CN118272339A - Protein, preparation method and application thereof, biological material and application thereof - Google Patents
Protein, preparation method and application thereof, biological material and application thereof Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
Abstract
The application relates to the technical field of reverse transcriptase, and particularly discloses a protein, a preparation method and application thereof, a biological material and application thereof. The protein is obtained by mutating MMLV reverse transcriptase, and the mutation comprises the following steps: glycine at position 40 of the amino acid sequence of the MMLV reverse transcriptase is mutated to cysteine, and arginine at position 253 is mutated to cysteine; the amino acid sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 2; the protein has higher reverse transcription capacity and DNA polymerization capacity; the protein has high sensitivity.
Description
Technical Field
The application relates to the technical field of reverse transcriptase, in particular to a protein, a preparation method and application thereof, a biological material and application thereof.
Background
Reverse transcriptase is a tool enzyme commonly used in medical molecular diagnosis and scientific research, and cDNA is synthesized by taking RNA as a template under the catalysis of reverse transcriptase. Reverse transcriptase plays an important role in the field of modern biotechnology and is widely applied to various scenes such as transcriptome analysis, pathogen detection and the like. The reverse transcriptase of moloney murine leukemia virus (Moloney murine leukemia virus, MMLV) is the most commonly used reverse transcriptase currently on the market. MMLV reverse transcriptase was discovered Edward Scolnick in 1970 and demonstrated to have reverse transcription activity for synthesizing DNA using RNA as a template, RNaseH activity for degrading RNA in RNA-DNA hybrid strand, and polymerase activity for synthesizing DNA using DNA as a template, which can perform cDNA synthesis using single-stranded RNA, DNA, or RNA-DNA hybrid as a template.
MMLV reverse transcriptase has strong reverse transcription activity, and the expression and purification process is easy to industrialize. Although MMLV reverse transcriptase has better comprehensive performance compared with other reverse transcriptases, the wild type thereof has the problems of poor heat stability, low sensitivity, insufficient amplification efficiency, few synthetic products and the like, thereby limiting the application of the MMLV reverse transcriptase in different fields.
Therefore, it is important to develop MMLV reverse transcriptase with high amplification efficiency and high sensitivity.
Disclosure of Invention
In order to improve the reverse transcription efficiency of MMLV reverse transcriptase, the application provides a protein, a preparation method and application thereof, a biological material and application thereof.
In a first aspect, the present application provides a protein, which adopts the following technical scheme:
a protein obtained by mutating MMLV reverse transcriptase, said mutating comprising: glycine at position 40 of the amino acid sequence of the MMLV reverse transcriptase is mutated to cysteine, and arginine at position 253 is mutated to cysteine; the amino acid sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 2.
By adopting the technical scheme, the application mutates glycine (G) at the 40 th position of MMLV reverse transcriptase into cysteine (C) and mutates arginine (R) at the 253 th position into cysteine (C) so as to form disulfide bonds, thereby improving the reverse transcription capacity of the MMLV reverse transcriptase mutant. The constructed MMLV reverse transcriptase mutant has the advantages of high amplification efficiency, convenience in operation, capability of stably completing the genome amplification of single cell level and the like, and the cost is obviously reduced.
In a second aspect, the present application provides a method for preparing the protein, which adopts the following technical scheme:
A method for producing the above protein, comprising the step of expressing a nucleic acid molecule encoding the protein in an organism to obtain the protein; the gene for encoding the protein comprises an MMLV reverse transcriptase gene, and the gene sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 1.
Alternatively, the organism is a microorganism, a plant or a non-human animal.
Alternatively, when a nucleic acid molecule encoding the protein is expressed in an organism, the method comprises the steps of: introducing a nucleic acid molecule encoding said protein into a recipient microorganism to obtain a recombinant microorganism capable of expressing said protein, culturing said recombinant microorganism, and expressing said protein.
Alternatively, a nucleic acid molecule encoding the protein or a fusion protein comprising the protein is inserted into a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into a recipient microorganism; the plasmid vector is selected from any one of pZJ plasmid, pZJ plasmid and pZJ plasmid.
Alternatively, the recipient microorganism is an escherichia bacterium.
In a third aspect, the present application provides a biomaterial, which adopts the following technical scheme:
a biomaterial, which is any one of B1) to B4):
b1 A nucleic acid molecule encoding the protein of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1);
b4 A recombinant microorganism comprising a nucleic acid molecule according to B1).
In B1, the biological material may be a nucleic acid molecule encoding only the above protein, or may be a nucleic acid molecule encoding a fusion protein comprising the above protein.
Alternatively, the nucleic acid molecule encoding the protein is: the GGT at the 118 th to 120 th positions of the MMLV reverse transcriptase gene sequence is replaced by TGC, and the CGT at the 757 th to 759 th positions is replaced by TGC; the gene sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 1.
In a fourth aspect, the present application provides an application of the protein or the biological material, which adopts the following technical scheme:
Use of the protein described above or the biological material described above, in any one of 1) to 4):
1) As an RNA reverse transcriptase, catalyzing reverse transcription of RNA;
2) For RNA sequencing or whole genome sequencing;
3) Preparing a product for catalyzing reverse transcription of RNA;
4) Preparing a product for RNA sequencing or whole genome sequencing;
the application of the biological material is any one of 5) to 8):
5) For catalyzing RNA reverse transcription;
6) For RNA sequencing or whole genome sequencing;
7) Preparing a product for catalyzing reverse transcription of RNA;
8) Products for RNA sequencing or whole genome sequencing were prepared.
In a fifth aspect, the present application provides a DNA amplification method, which adopts the following technical scheme:
A DNA amplification method comprising the step of amplifying DNA using the above protein as a DNA polymerase.
In the above-described embodiment, the DNA amplification may be performed with only the above-described protein, or with a fusion protein containing the above-described protein.
In summary, the application has the following beneficial effects:
1. The application provides a protein, which is obtained by mutating MMLV reverse transcriptase, in particular to mutating the 40 th and 253 th positions of an amino acid sequence of the MMLV reverse transcriptase so as to form disulfide bonds, thereby improving the reverse transcription activity and the sensitivity of the MMLV reverse transcriptase mutant.
2. The protein obtained by the application has excellent reverse transcription activity and DNA polymerase activity, and when the protein is used for reverse transcription or DNA polymerization, the efficiency is obviously improved, and the cost is further reduced; can also be used for preparing various products related to reverse transcription and DNA polymerization, and the related products with excellent performance are obtained.
Drawings
FIG. 1 is a schematic diagram of the structure of the pZJ.sup.205 plasmid prepared;
FIG. 2 is a standard curve of fluorescent quantitative detection;
FIG. 3 is the reverse transcription capacity of MMLV reverse transcriptase and MMLV reverse transcriptase mutants;
FIG. 4 is the reverse transcription ability of MMLV reverse enzyme mutants of recombinant E.coli eZJ283 in different buffers;
FIG. 5 is a comparison of the copy number of MMLV reverse enzyme mutant D524AG40CR253C of recombinant E.coli eZJ with commercial Vazyme and commercial Thermo.
Detailed Description
The application is described in further detail below with reference to the drawings and examples, in which: the following examples, in which no specific conditions are noted, are conducted under conventional conditions or conditions recommended by the manufacturer, and the raw materials used in the following examples are commercially available from ordinary sources except for the specific descriptions.
Raw material information
1. Reagent: the reagents involved in the present application are all commercially available reagents of a reagent grade or more.
Wherein, tryptone, yeast extract, naCl, plasmid extraction kit, gel recovery kit and all restriction endonucleases are all from Shanghai Bioengineering company.
PRIMESTAR MAX DNA polymerase, solution I ligase and pET-21c (+) vector were purchased from da Lian Bao Bio Inc. KOD Plus Neo DNA polymerase from TOYOBO; pET-28a (+) vector was purchased from Beijing Bai Albo technologies Co., ltd; KOD Plus Neo polymerase was purchased from Shanghai Toyobo Biotechnology Co.
Q5 High-FIDELITY DNA polymerase was purchased from British Biotechnology Co.Ltd.
2. Strains:
As a host bacterium used in DNA manipulation, E.coli BL21 (ESCHERICHIA COLI BL, available from Stratagene, calif.) strain, luria-Bertani (abbreviated as LB) medium containing 100. Mu.g/mL ampicillin was used as a medium for culturing E.coli BL21.
LB medium is used for induction expression medium; the LB medium comprises the following components: 5g/L yeast extract, 10g/L tryptone, 10g/L NaCl.
Constructing a plasmid:
1. pZJ203 plasmid construction:
1) And (3) carrying out codon optimization on the MMLV wild-type gene to obtain an MMLV reverse transcriptase gene fragment, wherein the D524A site mutation (therefore also called as a codon optimized MMLV-D524A gene) in the MMLV reverse transcriptase gene fragment, and the sequence of the MMLV reverse transcriptase gene fragment is shown as SEQ ID No. 1.
2) Cleaving pET-28a (+) vector (available from Beijing Bai Albo technology Co., ltd.) with restriction enzymes BamHI and Xho I to obtain a cleavage product of pET-28a (+) vector; cutting the MMLV reverse transcriptase gene fragment by restriction enzymes BamHI and Xho I to obtain a cut product of the MMLV reverse transcriptase gene fragment; the cleavage product of the pET-28a (+) vector and the cleavage product of the MMLV reverse transcriptase gene fragment are connected to obtain pZJ.A pZJ.203 plasmid, which is also called as MMLV-D524A gene recombinant expression vector.
In the sequence SEQ ID No.1, the 1 st to 2049 th positions are the MMLV-D524A gene after codon optimization, the expression product is MMLV reverse transcriptase, and the amino acid sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 2.
2. PZJ204 plasmid construction:
The pZJ plasmid differs from the pZJ plasmid only in that: the codon-optimized MMLV-D524A gene contained in pZJ203 plasmid is replaced by MMLV-D524A-G40C gene, and other nucleotides are kept unchanged.
The MMLV-D524A-G40C gene sequence specifically comprises: the GGT (G codon) at positions 118-120 of the sequence of the codon-optimized MMLV-D524A gene (also the MMLV reverse transcriptase gene fragment) is replaced by TGC (C codon), and other nucleotides are unchanged.
PZJ204 the MMLV-D524A-G40C gene was expressed in the plasmid, and the expression product was MMLV polymerase mutant D524AG40C. The amino acid sequence of MMLV polymerase mutant D524AG40C is that G at the 40 th position of the amino acid sequence of MMLV-D524A reverse transcriptase is replaced by C, and other amino acid residues are unchanged.
3. PZJ205 plasmid construction:
pZJ205 plasmid differs from pZJ plasmid only in that: the MMLV-D524A-G40C gene contained in pZJ204 plasmid was replaced with the MMLV-D524A-G40C-R253C gene, and the other nucleotides remained unchanged. A schematic structure of pZJ205,205 plasmid is shown in FIG. 1.
The MMLV-D524A-G40C-R253C gene sequence is shown as SEQ ID No.3, and the specific mutation is as follows: the CGT (R codon) at positions 757-759 of the MMLV-D524A-G40C gene sequence was replaced with TGC (C codon) with the other nucleotides unchanged.
PZJ205 plasmid expresses MMLV-D524A-G40C-R253C gene, and the expression product is MMLV polymerase mutant D524AG40CR253C (MMLV-D524A-G40C-R253C gene sequence is also MMLV polymerase mutant D524AG40CR253C gene sequence). The amino acid sequence of MMLV polymerase mutant D524AG40CR253C is shown in SEQ ID No.4, specifically: the MMLV polymerase mutant D524AG40C has 253 rd R replaced by C, and other amino acid residues are unchanged.
Examples
Example 1 protein
A protein which is MMLV reverse transcriptase obtained by mutating MMLV reverse transcriptase; wherein the mutation is as follows: glycine at position 40 in the amino acid sequence of MMLV reverse transcriptase is mutated to cysteine and arginine at position 253 is mutated to cysteine. Wherein the amino acid sequence of MMLV reverse transcriptase is shown as SEQ ID No. 2.
A preparation method of protein comprises the following steps:
1. Plasmid preparation:
1.1, entrusting the Shanghai Biotechnology company to synthesize MMLV reverse transcriptase gene fragment (namely MMLV-D524A gene after codon optimization), and inserting the gene into pET-28a (+) vector to construct pZJ plasmid. The pZJ plasmid was extracted and the extracted pZJ plasmid was used as a template, and PCR was performed using KOD Plus Neo DNA polymerase (TOYOBO), MMLV-G40C-F primer (gene sequence shown as SEQ ID No.5, CTTGGGCTGAAACCGGTGGTATGTGCCTGGCTGTTCGTCAGGCTCCGC) and MMLV-G40C-R primer (gene sequence shown as SEQ ID No.6, AGCGGGATGATCAGCGGAGCCTGACGAACAGCCAGGCACATACCACCGG), and the amplified product was digested with Dpn I to obtain a site-mutated plasmid, designated as pZJ plasmid.
The specific operation steps of mutant plasmid acquisition are as follows:
① PCR systems and procedures for amplifying whole plasmids using pZJ.sup.203 plasmids as templates are shown in tables 1 and 2.
TABLE 1
| Reagent(s) | Dosage of |
| 10 XKOD Plus Neo polymerase buffer | 20μL |
| 25mM MgSO4 | 12μL |
| 2MM dNTP mixture | 20μL |
| 10 Mu M MMLV-G40C-F primer | 3μL |
| 10 Mu M MMLV-G40C-R primer | 3μL |
| PZJ203 plasmid 203 | 0.4μL |
| KOD Plus Neo polymerase | 2μL |
| ddH2O | Make up to 200 mu L |
TABLE 2
| Step 1 | 94℃ | 2min |
| Step 2 | 98℃ | 10sec |
| Step 3 | 50℃ | 30sec |
| Step 4 | 68℃ | 7min |
| Step 5 | Returning to step 2 | 30 Cycles |
| Step 6 | 68℃ | 8min |
| Step 7 | 4℃ | Maintenance of |
② And (3) carrying out enzyme digestion on the amplified product by using Dpn I, overnight at 37 ℃, and recovering the product to obtain the plasmid containing the point mutation. The cleavage system for the amplified products is shown in Table 3.
TABLE 3 Table 3
| Amplification product | 1pmol |
| 10×buffer Tango | 10μL |
| Dpn I | 5μL |
| ddH2O | Make up to 100 mu L |
1.2, Extracting pZJ, taking the extracted pZJ plasmid as a template, carrying out PCR by using KOD Plus Neo DNA polymerase (TOYOBO), MMLV-R253C-F primer (gene sequence is shown as SEQ ID No.7, ACCCTGGGTAACCTGGGTTACTGCGCTTCTGCTAAAAAAGCTCAGATCTG) and MMLV-R253C-R primer (gene sequence is shown as SEQ ID No.8, TTTTTTAGCAGAAGCGCAGTAACCCAGGTTACCCAGGGTCTGCAGCAGAG), and obtaining 2 site-mutated plasmid after the amplified product is subjected to Dpn I digestion treatment, which is named pZJ plasmid.
The specific operation steps of mutant plasmid acquisition are as follows:
① PCR systems and procedures for amplifying whole plasmids using the corresponding plasmids as templates are shown in tables 4 and 5.
TABLE 4 Table 4
| Reagent(s) | Dosage of |
| 10 XKOD Plus Neo polymerase buffer | 20μL |
| 25mM MgSO4 | 12μL |
| 2MM dNTP mixture | 20μL |
| 10 Mu M MMLV-R253C-F primer | 3μL |
| 10 Mu M MMLV-R253C-R primer | 3μL |
| PZJ203 plasmid 203 | 0.4μL |
| KOD Plus Neo polymerase | 2μL |
| ddH2O | Make up to 200 mu L |
TABLE 5
| Step 1 | 94℃ | 2min |
| Step 2 | 98℃ | 10sec |
| Step 3 | 50℃ | 30sec |
| Step 4 | 68℃ | 7min |
| Step 5 | Returning to step 2 | 30 Cycles |
| Step 6 | 68℃ | 8min |
| Step 7 | 4℃ | Maintenance of |
② And (3) carrying out enzyme digestion on the amplified product by using Dpn I, overnight at 37 ℃, and recovering the product to obtain the plasmid containing the point mutation. The cleavage system for the amplified products is shown in Table 6.
TABLE 6
| Amplification product | 1pmol |
| 10×buffer Tango | 10μL |
| Dpn I | 5μL |
| ddH2O | Make up to 100 mu L |
2. Construction of recombinant E.coli
The plasmids are respectively transformed into host strain escherichia coli BL21 cells, and the specific construction process is as follows:
the pZJ plasmid is transformed into BL21 escherichia coli, so that the strain is recovered to have the Kana resistance function, and the function of expressing MMLV reverse transcriptase is newly increased. Positive clones were selected on Kana-resistant medium and verified by sequencing and the resulting positive strain was designated recombinant e.coli eZJ-282. Wherein, the formula of the culture medium containing Kana resistance is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl.
The pZJ plasmid containing the G40C site and the R253C site mutation is transformed into BL21 escherichia coli, so that the strain recovers the function of Kana resistance and the function of expressing MMLV reverse transcriptase is newly increased. Positive clones were selected on Kana-resistant medium and verified by sequencing, and the resulting positive strain was designated recombinant E.coli eZJ. Wherein, the formula of the culture medium containing Kana resistance is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl.
The specific construction method is as follows:
① Taking out a tube (100 μl) of competent bacteria from a freezer at-70deg.C, immediately heating with finger to melt, and inserting into ice for 5-10min.
② Add 5. Mu.L of the ligated plasmid mix (DNA content no more than 100 ng), gently shake and place on ice for 20min.
③ Slightly shaking, adding into water bath at 42deg.C for 45s, heat shock, quickly returning to ice, and standing for 3-5min.
④ Add 500. Mu.L LB liquid medium (without antibiotics) into each tube in the super clean bench, mix gently, then fix to the spring rack of the shaker and shake for 1h at 37deg.C. The formula of the liquid LB culture medium is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl.
⑤ Taking 100-300 mu L of the transformation mixed solution in an ultra-clean workbench, respectively dripping the transformation mixed solution into solid LB flat plate culture dishes containing proper antibiotics, and uniformly coating the solid LB flat plate culture dishes by using a glass coating rod burnt by an alcohol lamp.
⑥ Marking on the coated culture dish, placing in a 37 deg.C constant temperature incubator for 30-60min until the surface liquid is permeated into the culture medium, and placing in the 37 deg.C constant temperature incubator overnight after inversion.
⑦ Picking up the clone on the plate, culturing in liquid LB culture medium to obtain monoclonal, and carrying out sequencing verification on the monoclonal to obtain the strain containing the correct gene sequence (namely containing the corresponding target gene sequence), namely the positive strain. The formula of the liquid LB culture medium is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl.
3. Protein expression
The specific method for protein expression comprises the following steps:
1) Resuscitates the strain on LB plates and cultures at 37℃overnight. Control strain: recombinant E.coli eZJ to 282. Experiment strain: recombinant E.coli eZJ. The formula of the LB plate is as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl and 15g/L of agar.
2) The single clone on LB plate is picked up separately, and cultured overnight at 37 ℃ and 250rpm in 5mL liquid LB culture medium. The formula of the liquid LB culture medium is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl.
3) Preparing 42 bottles of 200mL LB culture medium and sub-packaging the LB culture medium into 1L conical triangular flasks. The formula of the LB culture medium is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl. Sterilizing for standby.
4) Inoculating the overnight culture in the step 2) into 200mL of LB culture medium, wherein the volume ratio of the overnight culture to the LB culture medium is 1:100, and then culturing at 37 ℃ and 250 rpm; each strain was repeated 3 times.
5) And step 4), when the culture is carried out until OD 600 = 0.6, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.5mM for induced expression, specifically, carrying out induced expression at 18 ℃ for 18h, collecting bacteria, and crushing the bacteria, thus obtaining the protein expression product. Wherein the control group is MMLV reverse transcriptase, and the experimental group is MMLV polymerase mutant D524AG40CR253C.
4. Protein purification
The protein purification steps are as follows:
All reagents used below were pre-chilled at 4 ℃ and the experimental procedure below was guaranteed to be performed on ice.
1) The strain expressing the protein (i.e. "3, step 4 in protein expression) taken out of the-80℃refrigerator was cultured until 18h induced") was treated with 20mL of lysis buffer (its specific composition is: the strain was washed once with 20mM Tris-HCl (pH 8.0), 300mM NaCl and 20mM imidazole (imidazole)), and then after centrifugation at 6000 Xg at 4℃for 5min, precipitated cells were obtained, and then the cells were suspended in 20mLlysis buffer.
2) Lysing the cells of step 1) by ultrasound in an ice bath using a 1.5s pulse at 30% intensity to obtain a lysate. Wherein, the ultrasonic intensity is 30%; ultrasound for 1.5s, stopping for 5s; run for 45min.
3) Centrifuging the lysate of the step 2) at 10000g and 4 ℃ for 40min after the crushing is finished, precipitating cell fragments at 4 ℃, and taking supernatant for protein purification.
4) The eluate (20 mM Tris-HCl (pH 8.0), 300mM NaCl and 250mM imidazole) was purified by adding 500. Mu.L of Ni-NTA matrix, 10mL of ddH 2 O,10mL of Lysis buffer, protein supernatant from step 3), 10mL Wash buffer I,10mL Wash buffer II,3mL to an affinity column. The Wash buffer I comprises the following components: 20mM Tris-HCl (pH 8.0), 300mM NaCl and 20mM imidazole; the Wash buffer II comprises the following components: 20mM Tris-HCl (pH 8.0), 300mM NaCl and 60mM imidazole; the components of the purified eluent are: 20mM Tris-HCl (pH 8.0), 300mM NaCl and 250mM imidazole.
5) Finally, the eluted protein of step 4) was collected with a 1.5mL centrifuge tube, and 10 tubes were collected in total, 200. Mu.L each.
6) Mixing 10 μl of the protein of step 5) with 2×protein loading buffer, heating at 99deg.C for 10min, centrifuging at 12000rpm at room temperature for 2min, and detecting with 10% SDS-PAGE gel.
7) And 6) preserving the residual protein sample at 4 ℃ for standby.
8) Boiling the dialysis bag buffer I and the dialysis bag buffer II. The buffer I comprises the following components: 2% NaHCO 3,1mM EDTA-Na2·2H2 O; the buffer II comprises the following components: 1mM EDTA-Na 2·2H2 O.
9) Putting the dialysis bag into a boiled dialysis bag buffer I, boiling for 10min, and thoroughly washing with ultrapure water; and putting into a dialysis bag buffer II, boiling for 10min, and thoroughly washing with ultrapure water.
10 And D), placing the dialysis bag treated in the step 9) at 4 ℃ for standby.
11 After running out the 10% SDS-PAGE gel of step 6), combining the tubes with the determined proteins, adding the tubes into the dialysis bag for preservation of step 10), sealing the opening by a clip, and taking care to open the chromatography cabinet at 4 ℃ in advance for cooling.
12 Placing the dialysis bag sealed in step 11) into a large beaker containing a rotor and about 500mL storage buffer a; the storage buffer comprises the following components: 20mM Tris-HCl (pH 8.0), 300mM NaCl,0.5% TritonX-100, 10% glycerol.
13 Place step 12) beaker on a magnetic stirrer in a chromatography cabinet at 4 ℃.
14 Standing the large beaker of step 13) overnight.
15 The next morning and noon to replace the storage buffer in the beaker of step 14) twice. A total of 3 times of dialysis, 4 hours each.
16 Recovering the dialyzed protein to obtain purified protein, and storing at 4deg.C.
Performance detection
1. Protein concentration determination
The specific method comprises the following steps:
1) The sample (i.e., the recovered protein of the above protein purification step 17) was diluted with distilled water at appropriate times, specifically 10-fold, 20-fold and 40-fold, respectively.
2) A1.5 mL centrifuge tube was added to each solution as shown in Table 7, wherein tube 0 was a blank.
TABLE 7 BSA Standard protein solution Components
| Pipe number | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Distilled water (mu L) | 1000 | 995 | 990 | 985 | 980 | 975 | 970 | 950 | 900 |
| 1mg/mL BSA(μL) | 0 | 5 | 10 | 15 | 20 | 25 | 30 | 50 | 100 |
| BSA concentration (μL/mL) | 0 | 5 | 10 | 15 | 20 | 25 | 30 | 50 | 100 |
3) 30 1.5ML centrifuge tubes were taken and divided into standard and sample groups.
Standard group: 18 1.5mL centrifuge tubes were set up for each standard with 2 replicates, and each tube was added with 100. Mu.L of the corresponding concentration BSA standard protein solution prepared in step 2), designated as tube numbers 0-9 according to Table 7.
Sample group: 12 1.5mL centrifuge tubes are divided into 3 repeated groups, the same repeated centrifuge tube in different groups has the same serial number, and 100 mu L of sample diluents with different concentrations diluted in the step 1) are respectively added into the two tubes with different serial numbers, and the sample dilutions are respectively 0 times, 10 times, 20 times and 40 times.
4) Step 3) each tube was added with 1mL of Bradford working fluid (Shanghai Bioengineering Co., C503031) and mixed rapidly.
5) After the centrifuge tube treated in the step 4) was reacted at room temperature of 25℃for 10 minutes, the A595 value of each tube was measured on a spectrophotometer with the tube No. 0 as a blank.
6) Standard curves were plotted in Microsoft Excel software with the mean value of each tube a595 of the standard group on the ordinate and the corresponding protein concentration on the abscissa.
7) According to the average value of the A595 values of two identical sample dilutions, the protein concentration of the diluted sample is calculated on a standard curve, the final sample protein concentration is calculated by selecting a sample with proper dilution, and the original sample protein concentration is calculated by dilution multiple.
2. Enzyme activity detection
The specific method comprises the following steps:
1) Reverse transcription reaction-acquisition of Experimental group cDNA
The reaction system is shown in Table 8, and the total amount is 20. Mu.L. Reacting the reaction system at 37 ℃ for 30min, and then reacting at 85 ℃ for 5sec; wherein water is used as a negative control instead of MS2 RNA, and the whole system has no template.
Information on reagents used in reverse transcription reactions: the components of 5× MMLV reaction Buffer are: 250mM Tris-HCl (pH=8.3), 340mM KCl and 15mM MgCl 2;
10mM dNTP mix (Bio), RNase inhibitor (Takara), MS2 RNA (Roche) and RNase-free ddH 2 O (Takara).
TABLE 8
2) Fluorescent quantitative PCR
Detecting the quantity of the substrate cDNA template in the step 1) by using a real-time fluorescence quantitative PCR method, and further obtaining the relative activity of reverse transcriptase. PUC-Human actin beta (Huikang biosynthesis, copy number 7.35X106 copies/. Mu.L) was used as a standard plasmid. Performing fluorescence quantitative PCR by 10 2、103、104、105 and 10 6 times dilution to standard curve; the reaction system is shown in Table 9. Wherein the fluorescent quantitative PCR kit is Roche FASTSTART UNIVERSAL SYBR GREEN MASTER (Rox).
TABLE 9
| 2×SYBR GreenMaster | 10μL |
| Primer mix (10 uM) | 0.8μL |
| CDNA (5000 times dilution) | 1μL |
| RNase-free ddH2O | 8.2μL |
The standard curve samples were run in parallel in three wells, and cDNA was changed to RNase-free ddH 2 O in the negative control, and amplified on a LightCycler480System type fluorescent quantitative PCR apparatus. The cycle parameters in the amplification procedure were: 15sec at 95 ℃,15 sec at 60 ℃,15 sec at 72 ℃ and 40 cycles.
The resulting standard curve is shown in figure 2.
3) The experimental group enzyme (i.e., the recovered protein of the above protein purification step 16) was reacted according to the above system, and the measured CT value was substituted into a standard curve to compare the enzyme activities, and the results are shown in FIG. 3.
As can be seen from the results of FIG. 3, the MMLV reverse transcriptase mutant D524AG40CR253C prepared by the method has significantly higher reverse transcription capacity than MMLV reverse transcriptase.
3. Optimization of storage buffer system
The experiment in this section verifies the enzyme activity effect of different storage buffers on MMLV reverse transcriptase mutant D524AG40CR253C, and the result shows that the enzyme activity of MMLV reverse transcriptase mutant D524AG40CR253C is highest when the storage buffers contain 400mM NaCl.
The specific experimental method comprises the following steps:
1) Resuscitates the strain recombinant E.coli eZJ283 on LB medium plates and cultures at 37℃for 1 day.
2) The individual clones were picked up separately and inoculated into 5mL of liquid LB medium at 37℃and 250rpm overnight.
3) Preparing 6 bottles of 50mL LB culture medium and sub-packaging the culture medium into 250mL conical triangular flasks. The formula of the LB liquid medium is as follows: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl. Sterilizing for standby.
4) And (3) inoculating the overnight culture in the step (2) into 200mL of LB medium, wherein the volume ratio of the overnight culture to the LB medium is 1:100, and culturing at 37 ℃ and 250 rpm. Each strain was repeated 3 times.
5) Step 4) culturing until OD 600 = 0.6, adding IPTG with a final concentration of 0.5mM to induce expression, specifically inducing expression at 18 ℃ for 18h, collecting bacteria, crushing the bacteria, purifying the protein, and purifying the protein. Proteins were split into five portions for dialysis, and dialyzed against storage buffers of different salt concentrations of 100mM, 200mM, 300mM, 400mM and 500mM, respectively.
6) Detecting the enzyme activity and yield of the purified protein of step 5). The method for detecting the enzyme activity is as described above. The method for detecting the yield is the same as the protein concentration measurement method described above.
As shown in FIG. 4, the cDNA copy number of recombinant E.coli eZJ at a salt concentration of 400mM NaCl was 8.4X10. 10 11 copies/. Mu.L, and the copy number was the highest, i.e., the enzyme activity was the highest.
1. Sensitivity comparison
The specific experimental method comprises the following steps:
1. Reverse transcription reaction-acquisition of Experimental group cDNA
The reaction system is as in table 10 (Vazyme commercial enzyme,III REVERSE TRANSCRIPTASE, source: nanjinopran organism, cat No.: R302-01-AB, unit specification: 10000U (200U/. Mu.L), table 11 (Thermo commercial enzyme, superScript III REVERSE TRANSCRIPTASE, origin: thermo FISHER SCIENTIFIC, cat# 18080093, unit specification: 2000U (200U/. Mu.L) and Table 8 (eZJ 283) in total, 20. Mu.L. This reaction system was reacted at 37℃for 30min and then at 85℃for 5sec, wherein water was used as a negative control instead of MS2 RNA, and the whole system was template-free.
Table 10Vazyme shows the reaction system of reverse transcriptase
| 5×HiScript III Buffer | 4μL |
| DTT(100mM) | 2μL |
| dNTP Mix(10mM each) | 1μL |
| Primer mix (1 uM) | 0.2μL |
| RNase inhibitor(40U/μL) | 0.5μL |
| MS2 RNA | 1μL |
| HiScript III Reverse Transcriptase(200U/μL) | 1μL |
| RNase-free ddH2O | 10.3μL |
Table 11 Thermo shows the reaction system of reverse transcriptase
2) Fluorescent quantitative PCR (Experimental methods same 2 fluorescent quantitative PCR)
This section of the experiment verifies the sensitivity of the MMLV reverse transcriptase mutant D524AG40CR253C of recombinant E.coli eZJ, and the result shows that the sensitivity of the MMLV reverse transcriptase mutant D524AG40CR253C produced by recombinant E.coli eZJ283 is superior to that of Vazyme commercial enzyme and Thermo commercial enzyme.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (10)
1. A protein obtained by mutating MMLV reverse transcriptase, said mutating comprising: glycine at position 40 of the amino acid sequence of the MMLV reverse transcriptase is mutated to cysteine, and arginine at position 253 is mutated to cysteine; the amino acid sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 2.
2. A method for producing the protein according to claim 1, comprising the step of expressing a nucleic acid molecule encoding the protein in an organism to obtain the protein; the gene for encoding the protein comprises an MMLV reverse transcriptase gene, and the gene sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 1.
3. The method for producing a protein according to claim 2, wherein the organism is a microorganism, a plant or a non-human animal.
4. The method for producing a protein according to claim 2, wherein the nucleic acid molecule encoding the protein is expressed in an organism, comprising the steps of:
introducing a nucleic acid molecule encoding said protein into a recipient microorganism to obtain a recombinant microorganism capable of expressing said protein, culturing said recombinant microorganism, and expressing said protein.
5. The method for producing a protein according to claim 4, wherein a nucleic acid molecule encoding the protein or a fusion protein containing the protein is inserted into a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into a recipient microorganism; the plasmid vector is selected from any one of pZJ plasmid, pZJ plasmid and pZJ plasmid.
6. The method for producing a protein according to claim 4, wherein the receptor microorganism is an Escherichia bacterium.
7. A biomaterial characterized in that the biomaterial is any one of B1) to B4):
b1 A nucleic acid molecule encoding the protein of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1);
B4 A recombinant microorganism comprising a nucleic acid molecule according to B1).
8. The biological material according to claim 7, wherein the nucleic acid molecule encoding the protein is: the GGT at the 118 th to 120 th positions of the MMLV reverse transcriptase gene sequence is replaced by TGC, and the CGT at the 757 th to 759 th positions is replaced by TGC; the gene sequence of the MMLV reverse transcriptase is shown as SEQ ID No. 1.
9. Use of a protein according to claim 1 or a biomaterial according to any one of claims 7 to 8, wherein the use of the protein is any one of 1) to 4):
1) As an RNA reverse transcriptase, catalyzing reverse transcription of RNA;
2) For RNA sequencing or whole genome sequencing;
3) Preparing a product for catalyzing reverse transcription of RNA;
4) Preparing a product for RNA sequencing or whole genome sequencing;
the application of the biological material is any one of 5) to 8):
5) For catalyzing RNA reverse transcription;
6) For RNA sequencing or whole genome sequencing;
7) Preparing a product for catalyzing reverse transcription of RNA;
8) Products for RNA sequencing or whole genome sequencing were prepared.
10. A DNA amplification method comprising the step of amplifying DNA using the protein of claim 1 as a DNA polymerase.
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