Mutant gene, recombinant protein, plasmid, recombinant bacterium, preparation method and application

Abstract

The invention discloses a mutant gene, a recombinant protein, a plasmid, a recombinant bacterium, a preparation method and application, and belongs to the technical field of creatininase production. According to the invention, codon optimization is carried out on CDS of a creatininase gene sequence (GenBank: AF170566.3) from Pseudomonas putida, enzyme active sites translated by the optimized gene sequence are identified, and the overall catalytic activity of the creatininase after translation is improved by mutating the active site gene sequence. And inserting the mutated gene sequence into an initial plasmid to construct a recombinant plasmid, and transferring the recombinant plasmid into escherichia coli to prepare the recombinant bacterium. According to the constructed recombinant bacteria, the yield and the enzyme activity of the creatininase in the fermentation process are greatly increased, the high-purity recombinant creatininase can be obtained through two-step purification, the cost can be reduced, and the recombinant bacteria have important significance on development of a renal function fine detection reagent.

Description

Technical Field

[0001] The present invention relates to the technical field of creatinase production, and in particular to a mutant gene, a recombinant protein, a plasmid, a recombinant bacterium, and a preparation method and application thereof. Background Art

[0002] Creatinine is formed by spontaneous and irreversible metabolic processes of creatine phosphate in human muscle. When kidney function is normal, the concentration of creatinine in the blood is maintained at a low level, while when kidney function is insufficiency, creatinine will accumulate in the serum. Therefore, the creatinine content in serum is an important indicator reflecting kidney function. For the detection of creatinine, the most widely used is the alkaline picric acid method, but there are problems of poor reaction specificity and limited sensitivity. It is gradually being replaced by the enzyme method (creatinine enzyme coupled to sarcosine oxidase method), among which creatinase, creatinase and sarcosine oxidase are three key enzymes. Creatinase reversibly hydrolyzes creatinine to generate creatine, creatinase catalyzes the hydrolysis of creatine to produce sarcosine and urea, and sarcosine is hydrolyzed by sarcosine oxidase to generate formaldehyde, glycine and H2O2. Coupled with the Trinder reaction, the creatinine content in the sample is calculated by colorimetric determination. The enzymatic method has the characteristics of strong anti-interference ability, good specificity, wide linear range and high sensitivity. Most commercial kits use the creatininase-coupled sarcosine oxidase method and have been increasingly used in clinical practice.

[0003] Creatinase is a key enzyme used in enzymatic creatinine detection kits. However, the key enzymes in enzymatic kits are currently still mainly imported. The fermentation yield of domestic enzyme raw materials is low and the technical stability is poor, which has caused the production cost of creatinine detection kits to remain at a high level. In order to reduce production and sales costs and reduce the burden of patient testing, it is particularly important to prepare creatinase production strains with high production activity and increase the expression and enzyme activity of creatinase to promote its widespread application in creatinine detection kits. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a mutant gene, recombinant protein, plasmid, recombinant bacteria, preparation method and application. The present invention aims to use codon optimization technology and gene mutation technology to synthesize a gene sequence-optimized creatinase gene, construct a recombinant expression vector, and thus obtain a recombinant strain of Escherichia coli that efficiently expresses recombinant creatininase. Escherichia coli (E. coli) has a clear genetic metabolic network, simple culture experimental conditions, easy production operation, and low fermentation cost. It is one of the most popular exogenous expression hosts. Adopting Pseudomonas putidaThe cah gene sequence from the original source was optimized according to the codon preference of the Escherichia coli host bacteria, and the enzyme activity was improved by identifying the active site and mutating the active site gene, thereby achieving the soluble expression of the exogenous creatinine enzyme gene in Escherichia coli. By studying the fermentation conditions and enzymatic properties of the recombinant bacteria, the preliminary scale-up production under culture conditions was completed, in order to provide a theoretical basis and technical support for the industrial production of creatinine enzyme.

[0005] To achieve the above object, the present invention adopts the following technical solution: In a first aspect of the present invention, a mutant gene is provided. The mutant gene differs from the CDS of the cah sequence with GenBank number AF170566.3 in that a G→C mutation occurs at the 323rd base position. The nucleotide sequence of the mutated gene is shown in SEQ ID NO:3.

[0006] The second aspect of the present invention provides a recombinant protein, which is expressed by the mutant gene according to claim 1, and is different from the protein expressed by the cah sequence with GenBank number AF170566.3 in that the 108th amino acid is mutated from arginine to proline, and the amino acid sequence of the mutated protein is shown in SEQ ID NO:4.

[0007] The third aspect of the present invention provides a plasmid, wherein the plasmid comprises the mutant gene.

[0008] The fourth aspect of the present invention provides a recombinant bacterium, wherein the recombinant bacterium comprises the mutant gene or the plasmid.

[0009] The fifth aspect of the present invention provides a method for preparing the recombinant bacteria, comprising the following steps: (1) The cah gene sequence is optimized, the gene active site is mutated, and then inserted into the initial plasmid to obtain a recombinant plasmid; (2) The recombinant plasmid can be transformed into Escherichia coli to obtain recombinant bacteria.

[0010] Furthermore, step (1) includes the following process: Will Pseudomonas putida The cah sequence of the source is optimized, and the enzyme active gene site is identified, and the active site is mutated to obtain the optimized gene; the optimized gene sequence is amplified, and BamHI and XhoⅠ restriction sites are introduced to obtain the amplified cah fragment; the initial plasmid and the amplified cah fragment are double-digested with BamHI and XhoⅠ enzymes, and after digestion and purification, a connection vector product and a site-mutated cah fragment product can be obtained respectively; the vector product and the site-mutated cah fragment product are connected to construct a recombinant plasmid.

[0011] Furthermore, the initial plasmid is pET-28a(+); and the Escherichia coli is Escherichia coli BL21(DE3).

[0012] In some embodiments of the present invention, the cah sequence after codon and gene site mutation optimization is inserted between BamHI and XhoⅠ of plasmid pET-28a(+).

[0013] Furthermore, in step (2), the transformation is carried out by heat shock method.

[0014] The sixth aspect of the present invention provides the use of the recombinant bacteria in the preparation of recombinant creatinine.

[0015] Furthermore, the recombinant bacteria are cultured in a fermentation medium, and the inducer isopropyl-β-D-thiogalactoside is added for induction culture. After the culture is completed, the bacteria are collected by centrifugation, and the recombinant creatinine enzyme is obtained by lysis and purification; the induction culture conditions are 25-35°C, 0.1-1mM IPTG induction for 6-18h; the purification method is a two-step purification, first purification with Ni-NTA, and then purification with ion exchange chromatography.

[0016] Beneficial effects of the present invention: The present invention introduces the creatinase gene after codon optimization and gene site mutation into competent Escherichia coli through a recombinant plasmid to construct a recombinant strain, and the preparation method is simple. The recombinant Escherichia coli strain provided by the present invention can efficiently express recombinant creatinase, and high-purity recombinant creatinase can be obtained by two-step affinity purification, which greatly improves the yield of creatinase and reduces the preparation cost of enzyme raw materials. The enzyme specific activity of the site-directed mutation group is increased to 130.3% relative to the enzyme specific activity of the non-mutated group, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the molecular map of the pET-C recombinant plasmid constructed in Example 1 of the present invention.

[0018] Figure 2 This is a graph showing the expression efficiency of creatinase at different times. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0020] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0021] The preparation method of the recombinant Escherichia coli of the present invention is: Pseudomonas putida The CDS of the source cah sequence (GenBank: AF170566.3) was codon optimized and gene site mutated, inserted into the initial plasmid to obtain a recombinant plasmid, and then the recombinant plasmid was transformed into competent Escherichia coli.

[0022] In the recombinant Escherichia coli described in the present invention, the initial plasmid includes but is not limited to plasmid pET-28a(+); in one embodiment of the present invention, the initial plasmid is pET-28a(+), and the cah gene sequence is inserted between the BamHⅠ and XhoⅠ sites of the plasmid pET-28a(+), and the resulting recombinant plasmid is recorded as pET-C (commissioned by GenScript Corporation for technical synthesis).

[0023] The experimental materials used in the examples of the present invention but not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels.

[0024] Example 1: Construction of recombinant bacteria Embodiment The recombinant bacteria that efficiently express recombinant creatinase were constructed by the following experimental steps: (1) Mutant genes and recombinant proteins The cah sequence (GenBank: AF170566.3) was codon optimized to identify its enzyme activity catalytic site (GCACGGCGCGCGTCAGCT). Using gene site-directed mutagenesis technology, the CGG gene site at the 322-324 base position of the CDS sequence was site-directed mutated to CCG, that is, a G→C mutation occurred at the 323 base position, and a mutant gene was obtained. The gene sequence before mutation was SEQ ID NO:1, and the gene sequence after mutation was SEQ ID NO:3; from the amino acid level, the mutation caused the 108th amino acid of the expression site to change from arginine to proline, thereby obtaining a recombinant protein, the amino acid sequence before mutation was SEQ ID NO:2, and the amino acid sequence after mutation was SEQ ID NO:4.

[0025] (2) Construction of recombinant plasmid pET-C The cah gene sequence that has undergone sequence mutation and codon optimization is subsequently used as the sequence of the site-directed mutation group; the cah gene sequence that has not undergone sequence mutation but has undergone the same codon optimization is subsequently used as the sequence of the non-mutation group; the sequences of the site-directed mutation group and the non-mutation group are taken and amplified separately.

[0026] The subsequent processing steps are the same for the sequences of the site-directed mutation group and the sequences of the non-mutation group, that is, BamHI and XhoⅠ restriction sites are introduced to obtain the amplified cah fragment; the vector (pET-28a(+)) and the amplified cah fragment are double-digested with BamHI and XhoⅠ, respectively, and then after digestion and purification, the vector product and the cah fragment product are obtained respectively; the vector product and the cah fragment product are connected to construct the recombinant plasmid pET-C. The map of the obtained recombinant plasmid pET-C is shown in Figure 1 The recombinant plasmid pET-C was added to the competent bacteria of Escherichia coli DH5α for heat transformation, and the strains were cultured after transformation. The recombinant plasmids of the non-mutated group and the site-directed mutation group with correct sequencing were obtained by colony PCR.

[0027] (3) Preparation of recombinant bacteria Take out the competent E.coli BL21 (DE3) from the -80℃ refrigerator and place it on ice for 10 minutes; add 2μL of the recombinant plasmid of the non-mutated group and the recombinant plasmid of the site-directed mutation group to 200μL of E. coli with strong chemical activity, and let it stand on ice for 30 minutes; heat shock the mixture in a 42℃ water bath for 90 seconds, and then immediately put it on ice for 2 minutes; add 700μL LB liquid culture medium, and shake at 37℃ for 1h; centrifuge the obtained bacterial liquid at 6000×g for 6 minutes at room temperature, remove the supernatant; add 100μL LB liquid culture medium, spread it on the LB[Kan] plate, and incubate it in a shaker at 37℃ overnight. After a single colony grows on the plate, mark the single colony with good growth on the plate. The experimental results show that the recombinant bacteria with high expression of recombinant creatinase containing the site-directed mutated cah sequence or the unmutated cah sequence have been successfully constructed and are ready for use.

[0028] Example 2: Optimization of induced fermentation conditions of recombinant bacteria LB[Kan] was used to inoculate 3 mL of the culture medium containing the two recombinant bacteria prepared in Example 1. A medium equivalent to 1 mL and 1 unit OD 600 Place 2% of the overnight bacterial solution into 1.5 mL centrifuge tubes and immediately aliquot. Inoculate 2% of the overnight bacterial solution into 10 mL LB[Kan] medium until the bacteria reach mid-logarithmic growth (OD 600 =0.4~0.6). Add the expression inducer IPTG to a specific concentration and continue culturing at a specific temperature. Use different time points during the induction period. 600Transfer the E. coli culture to a 1.5 mL centrifuge tube. Centrifuge the 1.5 mL centrifuge tube at 8000 r / pm for 2 minutes to remove the culture supernatant. Resuspend the cells in 50 μL 1× SDS-PAGE Sample Loading Buffer and treat at 95°C for 5 minutes. Centrifuge at 10000 r / pm for 2 minutes and store at -20°C. Load all prepared samples onto the gel. Reheat the samples to be tested to 95°C, take 10 μL of the suspension, and load it onto a 15% SDS-polyacrylamide gel.

[0029] According to the above operation, 0.6 mM IPTG was used to induce creatinase expression, and samples were taken at 2, 4, 8 and 12 h of culture and processed by SDS-PAGE.

[0030] Figure 2 The results showed that when E. coli was incubated at 30°C and the concentration was 0.6 mM IPTG, the optimal induction condition was 8 hours, and the production efficiency was the highest. Therefore, the subsequent induction conditions can be optimized to 0.6 mM IPTG and the induction time is 8 hours.

[0031] Example 3: Induced expression of creatinase by recombinant bacteria Take the two recombinant bacteria prepared in Example 1, the one containing the unmutated cah sequence is recorded as the unmutated group, and the one containing the mutated cah sequence is recorded as the site-directed mutation group, and express them under the following induction conditions: 30°C, 0.6mM IPTG for 8h. After the induction culture is completed, the Escherichia coli cells are collected by centrifugation, and the wet weight of the bacterial precipitate is weighed on a balance. Freeze overnight at -80°C, and then lyse the Escherichia coli cells. Resuspend the particles with binding / washing buffer. Turn on the circulating cooler, and when the temperature is lower than 4°C, use an ultrasonic bacterial crusher to crush the Escherichia coli cells until the liquid is clearly clear. Stop crushing. Centrifuge the crushed cell liquid at 10000×g for 20 minutes at 4°C. The precipitate is the insoluble part, which is discarded; the supernatant is the soluble part, that is, the crude enzyme solution containing creatinase. Use a high concentration of imidazole to elute the creatinase (NI-NTA affinity chromatography) for purification.

[0032] In order to further improve the purity of the enzyme, the product creatinase is purified in two steps. The specific steps are firstly to purify by NI-NTA affinity chromatography, and then to further purify and desalt the creatinase by ion exchange chromatography. After the two-step purification, the purity of the creatinase is increased to more than 95.0%.

[0033] The activity of the obtained creatinase was tested. The definition of enzyme activity unit is: the amount of enzyme required to catalyze the reaction to generate 1μmol of creatine per minute; the enzyme specific activity refers to the enzyme activity per milligram of enzyme protein. The test results of enzyme activity: after two-step chromatography purification, the enzyme specific activity of the obtained creatinase was: 162.74U / mg for the unmutated group and 212.03U / mg for the site-directed mutation group. The enzyme specific activity of the site-directed mutation group was increased to 130.3% relative to the enzyme specific activity of the unmutated group.

[0034] In summary, the present invention constructs a recombinant expression vector by codon-optimized and gene site-mutated creatininase genes, thereby obtaining a recombinant Escherichia coli strain that efficiently expresses recombinant creatininase, which greatly increases the yield and activity of creatininase during the fermentation process.

[0035] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A mutant gene, characterized in that: The mutant gene differs from the CDS of the cah sequence with GenBank number AF170566.3 in that a G→C mutation occurs at the 323rd base position, and the nucleotide sequence of the mutated gene is shown in SEQ ID NO:

3.

2. A recombinant protein, characterized in that The recombinant protein is expressed by the mutant gene according to claim 1, and is different from the protein expressed by the cah sequence with GenBank number AF170566.3 in that the 108th amino acid is mutated from arginine to proline, and the amino acid sequence of the mutated protein is shown in SEQ ID NO:

4.

3. A plasmid, characterized in that The plasmid comprises the mutant gene according to claim 1.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the mutant gene according to claim 1 or the plasmid according to claim 3.

5. The method for preparing the recombinant bacteria according to claim 4, characterized in that: The following steps are involved: (1) The cah gene sequence is optimized, the gene active site is mutated, and then inserted into the initial plasmid to obtain a recombinant plasmid; (2) The recombinant plasmid can be transformed into Escherichia coli to obtain recombinant bacteria.

6. The method for preparing the recombinant bacteria according to claim 5, characterized in that: Step (1) includes the following process: Will Pseudomonas putida The cah sequence of the source is optimized, and the enzyme active gene site is identified, and the active site is mutated to obtain the optimized gene; the optimized gene sequence is amplified, and BamHI and XhoⅠ restriction sites are introduced to obtain the amplified cah fragment; the initial plasmid and the amplified cah fragment are double-digested with BamHI and XhoⅠ enzymes, and after digestion and purification, a connection vector product and a site-mutated cah fragment product can be obtained respectively; the vector product and the site-mutated cah fragment product are connected to construct a recombinant plasmid.

7. The method for preparing the recombinant bacteria according to claim 6, characterized in that: The initial plasmid is pET-28a(+); the Escherichia coli is Escherichia coli BL21(DE3).

8. The method for preparing the recombinant bacteria according to claim 5, characterized in that: In step (2), transformation is performed using a heat shock method.

9. Use of the recombinant bacteria according to claim 4 in the preparation of recombinant creatinase.

10. The use according to claim 9, characterized in that: The recombinant bacteria are cultured in a fermentation medium, and the inducer isopropyl-β-D-thiogalactoside is added for induction culture. After the culture is completed, the bacteria are collected by centrifugation, and the recombinant creatinine enzyme is obtained by lysis and purification. The induction culture conditions are 25-35°C and 0.1-1mM IPTG induction for 6-18h. The purification method is a two-step purification, first using Ni-NTA for purification, and then using ion exchange chromatography for purification.

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