CN116789853A - Stable recombinant cTnI-C complex and application and preparation thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a stable recombinant cTnI-C complex and application and preparation thereof. The truncated cTnI is utilized to couple the full-length cTnC through 4-segment repeated linker (GGGGS) to form the cTnI-C complex, and the recombinant cTnI-C complex has high stability and high antigen reactivity and can be used as a calibrator; and the purification adopts a unique purification method, and can separate the dimer to obtain purer cTnI-C monomer protein, thereby reducing the batch difference in the production process and having more manufacturability.

Description

Stable recombinant cTnI-C complex and application and preparation thereof

Technical Field

The invention relates to the technical field of biology, in particular to a stable recombinant cTnI-C complex and application and preparation thereof.

Background

Troponin complex is a muscle protein involved in the regulation of calcium-dependent muscle contraction, playing an important role in the contraction process of cardiac and skeletal muscles, and is composed mainly of 3 subunits, troponin I (TnI), troponin T (TnT) and troponin C (TnC), respectively. Of these, tnI is mainly responsible for inhibiting myosin atpase activity, and in the absence of ca2+, tnI inhibits actin formation. The N-terminus of TnT regulates the interaction of troponin complex and the filament, while the C-terminus interacts with TnI and TnC in the presence of Ca2+. TnC acts as a Ca2+ binding subunit, primarily to regulate muscle contraction and stabilize cardiac troponin I (cTnI) (Gomes A V, potter J D, szczesna-Cordary D. The Role of Troponins in Muscle Contraction [ J ]. Iub.Life, 2010, 54 (6): 323-333).

In the human body, skeletal muscle type troponin (sTn) and cardiac muscle type troponin (cTn) can be classified according to the difference of the respective subunits, and the skeletal muscle type of TnC and the cardiac muscle type are the same, and three subtypes, namely, fast skeletal troponin I and T, slow skeletal troponin I and T, and cardiac muscle troponin I (cTnI) and cardiac muscle troponin T (cTnT) exist. cTnI is expressed only in myocardial tissue with high heart specificity and 40% difference in skeletal muscle type sequences (Bodor G S, porterfield D, voss E M, et al cardioac troponin-I is not expressed in fetal and healthy or diseased adult human skeletal muscle tissue [ J ]. Clinical Chemistry, 1995, 41 (12 Pt 1): 1710-1715). cTnT is somewhat less specific and is expressed in some diseased skeletal muscle in addition to myocardial tissue (Jaffe a S, vasile V C, milone M, et al Diseased skeletal muscle: a noncardiac source of increased circulating concentrations of cardiac troponin T. [ J ]. Journal of the American College of Cardiology, 2011, 58 (17): 1819-1824).

Early diagnosis and treatment of Acute Myocardial Infarction (AMI) are of great importance for reducing the risk of death, but the diagnosis of AMI is very dependent on the detection of myocardial tissue markers, which were clinically used before the 80 s of the 20 th century, including creatine phosphokinase (CK) and its isozymes (CK-MB), myoglobin (MB), etc., but according to clinical data, it was shown that the specificity of these markers was not high enough, the rise time was late and the duration was short, severely restricting the early diagnosis and treatment work of heart diseases. In the early 90 s of the 20 th century, the U.S. FDA approved cTnI for clinical AMI diagnosis, the release form of the cTnI was similar to CK-MB, and the increase of the cTnI value in blood can be detected after the myocardial injury for 4-6 hours, but the increase time of the cTnI can last for 6-10 days and is much longer than the duration time of CK-MB, so that the window period of heart injury disease diagnosis is greatly prolonged; in addition, cTnI is only expressed in myocardial tissue, is not affected by skeletal muscle injury, and has unique myocardial specificity. Therefore, cTnI is also known as a gold standard for myocardial damage disease detection.

At present, the cTnI detection kit used for clinical detection in China is greatly dependent on import, and because the cTnI monomers of natural and recombinant sources are extremely easy to degrade, transportation and storage are extremely difficult, the use of the cTnI high-specificity cardiac markers in clinical diagnosis is greatly limited. In blood of a patient with myocardial injury, cTnI exists in a form of troponin I-C complex (cTnI-C), the C end of the TnC can form an alpha helix structure with 33-80 amino acids of the cTnI so as to play a role in stabilizing the cTnI, but the extraction of natural cTnI-C also has a plurality of difficulties, such as lack of experimental materials, high cost, complex operation, low yield and the like. Along with the rapid development of genetic engineering technology, a plurality of domestic and foreign scholars try to express cTnI-C in vitro, but most of the research work is still in an experimental stage at present, only a few commercial recombinant cTnI-C complexes exist abroad, but the price is still high, the stability is poor, and the standard products used for clinical diagnosis are still limited.

Disclosure of Invention

In view of the problems existing in the background art, the invention provides a recombinant cTnI-C complex with high stability and a purification method thereof

The method comprises the following steps:

a stable recombinant cTnI-C complex has an amino acid sequence shown in SEQ ID No. 1.

In another aspect, the invention provides a gene for encoding the recombinant cTnI-C complex, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.

In another aspect, the present invention provides a recombinant vector comprising the above gene. Preferably, the recombinant vector is selected from the group consisting of pET28a, pET22b, pET32a, pCold II or pET21a.

In another aspect, the present invention provides a recombinant bacterium comprising the above gene or the above recombinant vector. Preferably, the strain in the recombinant is selected from BL21 (DE 3), BL21 (DE 3) -ER2566 or BL21 (DE 3) -T7.

In another aspect, the invention also provides a method for preparing the recombinant cTnI-C complex, which comprises the following steps:

(1) Constructing recombinant bacteria expressing the genes;

(2) And (3) carrying out induced expression on the recombinant bacteria in the step (1) to obtain the recombinant cTnI-C complex.

In another aspect, the invention also provides a calibrator comprising the recombinant cTnI-C complex.

On the other hand, the invention also provides a diagnostic kit which comprises the recombinant cTnI-C complex or the calibrator.

In another aspect, the invention also provides a purification eluent of the recombinant cTnI-C complex, wherein the purification is column purification, and the eluent contains DTT.

The invention relates to purification of a recombinant cTnI-C complex, which is obtained by inducing recombinant bacteria to express.

The recombinant cTnI-C complex obtained after induced expression refers to a recombinant cTnI-C complex obtained by crushing recombinant bacteria after induced expression, centrifugally collecting supernatant and filtering.

On the other hand, the invention also provides a purification method of the recombinant cTnI-C complex, which comprises the following steps:

(1) Affinity chromatography;

(2) Ion exchange chromatography;

wherein the filler used for the affinity chromatography contains cobalt sulfate.

The beneficial effects are that:

(1) The recombinant cTnI-C complex has high stability and high antigen reactivity, and can be used as a calibrator;

(2) According to the method for purifying the recombinant cTnI-C complex, the cobalt column is adopted to replace the traditional Ni column, so that the purity of the product is higher;

(3) The specific reducing agent DTT is added into the eluent, so that the dimer can be separated to obtain purer cTnI-C monomeric protein, and the batch difference in the production process is reduced, and the manufacturability is improved.

Drawings

Fig. 1: electrophoretic identification result of cTnI-C induced expression;

fig. 2: purifying and identifying the cTnI-C;

fig. 3A: electrophoresis identification results of the purified product obtained when no DTT is added to the purified eluent;

FIG. 3B shows the result of the electrophoretic identification of the purified product obtained after 1mM DTT was added to the purification eluate.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

As used herein, the terms "comprising," "including," "having," "can" and variations thereof are intended to be open-ended terms, or words that do not exclude the possibility of additional acts or structures.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

EXAMPLE 1 construction of cTnI-C Complex expression Strain

According to the protein sequences of cTnI and cTnC published by Uniprot database, the 28 th-110 th amino acid residues of the human cTnI protein sequence are connected with (GGGGS) 4 and the full-length cTnC amino acid sequence to obtain a spliced amino acid sequence.

And (3) adding BamH I enzyme cutting sites at the N end and HindIII enzyme cutting sites at the C end of the DNA sequence corresponding to the splicing sequence, optimizing according to the codon preference of the E.coli of an expression host, carrying out gene synthesis by Shanghai JieRui bioengineering limited company, connecting the gene synthesis to an expression vector pET28a by using the enzyme cutting sites BamH I and HindIII, and then converting the gene into an expression strain BL21 (DE 3).

In other embodiments, the vector may also be selected from pET22b, pET32a, pCold II or pET21a.

The expression strain may also be selected from BL21 (DE 3) -ER2566 or BL21 (DE 3) -T7.

The amino acid sequence of cTnI-C is shown as SEQ ID No.1, and the nucleotide sequence is shown as SEQ ID No. 2.

EXAMPLE 2 screening of highly expressed engineering strains

10 strains of the constructed expression strains are respectively inoculated into LB+Kana test tubes, cultured at 37 ℃ at 200-250 rpm for overnight, then inoculated into TB+Kan culture medium according to the inoculum size of 1 percent, cultured at 37 ℃ at 250rpm for 1.8-2.2 hours until OD600 is 0.6-0.8, 1mL of bacterial liquid before induction is reserved as a control, and then 0.5mM IPTG with the final concentration is added for induction expression for 6 hours at 25 ℃ at 200-250 rpm.

The supernatant was removed by centrifugation from 1mL of each of the bacterial solutions before and after induction, and the supernatant was resuspended in 500. Mu.L of 1 XPBS, and then subjected to SDS-PAGE electrophoresis to identify, stained with Coomassie Brilliant blue, and the electrophoretic bands were analyzed after decolorization. The induced band has an obvious band at the position of the predicted molecular weight, the band before induction has no obvious band at the position of the same molecular weight, and the strain with the highest expression quantity is selected as the expression engineering strain.

After the induced thalli are resuspended by 1 XPBS and then are ultrasonically crushed, supernatant and sediment are respectively identified by SDS-PAGE after centrifugation, and the result shows that the recombinant protein is mainly present in the supernatant, thus proving the soluble protein when the recombinant protein exists.

EXAMPLE 3 high Density upper tank fermentation of engineering bacteria

(1) Preparation of seed liquid in upper tank

One engineering bacterium is taken in a biosafety cabinet, streaked on a LB solid culture medium plate containing the kana resistance by an inoculating loop, the plate is sealed and then is inverted into a biochemical incubator at 37 ℃ for culturing 16-24 h.

Single colonies are picked on an LB solid medium plate by using a sterile inoculating loop, inoculated into 25 mL of LB liquid medium which is added with 25 mu L of kanamycin mother solution (50 mg/mL) in advance, placed in a constant temperature culture shaker, and cultured at 37 ℃ at 200-250 rpm for 4-10 h to serve as primary seed liquid.

Into 2 bottles, 500. Mu.L of kanamycin mother solution (50 mg/mL) and 500 mL of LB liquid medium are added, and 25-100. Mu.L of primary seed solution is inoculated. And placing the inoculated LB liquid culture medium into a constant temperature culture oscillator, and culturing at 37 ℃ and 200-250 rpm for 6-10 h serving as seed liquid for the upper tank.

(2) Fermentation in upper tank

Pouring the seed liquid in the upper tank into a fermentation tank. The ammonia water feeding bottle is connected into the fermentation tank through the feeding port after passing through the acid-base feeding pump, and the pH value is controlled to be 6.8-7.2 by automatic feedback feeding ammonia water. When the dissolved oxygen of the fermentation tank is lower than 30%, firstly adjusting the stirring rotating speed, lifting 200 rpm (up to 750 rpm) each time, adjusting ventilation volume after the stirring rotating speed reaches 750 rpm, lifting 10L/min (up to 30L/min) each time, lifting pressure by 0.02 mPa (up to 0.08 mPa) each time when the ventilation volume is lifted each time, and maintaining the dissolved oxygen of the fermentation tank by 25% -150%. When the concentration of bacteria in the fermentation tank is OD600 = 15-20, the temperature is reduced to 25 ℃, 5mM IPTG with the final concentration is added for induction, fermentation is stopped after 6 hours of induction, fermentation liquor is discharged, and the bacteria are collected by centrifugation.

(3) Results of induction

The induction results are shown in FIG. 1, wherein lane M is maker, and lanes 1-4 are staining results of the expressed bacteria samples induced for 0, 2, 4, and 6 hours after SDS-PAGE electrophoresis, respectively. Taking induction 0h as a negative control, sampling at 2h, 4h and 6h respectively for SDS-PAGE identification, and taking the target protein as an arrow, wherein the graph obtains high expression of cTnI-C.

EXAMPLE 4 purification of troponin I-C Complex

(1) Resuspension and disruption of the thallus

50 g thalli are weighed by a balance in a beaker, and 1 XPB is added according to the feed liquid ratio of 1:8 and stirred until no obvious thalli block exists. Then, the bacterial body weight suspension was added to a high-pressure homogenizer, and the bacterial liquid after being crushed was centrifuged and the supernatant was collected by circulating for 3 times, and was filtered with a 0.45 μm filter membrane.

(2) Affinity chromatography

The cobalt column was connected to a purifier with a full flow rate of 15 mL/min. And (3) balancing the chromatographic column by using a solution A (1 XPB), loading all the filtered supernatant in the step (1), balancing by using the solution A, washing impurities by using 5% of a solution B (1 XPB+0.5M imidazole) when the ultraviolet absorption value is reduced to be less than 50 mAu, and stopping washing impurities after the ultraviolet absorption value is peaked and is reduced to be less than 50 mAu, wherein the part is not collected. The gradient of the proportional valve was adjusted to 40% B to elute the protein of interest, samples were collected in product bottles and 1M DTT mother liquor was added to the eluate to a final concentration of 1 mM.

(3) Ion exchange chromatography

The Q column was connected to a purifier with a full flow rate of 10 mL/min. The column was equilibrated with solution A (1 XPB+1 mM DTT) and suspended after the UV absorption peak was stationary. And (3) loading all the eluent in the step (2), then continuing to balance by using the solution A, and starting to linearly elute after the ultraviolet absorption value is reduced to below 20 mAu. The gradient of the proportional valve is adjusted to be 0-100% of B liquid (1 XPB+ 1M NaCl+1 mM DTT) in 50 CVs, the ultraviolet absorption value after peak emergence is more than 50 mAu, the concentration of the B liquid at the moment is about 24%, the conductivity value is about 23 mS/cm, the collection is stopped at the bottom of the peak, and the sample is collected in a product bottle.

(4) Purification results

The cTnI-C purification result is shown in FIG. 2, wherein lane M is marker, lane 1 is supernatant, lane 2 is flow-through, lane 3 is wash, lane 4 is first-step elution, lane 5 is second-step elution of still, lane 6 is elution of non-still, and lane 7 is second-step wash

Supernatant: cell wall-broken supernatant of cTnI-C zymophyte;

and (3) flow through: the first step of affinity chromatography loading penetrating fluid;

washing: eluting by using 5% of B solution of affinity chromatography;

and (3) eluting in the first step: eluting by using 40% of B solution of affinity chromatography;

the purity of the reducing gel and the non-reducing gel of the eluent in the second step reaches more than 95%, which means that the monomer target protein cTnI-C is successfully purified, and the purity reaches more than 95%.

EXAMPLE 5 Effect of DTT in eluent

The purification steps were the same as in example 4 except that DTT was added or not added to the eluate, and the results are shown in FIG. 3A (DTT was not added) and FIG. 3B (DTT was added at a final concentration of not 1 mM).

Wherein lane M is marker, lane 1 is reduction, lane 2 is non-reduction, it can be seen that there is a distinct dimer (at position 70) after purification without the addition of DTT non-reducing gel, and the result of purification with the addition of DTT is monomer cTnI-C.

EXAMPLE 6 identification of cTnI-C antigen reactivity

Test reagent: cTnI finished product kit lotS210602

Protein concentration: 2.2mg/ml (BCA method)

Principle of protein concentration measurement by BCA method: peptide bonds in proteins will Cu ²⁺ Reduction to Cu ¹⁺ ，Cu ¹⁺ A stable violet-blue complex can be formed with bicinchoninic acid, with a high light absorption value at 562, 562 nm and proportional to protein concentration, from which protein concentration can be determined. The BCA protein determination method has the advantages of simpler operation, better stability of the reagent and the color complex formed by the reagent, almost no influence of interfering substances, high sensitivity (the trace detection can reach 0.5 mug/ml), and more flexible application.

S1 and S2 are self-contained calibrators of the kit, and RLU is a light value

Table 1 kit calibration

TABLE 2 Linear experiments

It can be seen that the cTnI-C complex has better antigen reactivity and linearity, and the cTnI-C complex can be used as a calibration quality control material.

EXAMPLE 7 cTnI-C Complex freeze thawing and accelerated stability characterization at 37 ℃

Placing cTnI-C complex stock solution at 37deg.C for 7 days and repeatedly freezing and thawing at-20deg.C for 5 times, diluting with diluent to detection range, and testing with finished product kit as follows

TABLE 3 stability test

It can be seen that the stability is not affected after the antigen stock solution is subjected to 37 ℃ acceleration for 7 days and repeated freeze thawing for 5 times at-20 ℃, and the cTnI-C complex can be used as a calibrator or a quality control product to be applied to diagnostic reagents.

While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims (10)

1. The stable recombinant cTnI-C complex is characterized in that the amino acid sequence of the cTnI-C complex is shown as SEQ ID No. 1.

2. A gene encoding the recombinant cTnI-C complex of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.

3. A recombinant vector comprising the gene of claim 2.

4. The recombinant vector of claim 3, wherein the recombinant vector is selected from the group consisting of pET28a, pET22b, pET32a, pCold II, and pET21a.

5. A recombinant bacterium comprising the gene of claim 2 or the recombinant vector of claim 3 or 4; preferably, the strain in the recombinant is selected from BL21 (DE 3), BL21 (DE 3) -ER2566 or BL21 (DE 3) -T7.

6. A method of preparing a recombinant cTnI-C complex as defined in claim 1, comprising the steps of:

(1) Constructing recombinant bacteria expressing the gene of claim 2;

(2) And (3) carrying out induced expression on the recombinant bacteria in the step (1) to obtain the recombinant cTnI-C complex.

7. A calibrator, comprising the recombinant cTnI-C complex of claim 1.

8. A diagnostic kit comprising the recombinant cTnI-C complex of claim 1 or the calibrator of claim 7.

9. A purification eluate for purifying a recombinant cTnI-C complex as defined in claim 1, wherein the purification is a column purification and the eluate comprises DTT.

10. A method of purifying a recombinant cTnI-C complex as defined in claim 1, comprising the steps of:

(1) Affinity chromatography;

(2) Ion exchange chromatography;

wherein the filler used for the affinity chromatography contains cobalt sulfate.