and outer tanks to check for leakage. At the same time, mix the sample with the loading buffer, boil at 100 °C for 5 min, 3000 rpm, 1 min, take the supernatant and load the sample.

5) At the beginning of electrophoresis, the voltage is 8V/cm gel. After the dye enters the separating gel, increase the voltage to 15V/cm gel. When the dye reaches the bottom of the separating gel, disconnect the power and remove the gel.

6) Soak the gel with staining solution and shake at room temperature for about 1h.

7) Soak the gel with decolorizing solution, shake slowly to decolorize until clear, take pictures, or seal in 20% glycerol.

2.2 IPTG-induced expression of recombinant human cTnI

1) Inoculate 50ul of glycerol inoculum into 5ml of K ⁺ resistant LB liquid medium, 37°C, 160rpm, 12-16h, so that the OD600 value reaches 0.6-0.8.

2) Add 5ml of the above bacterial liquid to 500ml of K ⁺ resistant LB liquid medium, 37°C, 230rpm, when the OD600 value reaches 0.6-0.8, add IPTG stock solution (final concentration 1mM), continue culturing.

3) Induction for 5-8 hours, 5000 rpm, centrifugation for 15 minutes, and the supernatant was discarded.

4) After resuspension in deionized water, centrifuge at 5000 rpm for 15 minutes, discard the supernatant, and freeze at -20°C.

2.3 Cleavage and washing of recombinant human cTnI inclusion body protein

1) Preparation of crude inclusion bodies: Take out 5 g of the stored induced bacteria, add 250 ml of bacterial lysis buffer to resuspend after thawing, sonicate in ice bath, ultrasonicate for 3 s, interval for 5 s, power 400 W, 20 min, then centrifuge at 6000 rpm for 20 min, discard the clear, and sampled to obtain crude cTnI inclusion bodies;

2) Washing of inclusion bodies: The crude inclusion bodies were resuspended in 300ml of 1% Triton X-100 washing solution and 2MUre washing solution in turn, washed with magnetic stirring for 1h and 0.5h at 4°C, and finally resuspended in Tris buffer. Centrifuge at 6000rpm for 20min, discard the supernatant and sample;

3) Dissolution of inclusion bodies: Add 0.1 g of wet weight inclusion bodies after washing into 50 ml of inclusion body dissolution buffer, overnight at 4°C, and the precipitation solution becomes transparent and clear, ready for use;

4) Perform denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on the samples in the washing process respectively to analyze the washing and dissolution of the target protein.

2.4 Purification of recombinant human cTnI

2.4.1 Dilution and renaturation of inclusion bodies (as shown in Figure 1)

Add 50ml of 0.1g wet weight inclusion body solubilization buffer into 1L of renaturation buffer by pulse, the whole process lasts 2-3h, renaturation with magnetic stirring in ice bath for 18-24h, pass through 0.22um membrane, and pass through 5KD ultrafiltration membrane After the packets were concentrated, they were dialyzed against 4°C dialysate (Tris: 20 mM pH=7.4) and finally loaded on a cation column.

The specific operation steps are as follows:

1) Equilibrate the cation column (5ml, CM-FF) with 5CVTris buffer, and adjust the UV value to zero;

2) The sample is loaded at 0.5-1ml/min, and the UV value is washed back to the baseline with Tris buffer after the end;

3) Elute with 30%, 100% 1M NaCl elution buffer, flow rate 5ml/min, pay attention to sample collection and sampling.

2.4.2 On-column renaturation of inclusion bodies (as shown in Figure 1)

The basic principle is: equilibrate the column with inclusion body dissolution buffer, and gradually increase the ratio of Tris buffer/inclusion body dissolution buffer to form a linear gradient of decreasing urea concentration. The renatured target protein is eluted by increasing the concentration of salt ions.

The specific operation steps are as follows:

1) Equilibrate the cation column (5ml, CM-FF) with 5CV of inclusion body solubilization buffer, and set the UV value to zero.

2) Pass 45ml of the denaturation solution containing 0.1g of inclusion bodies through a 0.22um microporous membrane, and load the sample at 0.5-1ml/min. After the end, use the inclusion body dissolution buffer to wash the UV value back to the baseline.

3) Gradually increase the ratio of Tris buffer/inclusion body dissolution buffer from 0 to 100%, flow rate 0.5ml/min, and complete within 2h.

4) Elute with 8%, 30%, and 80% 1M NaCl elution buffer, respectively, at a flow rate of 5 ml/min, and collect the second protein peak.

5) Finally, use 8M Ure elution buffer to elute the protein that failed to renature on the column, and pay attention to sampling during the whole process.

3. High-performance liquid chromatography analysis of recombinant troponin I (as shown in Figure 2)

Experimental conditions: mobile phase A: ultrapure water + 0.1% TFA; mobile phase B: acetonitrile; injection volume: 10 μL; stop time: 30 min; column temperature: 40 °C; mm×5 μm; flow rate: 0.2 mL/min; detector: DAD detector; mobile phase ratio is A:B=90:10.

The full wavelength scanning detection showed that the maximum absorption wavelength of recombinant troponin I was 210.4 nm. At this detection wavelength, the high-performance liquid chromatogram of the sample is shown in Figure 3. According to the peak area normalization method, the purity of the sample was 97.3%.

5. Stability assay of recombinant human cTnI (as shown in Figure 3)

The stability of the reference material refers to the property that the characteristic value of the reference material remains within the specified range under the specified time interval and environmental conditions. CTNI reference materials are stored in a freezer at -80°C and shipped under dry ice protection. Long-term stability testing of CTNI reference materials using chromatography, storage at -80°C for 24 months, analysis for 1 month, 3 months, 6 months, 9 months, 14 months and 18 months and 24 months Monthly change in CTNI reference material values. The short-term stability test adopts the ELISA method, and observes the changes of CTNI standard material values within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days at a temperature of 4°C and observes at a temperature of 25°C. Changes in CTNI reference material values at each time point at 1 hour, 2 hours, 12 hours and 24 hours. The stability test method is the same as for homogeneity. Short- and long-term stability of candidate CRMs was assessed by regression analysis as recommended in ISO Guide 35:2006.

Stability test results Figure 3 shows that the CTNI reference material can be stable for 24 hours at 25°C, 48 hours at 4°C, with obvious degradation within 48 hours, and more than 24 months at -80°C.

The above-mentioned embodiments merely describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. Variations and improvements should fall within the protection scope determined by the claims of the present invention.

sequence listing

<110> China Institute of Metrology

<120> A kind of stable troponin I standard substance and preparation method thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 210

<212> PRT

<213> Cardiac Troponin I

<400> 1

Met Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro

1 5 10 15

Ala Pro Ile Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu

20 25 30

Pro His Ala Lys Lys Lys Lys Ser Lys Ile Ser Ala Ser Arg Lys Leu Gln

35 40 45

Leu Lys Thr Leu Leu Leu Gln Ile Ala Lys Gln Glu Leu Glu Arg Glu

50 55 60

Ala Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys

65 70 75 80

Gln Pro Leu Glu Leu Ala Gly Leu Gly Phe Ala Glu Leu Gln Asp Leu

85 90 95

Cys Arg Gln Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr

100 105 110

Asp Ile Glu Ala Lys Val Thr Lys Asn Ile Thr Glu Ile Ala Asp Leu

115 120 125

Thr Gln Lys Ile Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu

130 135 140

Arg Arg Val Arg Ile Ser Ala Asp Ala Met Met Gln Ala Leu Leu Gly

145 150 155 160

Ala Arg Ala Lys Glu Ser Leu Asp Leu Arg Ala His Leu Lys Gln Val

165 170 175

Lys Lys Glu Asp Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg

180 185 190

Lys Asn Ile Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe

195 200 205

Glu Ser

210

<210> 2

<211> 81

<212> PRT

<213> cTnI fragment (Cardiac Troponin I)

<400> 2

Ala Thr Glu Pro His Ala Lys Lys Lys Ser Lys Ile Ser Ala Ser Arg

1 5 10 15

Lys Leu Gln Leu Lys Thr Leu Leu Leu Gln Ile Ala Lys Gln Glu Leu

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Glu Arg Glu Ala Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser

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Thr Arg Cys Gln Pro Leu Glu Leu Ala Gly Leu Gly Phe Ala Glu Leu

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Gln Asp Leu Cys Arg Gln Leu His Ala Arg Val Asp Lys Val Asp Glu

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Glu

Claims (10)

1. a preparation method of stable troponin I standard substance, it is characterized in that: on the basis of cTnI stable fragment amino acid sequence, transform BL21 (DE3) competent cell, the engineering bacteria of ultrasonic fragmentation induction expression obtains cTnI inclusion body, 1% Triton X-100, washed with 2M urea, dissolved in 8M urea, renatured and renatured by dilution on a CM-FF column to obtain the standard substance of troponin I; wherein, the cTnI stable fragment amino acid The sequence is selected from the fragment of the 30th-110th amino acid sequence in the cTnI protein sequence, and the cTnI protein sequence is shown in SEQ No: 1. 2. The method for preparing a stable troponin I standard substance according to claim 1, wherein the amino acid sequence of the cTnI stable fragment is as shown in SEQ No:2. 3. the preparation method of stable Troponin I standard substance according to claim 1, is characterized in that, comprises the following steps: (1) Preparation of human serum samples; (2) Preparation of stable cardiac troponin I standard substance: 1) Transformation of BL21(DE3) competent cells with recombinant plasmid and screening; 2) IPTG-induced expression of recombinant human cTnI; 3) lysis and washing of recombinant human cTnI inclusion body protein; 4) purification of recombinant human cTnI. 4. the preparation method of stable troponin I standard substance according to claim 3, is characterized in that: described step (1) is specially, the human serum sample of collection is carried out human immunodeficiency virus antibody (HIV), syphilis , Hepatitis B Surface Antigen (HBV), Hepatitis C Antibody (HCV) and a full set of routine biochemical tests; according to the cTnI content detected by conventional methods, the serum samples were divided into three concentration levels: low, medium and high, respectively recorded as 1-2-1 , 1-2-2, 1-2-3, the serum of each concentration level was mixed and filtered into a sterile filter bottle; the serum samples were divided and stored in a refrigerator at -80 degrees Celsius. 5. the preparation method of stable troponin I standard substance according to claim 3, is characterized in that: described recombinant plasmid transforms BL21 (DE3) competent cell and screening step is specifically, 1) Take out a tube of competent cells (BL21, 0.5ml) from the refrigerator and quickly place them on ice, and thaw the cells on ice for 20 minutes; 2) Add 1 ul of recombinant PET-32a plasmid DNA directly to the cells, then reset the tube to ice for 30 min; 3) 42 ℃ water bath for 60 seconds, do not shake, quickly put on ice for 5 minutes; 4) Add 900ul blank LB medium, 37°C, 160rpm, 1h, take 300ul LB plate coated with kanamycin; 5) Put the plate on the workbench for a few minutes to absorb the coated liquid, invert, and incubate at 37°C overnight; The next day, use a sterile inoculating loop to pick a single colony in good condition from the solid plate, inoculate it into 5ml kanamycin-resistant (K ⁺ ) LB liquid medium, 37°C, 230rpm, 12-18h, until about OD600. is 0.6-0.8, one tube is added with inducer IPTG (final concentration is 1mM), the other tube is not added as a control, 37 ° C, 230rpm, 5h, 5000rpm, centrifuge for 15min to collect bacteria, discard the supernatant, add 5ml of bacterial lysis buffer Liquid lysed bacteria, 5000rpm, 15min, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on the supernatant and precipitate to identify the expression of the target protein; screen out single colonies expressing the target protein; and analyze the solubility of the target protein, - The strain containing the recombinant plasmid pET-32a-cTnI (800ul bacterial solution + 200ul 80% glycerol) was stored at 20°C as a seed solution for future use. 6. the preparation method of stable Troponin I standard substance according to claim 5, is characterized in that: the concrete operation method of described SDS-PAGE is: 1) Fix the glass tank, use deionized water to check whether there is leakage, prepare 12% separation glue, mix it and inject it into the gap between the glass plates, and slowly add a few milliliters of deionized water to cover it; 2) The gel polymerization is completed in about 30 minutes at room temperature, pour out the covering water, prepare a 5% stacking gel, mix well and inject it into the upper end of the separating gel, and carefully insert a comb to avoid air bubbles; 3) Polymerization of concentrated gelatin at room temperature for about 30min, remove the comb for use; 4) Add 1x electrode buffer to both the inner and outer tanks to check for leakage. At the same time, mix the sample with the loading buffer, boil at 100 °C for 5 min, 3000 rpm, 1 min, take the supernatant and load the sample; 5) The voltage at the beginning of electrophoresis is 8V/cm gel. After the dye enters the separating gel, the voltage is increased to 15V/cm gel. When the dye reaches the bottom of the separating gel, disconnect the power and remove the gel; 6) Soak the gel with staining solution and shake it at room temperature for about 1h; 7) Soak the gel with decolorizing solution, shake slowly to decolorize until clear, take pictures, or seal in 20% glycerol. 7. the preparation method of stable troponin I standard substance according to claim 3, is characterized in that: the IPTG induction expression step of described recombinant human cTnI is specially: 1) Inoculate 50ul of glycerol inoculum bacteria into 5ml of K ⁺ resistant LB liquid medium, 37°C, 160rpm, 12-16h, so that the OD600 value reaches 0.6-0.8; 2) Add above-mentioned 5ml bacterial liquid to 500ml K ⁺ resistant LB liquid medium, 37°C, 230rpm, when the OD600 value reaches 0.6-0.8, add IPTG storage solution (final concentration 1mM), and continue to cultivate; 3) Induction for 5-8h, 5000rpm, centrifugation for 15 minutes, and discard the supernatant; 4) After resuspension in deionized water, centrifuge at 5000 rpm for 15 minutes, discard the supernatant, and freeze at -20°C. 8. the preparation method of stable troponin I standard substance according to claim 3, is characterized in that: the cracking of described recombinant human cTnI inclusion body protein, washing step are specifically, 1) Preparation of crude inclusion bodies: Take out 5 g of the stored induced bacteria, add 250 ml of bacterial lysis buffer to resuspend after thawing, sonicate in ice bath, ultrasonicate for 3 s, interval for 5 s, power 400 W, 20 min, then centrifuge at 6000 rpm for 20 min, discard the clear, and sampled to obtain crude cTnI inclusion bodies; 2) Washing of inclusion bodies: The crude inclusion bodies were resuspended in 300ml of 1% Triton X-100 washing solution and 2M Ure washing solution in turn, washed with magnetic stirring for 1h and 0.5h at 4°C, and finally resuspended in Tris buffer. , 6000rpm centrifugation for 20min, discard the supernatant and sample; 3) Dissolution of inclusion bodies: Add 0.1 g of wet weight inclusion bodies after washing into 50 ml of inclusion body dissolution buffer, overnight at 4°C, and the precipitation solution becomes transparent and clear, ready for use; 4) Perform denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on the samples in the washing process respectively to analyze the washing and dissolution of the target protein. 9. The preparation method of stable troponin I standard substance according to claim 3, is characterized in that: the purification step of described recombinant human cTnI comprises the dilution renaturation of inclusion bodies and the renaturation on the cation column of inclusion bodies; The dilution and renaturation of inclusion bodies is as follows: 50ml of 0.1g wet weight inclusion body dissolution buffer is added to 1L of renaturation buffer in pulses, the whole process lasts 2-3h, and the ice bath is magnetically stirred for renaturation for 18-24h, and after 0.22 um filter membrane, concentrated by 5KD ultrafiltration membrane package, dialyzed in 4 ℃ dialysate (Tris: 20mM pH=7.4), and finally loaded by cation column; The specific operation steps are as follows: 1) Equilibrate the cation column (5ml, CM-FF) with 5CVTris buffer, and adjust the UV value to zero; 2) The sample is loaded at 0.5-1ml/min, and the UV value is washed back to the baseline with Tris buffer after the end; 3) Elute with 30%, 100% 1M NaCl elution buffer, flow rate 5ml/min, pay attention to sample collection and sampling; The specific operation steps of the cation column renaturation of inclusion bodies are as follows: 1) Equilibrate the cation column (5ml, CM-FF) with 5CV inclusion body dissolution buffer, and adjust the UV value to zero; 2) Pass 45ml of 0.1g inclusion body denaturation solution through a 0.22um microporous membrane, load 0.5-1ml/min, and wash the UV value back to the baseline with inclusion body dissolution buffer after completion; 3) Gradually increase the ratio of Tris buffer/inclusion body dissolution buffer from 0 to 100%, flow rate 0.5ml/min, and complete within 2h; 4) Elute with 8%, 30%, and 80% 1M NaCl elution buffer, respectively, at a flow rate of 5 ml/min, and collect the second protein peak; 5) Finally, use 8M Ure elution buffer to elute the protein that failed to renature on the column, and pay attention to sampling during the whole process. 10. The stable troponin I standard substance obtained by the preparation method of any one of claims 1-9.

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