Recombinant cholesterol esterase, polynucleotide thereof, recombinant plasmid, expression system and application

Abstract

The invention provides a recombinant cholesterol esterase, polynucleotide thereof, a recombinant plasmid, an expression system and application. Belongs to the technical field of protein engineering, and particularly relates to a recombinant cholesterol esterase, the amino acid sequence of the recombinant cholesterol esterase is shown as SEQ ID No.1, and the nucleotide sequence for coding the recombinant cholesterol esterase is shown as SEQ ID No.2. The total length of the sequence of the recombinant cholesterol esterase provided by the invention is 482 amino acids, and the recombinant cholesterol esterase is derived from aspergillus niger and has the advantage of high thermal stability, the thermal stability deviation of the recombinant cholesterol esterase after 11 days of thermal storage at 37 DEG C and 30 days of airborne opening is less than 10%, and the recombinant cholesterol esterase has a wide application prospect in the field of in-vitro diagnostic reagents.

Description

Technical Field

[0001] The present invention relates to the field of protein engineering technology, and in particular to a recombinant cholesterol esterase, its polynucleotide, recombinant plasmid, expression system and application. Background Art

[0002] Cholesterol esterase (EC3.1.1.13) is an important steroidal esterase that can act on fatty acid esters formed by cholesterol and other sterols in aqueous media, and the reaction of catalyzing the hydrolysis of ester bonds generally does not involve cofactors. Cholesterol esterase is widely present in nature. In the early days, cholesterol esterase products were mainly derived from animal organs, such as liver, pancreas, adrenal glands, gonads, mammary glands, etc., but the method of extracting viscera was limited by the source of materials, and the purification step was difficult, which affected the stability of the enzyme preparation. After the 1970s, microbial fermentation to produce cholesterol esterase became the main trend. Many microorganisms such as Streptomyces, Pseudomonas, Bacillus, Lactobacillus, Thermoanaerobacterium, Micrococcus, Aspergillus, Spore Fungus, Saccharomyces, Candida, etc. have been identified as having the ability to synthesize cholesterol esterase. Studies have shown that cholesterol esterases from different sources have different characteristics in terms of molecular size and structure, substrate specificity, reaction kinetics, etc.

[0003] In 1974, cholesterol esterase was used as a key raw material for diagnostic reagents to measure cholesterol in human serum, which pioneered the industrial application of this enzyme. The clinical use of test kits containing cholesterol esterase to measure serum cholesterol has the advantages of simplicity, rapidity, precision, good specificity, suitability for automated instruments and conducive to standardization, and has gradually replaced optical, electrochemical, chemical and other methods. In medicine, the detection of serum cholesterol plays an extremely important role in the diagnosis and prevention of multiple diseases such as diabetes, cardiovascular and cerebrovascular diseases, hepatobiliary diseases, and kidney diseases.

[0004] Currently, the application of cholesterol esterase in in vitro diagnostic reagents has the problem of poor stability. Summary of the invention

[0005] The main purpose of the present invention is to provide a suitable enzyme source for a high-density lipoprotein cholesterol detection kit, thereby solving the problem of poor stability of the existing cholesterol esterase after using the detection reagent.

[0006] To achieve the above object, the present invention provides a recombinant cholesterol esterase in a first aspect, wherein the amino acid sequence of the recombinant cholesterol esterase is shown in SEQ ID No.1.

[0007] In view of the technical problem of poor stability of cholesterol esterase in the prior art, the present invention provides a recombinant cholesterol esterase, which is derived from Aspergillus niger and has a full-length sequence of 482 amino acids after modification. The N-terminus of the recombinant cholesterol esterase of the present invention has 6 histidine tags and can be used for purification and separation on a nickel column.

[0008] The second aspect of the present invention provides a polynucleotide, the sequence of which is shown in SEQ ID No. 2. The polynucleotide provided by the present invention is used to efficiently encode the recombinant cholesterol esterase described in the first aspect.

[0009] Preferably, the sequence of the polynucleotide is as shown in SEQ ID No.2.

[0010] The third aspect of the present invention provides a recombinant plasmid, which comprises the polynucleotide described in the second aspect and is capable of translating and expressing the recombinant cholesterol esterase described in the first aspect.

[0011] Preferably, the recombinant plasmid further contains hp4d hybrid promoter, Xpr2 terminator and Xpr2-pre signal peptide.

[0012] Preferably, the empty plasmid of the recombinant plasmid is pINA1317.

[0013] The fourth aspect of the present invention provides a recombinant cholesterol esterase expression system, wherein the recombinant cholesterol esterase expression system is a Yarrowia lipolytica Po1h strain that can stably secrete and express the recombinant cholesterol esterase shown in SEQ ID No.1.

[0014] Preferably, the recombinant cholesterol esterase expression system is integrated with a Zeta1-CEN1 expression cassette-Zeta2 fragment, and the nucleotide sequence of the Zeta1-CEN1 expression cassette-Zeta2 fragment is shown in SEQ ID No.3.

[0015] The fifth aspect of the present invention provides a method for preparing recombinant cholesterol esterase, wherein the preparation method comprises the steps of culturing the recombinant cholesterol esterase expression system described in the fourth aspect at a high density and then purifying the fermentation broth using a nickel column to obtain the recombinant cholesterol esterase.

[0016] More preferably, the above preparation method may specifically include the following steps: Step 1: synthesize the nucleotide sequence shown in SEQ ID No. 2 in vitro and integrate the sequence into the pINA1317 vector; Step 2: Recover the linearized nucleotide fragment of the recombinant cholesterol esterase, and transform the fragment into the Yarrowia lipolytica engineered bacteria, screen the positive transformants using a solid plate of a basic medium without adding uracil, and evaluate the level of cholesterol esterase expressed by the strain by the enzyme activity in the fermentation broth supernatant; Step 3: Amplify and culture the strain with high expression of cholesterol esterase; Step 4: Purify the recombinant cholesterol esterase from the yeast fermentation broth supernatant by nickel column affinity chromatography.

[0017] More preferably, the recombinant cholesterol esterase prepared above should be stored in a protective agent, which is a mixture of 5% mannitol and 2.5% salmon serum albumin.

[0018] More preferably, the recombinant cholesterol esterase prepared above is stored by freeze-drying.

[0019] The sixth aspect of the present invention provides a use of the recombinant cholesterol esterase described in the first aspect in preparing a high-density lipoprotein cholesterol detection kit.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. The recombinant cholesterol esterase provided by the present invention is derived from Aspergillus niger, has a high expression level in yeast and is easy to extract, and is suitable for use as an enzyme preparation in a high-density lipoprotein cholesterol detection kit; 2. The recombinant cholesterol esterase provided by the present invention has extremely excellent thermal stability. Its thermal stability deviation is less than 10% after 11 days of heat storage at 37°C and 30 days of airborne opening, and has broad application prospects in the field of in vitro diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the plasmid map of the recombinant expression vector pINA1317-CEN1 in a specific embodiment of the present invention; Figure 2 The SDS-PAGE electrophoresis result of the purified product in Example 4 of the present invention is shown in FIG. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0023] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0024] As described in the background art, the enzymatic properties of cholesterol esterase from different sources are not the same and cholesterol esterase as an enzyme preparation for a high-density lipoprotein detection kit has the problem of poor stability.

[0025] Based on this, a specific embodiment of the present invention provides a recombinant cholesterol esterase, the sequence of which is 482 amino acids in length, and its sequence is shown in SEQ ID No. 1. The recombinant cholesterol esterase is derived from Aspergillus ruber, and its N-terminus is connected to 6 histidine tags for nickel column purification.

[0026] More specifically, the nucleotide sequence encoding the recombinant cholesterol esterase provided in the above example is shown as SEQ ID No.2.

[0027] When different species translate the same amino acid sequence, due to the existence of synonymous codons and the influence of RNA abundance in the species, different species will show codon bias when translating. Therefore, the polynucleotide sequences encoding the recombinant cholesterol esterase provided by the present invention obtained by technicians in this field through existing codon optimization tools should all fall within the scope of protection of the present invention.

[0028] More specifically, a specific embodiment of the present invention further provides a recombinant plasmid, which contains the nucleotide sequence shown in SEQ ID No. 2 and can translate and express the recombinant cholesterol esterase shown in SEQ ID No. 1.

[0029] More specifically, a specific embodiment of the present invention further provides a recombinant cholesterol esterase expression system, which is used to secrete and express the recombinant cholesterol esterase shown in SEQ ID No.1.

[0030] More specifically, a specific embodiment of the present invention also provides a method for preparing recombinant cholesterol esterase using the recombinant cholesterol esterase expression system provided in the above embodiment, which specifically comprises the following steps: S1: In vitro synthesis of the nucleotide sequence shown in SEQ ID No.2; S2: The nucleotide sequence shown in SEQ ID No.2 is transferred into the pINA1317 vector. More specifically, the nucleotide sequence shown in SEQ ID No.2 is inserted between the two restriction enzyme sites SfiI and KpnI of pINA1317 to obtain a recombinant expression vector pINA1317-CEN1. The recombinant expression vector has a Zeta1-CEN1 expression cassette-Zeta2 fragment for efficient expression of recombinant cholesterol esterase. The nucleotide sequence of the Zeta1-CEN1 expression cassette-Zeta2 fragment is shown in SEQ ID No.3. The plasmid map of the recombinant expression vector pINA1317-CEN1 is shown in Figure 1 As shown; S3: The expression vector pINA1317-CEN1 was digested with NotI, and the fragment containing the cholesterol esterase gene was recovered by nucleic acid gel electrophoresis and gel cutting, and the purified fragment was transformed into Yarrowia lipolytica Po1h. + Type (uracil auxotrophic label reversion mutant) recombinant strain, label and pick Ura + The recombinant strain was fermented in 5 mL YPD liquid medium for 5-7 days, and the activity of recombinant cholesterol esterase in the fermentation supernatant was detected. The recombinant expression strain with the highest activity was preserved by freezing. S4: Streak the recombinant expression strain on the YPD plate, pick a single colony to inoculate and prepare the seed solution, transfer it to a 20 L fermenter at a 5% inoculation rate, and expand the recombinant expression strain at a high density; S5: collecting the centrifuged fermentation broth, purifying the supernatant with a Ni column affinity layer, and freeze-drying to obtain the recombinant cholesterol esterase.

[0031] More specifically, in step S3 of the above embodiment, a linearized fragment with Zeta1 / 2 sites at both ends can be obtained after single digestion with NotI.

[0032] More specifically, in step S4 of the above embodiment, the detection reagent for the activity of recombinant cholesterol esterase includes reagent R1 and reagent R2, wherein reagent R1 includes: 50 mM PBS buffer, 400 U / L cholesterol oxidase, and 2 mL / L Triton X-100; reagent R2 includes: 50 mM PBS buffer, 1000 U / L peroxidase, 1 mmol / L 4-aminoantipyrine, and 0.2 mM cholesterol linoleate.

[0033] More specifically, in step S5 of the above embodiment, the protective agent used for freeze-drying the recombinant cholesterol esterase is a mixture of 5% mannitol and 2.5% salmon serum albumin. Salmon serum albumin can effectively improve the protein structure stability of the recombinant cholesterol esterase during the preparation and storage process and reduce the enzyme activity loss rate.

[0034] The technical scheme of the present invention is further described below by specific examples. Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, the terms with conventional understandings are defined herein for the purpose of explanation or convenience of reference, and such definitions herein should not be understood to represent significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out according to the schemes and parameters given by the manufacturer.

[0035] Example 1 Screening of recombinant expression strains.

[0036] The nucleotide sequence shown in SEQ ID No.2 was designed and synthesized by a commercial company. The nucleotide sequence was also transferred into the expression vector pINA1317. The nucleotide sequence shown in SEQ ID No.2 was located between the SfiI and KpnI restriction enzyme sites in the multi-cloning site of the vector. The final recombinant plasmid was named pINA1317-CEN1.

[0037] The glycerol bacteria containing the aforementioned pINA1317-CEN1 expression vector were streaked on an LB plate containing 50 μg / mL kanamycin resistance, placed in a 37°C incubator overnight, and a single colony on the plate was picked up and placed in a 50 μg / mL kanamycin resistance LB liquid medium, shaken and cultured at 37°C and 220 rpm for 14-18 hours, and the target expression vector was extracted using a plasmid mini-extraction kit. NotI fast-cutting enzyme and corresponding buffer were added, and incubated at 37°C for 1.5 hours. The system of NotI digestion of the pINA1317-CEN1 recombinant expression vector is shown in Table 1.

[0038] Table 1

[0039] The target fragment and the impurity fragment containing the recombinant cholesterol esterase gene were separated from the products after enzyme digestion by nucleic acid gel electrophoresis, and the linearized fragment was extracted by gel recovery kit.

[0040] Pipette 10 μL of Yarrowia lipolytica liquid from the culture storage tube into 5 mL of YPD liquid medium, and culture it in a shaker at 30°C and 200 rpm for 24 hours. Pipette the bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, collect the bacterial sludge to a volume of 100 μL, wash twice with sterile water, add 0.6 mL of 0.1 M lithium acetate, incubate at 30°C for 1 hour, centrifuge at 4000 rpm for 1 min, discard the supernatant, and add 40 μL of 0.1 M lithium acetate to obtain competent cells.

[0041] Add 1 μg of linearized fragment and 50 μg of single-stranded salmon sperm DNA to 40 μL of prepared competent cells, mix well by pipetting, and incubate in a 30°C water bath for 15 min. Add 350 μL of 40% PEG4000 + 0.1M lithium acetate buffer and 16 μL of 1M dithiothreitol in sequence, and continue incubating in a 30°C water bath. After 1 hour, remove the centrifuge tube and place it in a 39°C metal bath for heat shock for 10 min. Add 0.6 mL of 0.1M lithium acetate and incubate in a 30°C water bath for 30-60 min. Take 100~200μL of bacterial liquid and spread it on a solid basal medium without leucine (20g / L glucose, 10% YNB: 1.7g amino acid-free yeast nitrogen source + 5g ammonium sulfate). After placing it in a 30℃ incubator for 4~5 days, take out the plate, pick 20 single colonies, and inoculate them into 5mL YPD liquid medium (10g yeast powder, 20g peptone, 50g glucose per liter of pure water), shake and culture at 30℃ and 200rpm for 120 hours, aspirate 200μL of fermentation liquid, centrifuge at 12000rpm, and keep the supernatant for qualitative activity measurement. The single colony with the highest activity is re-inoculated and cultured. When the yeast growth enters the exponential phase, 50% glycerol is added and stored in a -80℃ freezer.

[0042] Example 2 Recombinant cholesterol esterase activity assay.

[0043] The enzyme activity assay reagent of recombinant cholesterol esterase was prepared according to the formula shown in Table 2.

[0044] Table 2

[0045] The reaction parameters were set on the Mecon 880 biochemical analyzer: the detection wavelength was set to 546 nm, the sample volume to be tested was set to 10 μL, the reagent R1 volume was set to 150 μL, the reagent R2 volume was set to 150 μL, the reaction method was the rate method, and the reading points were 35 to 45. Water and 100 U / L cholesterol esterase were used as calibrants.

[0046] The test results after 120 hours of small-scale culture of 20 monoclonal clones are shown in Table 3.

[0047] Table 3

[0048] Example 3 High density culture.

[0049] Select the positive single colony numbered 3 in Table 3 and streak it onto a YPD solid plate. Culture it at 30°C for 3 days. Pick a single colony and inoculate it into 5 mL YPD liquid culture medium. Culture it at 30°C and 200 rpm with shaking for 24 hours. Expand the culture to 250 mL YPD culture medium at a 2% inoculation rate. Culture it at 30°C and 200 rpm with shaking for 24 hours. Transfer it to a fermenter with a tank volume of 20 L at a 5% inoculation rate. The fermentation tank parameters were set as follows: the fermentation tank liquid volume was 15 L, the culture medium used was YPD liquid culture medium, the stirring speed was 500~1000 rpm, the temperature was set to 28~30°C, the pH was adjusted to around 7.0 by adding ammonia water, the DO was controlled at 20~50%, and samples were taken every 24 hours to determine the wet weight of the bacteria and the enzyme activity of cholesterol esterase. Glucose was added at the same time, and the fermentation broth collected after 120 hours of fermentation was centrifuged. The supernatant volume was about 9 L, the wet weight of the bacteria was 48 g / 100 mL, and the enzyme activity of recombinant cholesterol esterase in the supernatant was detected to be 37500 U / L.

[0050] Example 4 Product purification.

[0051] The fermentation broth harvested in Example 3 was collected and centrifuged at 4°C and 4000 rpm for 30 min. The supernatant was concentrated by tangential flow ultrafiltration system to complete protein concentration and pigment removal. The concentration multiple was 4 to 5 times, and the replacement buffer was 20 mM Tris-HCl + 300 mM Nacl (pH 7.8).

[0052] The nickel column was equilibrated with 5-10 times the volume of filler 20mM Tris-HCl+300mM Nacl (pH7.8) buffer, the ultrafiltration concentrate was loaded onto the nickel column, and the nickel column was equilibrated with 20mM Tris-HCl+300mM Nacl (pH7.8) buffer until the UV detection was stable. The target protein was eluted with 20mM Tris-HCl+300mM Nacl+20mM imidazole, 20mM Tris-HCl+300mM Nacl+50mM imidazole, and 20mMTris-HCL+300mM Nacl+400mM imidazole gradients, and the elution peaks were collected. The total amount of eluent was 700mL, the enzyme activity was 140KU / L, and the SDS-PAGE electrophoresis results of the purified product were as follows: Figure 2 As shown, the target protein band is clear, mainly concentrated in the 400mM imidazole elution solution, and there is basically no impurity protein.

[0053] The 400 mM imidazole eluate containing the recombinant cholesterol esterase was placed in a dialysis bag, and dialyzed 3 to 4 times with 50 mM phosphate buffer (pH 7.8) as the dialysis fluid to obtain the pure recombinant cholesterol esterase.

[0054] Example 5 Effect of protective agents on the stability of recombinant cholesterol esterase protein.

[0055] According to Table 4, protective agent formulations 1 to 6 were prepared, and protective agent formulations 1 to 6 were mixed with 10 mL of the pure recombinant cholesterol esterase obtained in Example 4, respectively.

[0056] Table 4

[0057] The recombinant cholesterol esterase solution with the protective agent added was dispensed into a freezing bottle in a volume of 1 mL, and freeze-dried using a freeze dryer to obtain a lyophilized powder. 1 mL of sterile water was taken to reconstitute the lyophilized powder, and the relative deviation of the cholesterol esterase activity before and after lyophilization was recorded. The reconstituted solution was placed at 4°C and 42°C, and the relative deviation of the enzyme activity was detected on the 0th day and the 14th day. The results are shown in Table 5.

[0058] Table 5

[0059] It can be seen from Table 5 that both Formula 4 and Formula 6 with the addition of salmon serum albumin can effectively improve the stability of recombinant cholesterol esterase, and when 5% mannitol and 2.5% salmon serum albumin are used as lyophilization protectants, the enzyme activity freeze-drying loss is the lowest, and the long-term storage stability at 4°C and 42°C is the best.

[0060] Example 6 Application of recombinant cholesterol esterase in high-density lipoprotein cholesterol detection kit.

[0061] Prepare high-density lipoprotein cholesterol detection reagent RA. The ingredients of reagent RA are shown in Table 6.

[0062] Table 6

[0063] Prepare high-density lipoprotein cholesterol detection reagent RB, the components of which are shown in Table 7.

[0064] Table 7

[0065] The detection reagent RA / RB was used to detect high-density lipoprotein cholesterol quality control products (level 1 range: 0.89~1.33mmol / L, level 1 target value: 1.11mmol / L; level 2 range: 0.89~1.33mmol / L, level 2 target value: 1.93mmol / L) and 20 serum samples before and after 11 days of heat storage in a 37°C water bath and before and after 30 days of onboard opening. The specific data are shown in Tables 8 and 9.

[0066] Table 8

[0067] Table 9

[0068] As can be seen from Tables 8 and 9, the values ​​of the serum samples and the quality control products are basically consistent, proving that the recombinant cholesterol esterase provided by the present invention as a raw material for the high-density lipoprotein cholesterol detection kit has high stability and can meet the basic detection performance.

[0069] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A recombinant cholesterol esterase, characterized in that The amino acid sequence of the recombinant cholesterol esterase is shown in SEQ ID No.

1.

2. A polynucleotide, characterized in that The sequence of the polynucleotide is shown as SEQ ID No.

2.

3. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the polynucleotide according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that The recombinant plasmid also includes hp4d hybrid promoter, Xpr2 terminator and Xpr2-pre signal peptide.

5. The recombinant plasmid according to claim 3, characterized in that The empty vector plasmid of the recombinant plasmid is pINA1317.

6. A recombinant cholesterol esterase expression system, characterized in that: The recombinant cholesterol esterase expression system is a Yarrowia lipolytica Po1h strain that can stably secrete and express the recombinant cholesterol esterase shown in SEQ ID No.

1.

7. The recombinant cholesterol esterase expression system according to claim 6, characterized in that: The recombinant cholesterol esterase expression system is integrated with the Zeta1-CEN1 expression cassette-Zeta2 fragment, and the nucleotide sequence of the Zeta1-CEN1 expression cassette-Zeta2 fragment is shown in SEQ ID No.

3.

8. A method for preparing the recombinant cholesterol esterase according to claim 1, characterized in that: The preparation method comprises the following steps: after high-density culturing the recombinant cholesterol esterase expression system according to claim 6 or 7, purifying the fermentation liquid using a nickel column to obtain the recombinant cholesterol esterase.

9. The preparation method according to claim 8, characterized in that: The following steps are involved: Step 1: synthesize the nucleotide sequence shown in SEQ ID No. 2 in vitro and integrate the sequence into the empty vector plasmid; Step 2: Recover the linearized nucleotide fragment of the recombinant cholesterol esterase, and transform the fragment into the Yarrowia lipolytica engineered bacteria, screen the positive transformants using a solid plate of a basic medium without adding uracil, and evaluate the level of cholesterol esterase expressed by the strain by the enzyme activity in the fermentation broth supernatant; Step 3: Amplify and culture the strain with high expression of cholesterol esterase; Step 4: Purify the recombinant cholesterol esterase from the yeast fermentation broth supernatant by nickel column affinity chromatography.

10. Use of the recombinant cholesterol esterase according to claim 1 in preparing a high-density lipoprotein cholesterol detection kit.

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