Preparation method and application of reverse transcriptase from thermophilic bacteria
By preparing the thermostable reverse transcriptase from thermophilic bacteria Thermotoga caldifontis, the problem of poor thermal stability of existing reverse transcriptases was solved, and efficient RNA detection and cDNA synthesis were achieved under high temperature conditions.
Patent Information
- Application Number
- CN202510874203.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-09-23
AI Technical Summary
Existing reverse transcriptases have poor thermal stability, resulting in low efficiency in RNA detection and cDNA synthesis, making it difficult to carry out effectively under high temperature conditions.
By constructing a recombinant expression vector of the thermostable reverse transcriptase from the thermophilic bacterium Thermotoga caldifontis and expressing and purifying it in Escherichia coli, a high-temperature stable reverse transcriptase was prepared, which can be used to extend DNA and synthesize cDNA from RNA template chains that are still active at 50 degrees.
The prepared reverse transcriptase is still active at 50 degrees with a half-life of 1 hour. It can effectively synthesize cDNA at high temperatures, eliminate the inhibitory effects of RNA secondary structure, and improve the length and speed of RNA detection and cDNA synthesis.
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Abstract
Description
Technical Field
[0001] The present invention mainly relates to the technical field of bioengineering, and specifically relates to a preparation method and application of a reverse transcriptase derived from thermophilic bacteria. Background Art
[0002] Reverse transcriptase is essentially a DNA polymerase. However, unlike conventional DNA polymerases, which can only use a single DNA strand as a template, reverse transcriptase uses a single RNA strand as a template to synthesize a complementary DNA strand (also known as a cDNA strand). Reverse transcriptase has important applications in RNA detection and gene cloning. Reverse transcriptase can use both RNA and DNA as templates to synthesize complementary DNA strands. The DNA polymerase activity when using RNA as a template is called reverse transcriptase. Currently, the commonly used reverse transcriptase is MMLV reverse transcriptase, which has strong reverse transcriptase activity but poor thermal stability. Some reverse transcriptases currently developed with improved thermal stability have low activity, and there is a need to develop reverse transcriptases with improved thermal stability and high activity.
[0003] DNA and RNA detection are widely used in life sciences today. For example, Taq enzyme-based DNA amplification reactions are widely used in a variety of detections for viruses, microorganisms, and other organisms. However, RNA detection differs from DNA detection. Most DNA polymerases can only synthesize DNA using DNA as a template; RNA detection requires the RNA to be converted to DNA before it can be detected using enzymes such as Taq DNA polymerase. Summary of the Invention
[0004] Based on this, the purpose of the present invention is to provide a preparation method and application of reverse transcriptase derived from thermophilic bacteria to solve the technical problems raised in the above background technology.
[0005] To achieve the above object, the present invention provides the following technical solutions: A reverse transcriptase derived from thermophilic bacteria, the amino acid sequence of which is shown as sequence 1 in the sequence listing, and the nucleotide sequence of which is shown as sequence 2 in the sequence listing.
[0006] A method for preparing a thermostable reverse transcriptase comprises the following steps: S1. Construction of a recombinant expression vector for thermostable reverse transcriptase; S2. The recombinant expression vector is transferred into the host bacteria E. coli to obtain recombinant E. coli bacteria, and then cultured and induced to express; S3. The E. coli cells after induction culture were collected, crushed, and centrifuged to obtain a supernatant; S4. Affinity purification is performed on the supernatant to obtain the thermostable reverse transcriptase.
[0007] In summary, the present invention mainly has the following beneficial effects: The thermostable reverse transcriptase of the present invention is thermostable and remains active at 50°C, with a half-life of one hour at 50°C. It can extend DNA using RNA as a template at high temperatures to synthesize complementary DNA chains (cDNA). The thermostable reverse transcriptase is used to perform reverse transcription reactions at temperatures between 37°C and 50°C to synthesize cDNA, which can be used for RNA detection and cloning of cDNA-encoding genes. The high-temperature reverse transcription reaction eliminates the inhibitory effects of RNA secondary structure on cDNA synthesis, thereby increasing the length and speed of cDNA synthesis. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 This is the purification result of the thermostable reverse transcriptase of the present invention; Figure 2 is the reverse transcription reaction result of the thermostable reverse transcriptase of the present invention; Figure 3 The results of optimizing the optimal reaction conditions for the thermostable reverse transcriptase of the present invention are as follows; Figure 4 This is the thermal stability result of the thermostable reverse transcriptase. DETAILED DESCRIPTION
[0009] The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention.
[0010] Preparation and Application of Thermostable Reverse Transcriptase S1. Construction of prokaryotic recombinant expression vector Based on the protein sequence of Thermotoga caldifontis thermostable reverse transcriptase TcaRTase (sequence 1 in the sequence listing), the TcaRTase gene sequence was optimized according to the codon usage frequency of Escherichia coli. The optimized gene sequence is shown in sequence 2 in the sequence listing.
[0011] First, the optimized TcaRTase gene sequence was chemically synthesized and then cloned into the prokaryotic expression vector pET28a to construct a TcaRTase protein recombinant expression vector.
[0012] The pET28 vector and TcaRTase gene fragment were double-digested with NdeI and BamHI, and the digested product was recovered using a DNA product purification kit. The thermostable reverse transcriptase gene was recombined with the NdeI / BamHI double-digested pET28 using T4 DNA ligase. The recombinant product was transformed into DH5α competent cells, and colonies that tested positive for kanamycin resistance were selected for PCR verification. DNA sequencing of recombinant clones that tested positive for PCR was performed to verify the correctness of the thermostable reverse transcriptase gene sequence. Finally, a recombinant expression vector for the thermostable reverse transcriptase was successfully constructed.
[0013] S2. Prokaryotic expression of recombinant thermostable reverse transcriptase The prokaryotic recombinant expression vector for the thermostable reverse transcriptase TcaRTase was transformed into competent Escherichia coli BL21 (DE3) cells. The transformed culture was evenly spread onto solid LB plates containing 50 mg / ml kanamycin and incubated at 37°C for 16 hours. A single colony was transferred to 20 ml of LB liquid medium supplemented with 50 mg / ml kanamycin and incubated overnight at 37°C at 200 rpm. The 20 ml culture was then expanded to a 500 ml plate and incubated at 37°C at 200 rpm. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.1-1.0 mM and incubated at 20°C at 200 rpm for 18 hours to induce expression of the thermostable reverse transcriptase.
[0014] S3. Affinity Purification of Thermostable Reverse Transcriptase After induction, the E. coli cells were centrifuged at 8,000 rpm for 3 minutes to collect the cells. The cells were resuspended in 40 ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol) and disrupted by sonication. Ultrasonication was performed at 500 W for 4 seconds, followed by a 4-second pause, for a total of 30 minutes. The cells were then centrifuged at 10,000 rpm for 20 minutes at 4°C, and the supernatant was collected to obtain the crude recombinant protein.
[0015] The supernatant was added to a chromatography column containing 2 ml of Ni-NTA purification resin. The buffer was passed through the column to allow the six consecutive histidine affinity tags at the N-terminus of the thermostable reverse transcriptase to specifically bind to the nickel ions immobilized on the Ni-NTA resin. The resin was then washed with lysis buffer containing 20 mM imidazole to remove nonspecifically bound contaminants. The Ni-NTA resin was then eluted with 20 ml of elution buffer containing 250 mM imidazole, and the eluate, which contains the thermostable reverse transcriptase, was collected.
[0016] Replace the thermostable reverse transcriptase with a storage solution containing 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 150 mM NaCl, and 50% (v / v) glycerol and store at -20°C.
[0017] SDS-PAGE electrophoresis analysis and identification, the identification results are as follows Figure 1 shown.
[0018] S4. Reverse transcriptase activity assay The thermostable reverse transcriptase affinity-purified (Ni-NTA resin) in step S3 was used to assay reverse transcriptase and conventional DNA polymerase activity. The template used in the reverse transcription reaction was a 36nt single-stranded RNA (numbered DDC17), the template used for conventional DNA polymerase activity was a 45nt single-stranded DNA (numbered DDC7), and the primer was a 17nt primer (numbered DDC2) that paired with the 36nt single-stranded RNA and single-stranded DNA templates. The reaction buffer consisted of 50 mM Tris–HCl (pH 8.3), 150 mM NaCl, 1 mM MgCl2, 10mM DTT, 1 unit of nuclease inhibitor, the reaction temperature was 37 degrees, and the reaction time was 5 minutes. The polymerization product of the reverse transcriptase was a 36bp DNA-RNA hybrid chain, and the polymerization product of the DNA polymerase was a 45bp DNA double-stranded chain. The specific results are shown in [ 1 ]. Figure 2 .
[0019] S5. Optimization of the optimal reaction components for reverse transcriptase The thermostable reverse transcriptase affinity-purified (Ni-NTA resin) in step S3 was used to determine the conditions required for optimal reverse transcriptase activity. The template used in the reverse transcription reaction was a 36nt single-stranded RNA (numbered DDC17), and the primer was a 17nt primer (numbered DDC2) that paired with the 36nt single-stranded RNA and single-stranded DNA templates. The basic reaction buffer was 50 mM Tris–HCl (pH 6.0-10.0), 0-400 mM NaCl, 1 mM divalent metal ions (MgCl2, MnCl2, NiCl2, ZnCl2, CuCl2, CoCl2, CaCl2), 0-16 mM DTT, 1 unit of nuclease inhibitor, the reaction temperature was 37 degrees, and the reaction time was 5 minutes. After optimization, the optimal reaction components for TcaRTase were obtained as 50 mM Tris–HCl (pH 9.0), 100 mM NaCl, 1 mM MgCl2, and 1 mM DTT. The specific results are shown in the table. Figure 3 .
[0020] S6. Determination of optimal reaction temperature / thermostability of reverse transcriptase The thermostable reverse transcriptase affinity-purified (Ni-NTA resin) in step S3 was used to determine the reaction temperature and thermostability using the optimal reaction components determined in step S5. The reverse transcription reaction used a 36-nt single-stranded RNA template (designated DDC17), and the conventional DNA polymerase activity used a 45-nt single-stranded DNA template (designated DDC7). The primers used were 17-nt primers (designated DDC2) that matched the 36-nt single-stranded RNA and DNA templates. The reaction buffer consisted of 50 mM Tris–HCl (pH 9.0), 100 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 unit of nuclease inhibitor. The reaction temperatures were 30, 35, 40, 45, 50, 55, 60, 65, and 70°C, and the reaction time was 5 minutes. The thermal stability test was to heat the TcaRTase reverse transcriptase at 40, 50, 60, and 70 degrees for 15, 30, 45, 60, 75, and 90 minutes, and then measure the residual reverse transcriptase activity. The results showed that the optimal reaction temperature of TcaRTase was 50-55 degrees ( Figure 4 A), good thermal stability at 40-50 degrees, and begins to deactivate rapidly at 60 degrees or above ( Figure 4 , BE), the specific results are shown in Figure 4 .
[0021] Compared to existing technologies, the thermostable reverse transcriptase of the present invention possesses both high reverse transcriptase activity and high thermal stability. It can be used to synthesize complementary DNA (cDNA) using RNA as a template strand, leveraging its reverse transcriptase activity. The reverse transcriptase exhibits high thermal stability, exhibiting high activity at temperatures between 30 and 50°C, with a half-life of up to one hour at 50°C. The thermostable reverse transcriptase can be used in reverse transcription reactions to synthesize cDNA, which can be used for RNA detection and cloning of cDNA-encoding genes.
[0022] The above embodiments are only for illustrating the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the present invention.
Claims
1. A reverse transcriptase derived from a thermophilic bacterium, the amino acid sequence of which is shown in Sequence 1 in the sequence listing, and the nucleotide sequence of which is shown in Sequence 2 in the sequence listing.
2. A method for preparing a thermostable reverse transcriptase, which is applied to the reverse transcriptase of thermophilic bacteria according to claim 1, characterized in that: The following steps are involved: S1. Construction of a recombinant expression vector for thermostable reverse transcriptase; S2. The recombinant expression vector is transferred into the host bacteria E. coli to obtain recombinant E. coli bacteria, and then cultured and induced to express; S3. The E. coli cells after induction culture were collected, crushed, and centrifuged to obtain a supernatant; S4. Affinity purification is performed on the supernatant to obtain the thermostable reverse transcriptase.