Polypeptide ligand for improving thermal stability of MMLV reverse transcriptase and application thereof

Abstract

The invention discloses a polypeptide ligand for improving the thermal stability of MMLV reverse transcriptase and application of the polypeptide ligand, and relates to the technical field of biology. The invention specifically discloses a polypeptide ligand with an amino acid sequence as shown in SEQ ID NO: 1. The polypeptide ligand can obviously improve the thermal stability of MMLV RT. After being combined with the polypeptide ligand, the MMLV RT can effectively maintain the activity of the MMLV RT at 45-60 DEG C and maintain good activity in a freeze-thaw cycle, and the Ct value change is small after the MMLV RT is placed at 37 DEG C for more than 15 days, so that the polypeptide has a very good application prospect in industrial production and commercial application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a polypeptide ligand for improving the thermal stability of MMLV reverse transcriptase and its application. BACKGROUND

[0002] As a key enzyme preparation in the field of molecular diagnosis and genetic engineering, the importance of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is increasingly highlighted in biomedical research and clinical applications. This enzyme has dual catalytic properties, both synthesizing cDNA from RNA templates and extending DNA templates, and is a basic tool for core technologies such as RT-qPCR, high-throughput sequencing, and viral nucleic acid detection.

[0003] Studies have shown that the thermal stability defect of MMLV RT has become a key factor restricting its application performance. Although the enzyme can maintain more than 90% activity in the range of 37-42°C, its catalytic efficiency shows a sharp downward trend when the temperature rises to 45-50°C, and the half-life (t1 / 2) decreases from 60 minutes to less than 15 minutes. Therefore, it is important to provide a polypeptide ligand that has a thermal stability enhancing effect on MMLV RT. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a polypeptide ligand that can effectively improve the stability of MMLV RT.

[0005] The present application also provides a preparation method of the above-mentioned polypeptide ligand.

[0006] The present application also provides an enzyme complex comprising the above-mentioned polypeptide ligand.

[0007] The present application also provides a preparation method of the above-mentioned enzyme complex.

[0008] The present application also provides a biological material related to the above-mentioned polypeptide ligand.

[0009] The present application also provides applications of the above-mentioned polypeptide ligand, enzyme complex or biological material.

[0010] According to the first aspect of the present application, a polypeptide ligand is provided, which is any one of a1) to a3): a1) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; a2) a polypeptide obtained by substitution and / or deletion and / or addition of amino acid residues of the polypeptide of a1), which has more than 90% identity with the polypeptide of 1) and has the same activity; a3) a fusion polypeptide obtained by connecting a protein tag to the N-terminal or / and C-terminal of a1) or a2).

[0011] The polypeptide ligand according to the embodiments of the present application has at least the following beneficial effects: The polypeptide ligand of the embodiments can be electrostatically complementary to target and bind at the positively charged nucleic acid binding pocket of MMLV RT, significantly improving the thermal stability of MMLV RT. After binding to the polypeptide ligand, MMLV RT can effectively maintain MMLV RT activity at 45-60°C, maintain good activity in freeze-thaw cycles, and have small Ct value changes when placed at 37°C for more than 15 days, having a good application prospect in industrial production and commercial application.

[0012] According to some embodiments of the present application, the polypeptide ligand can comprise one or more protein tags. The protein tags can comprise a combination of the same protein tags, or a combination of different protein tags.

[0013] According to some embodiments of the present application, the protein tag comprises at least one of a protein tag facilitating solubilization, purification and detection of the polypeptide ligand. For example, the protein tag facilitating solubilization of the polypeptide ligand includes but is not limited to a nus tag or a maltose binding protein tag; the protein tag facilitating purification of the polypeptide ligand includes but is not limited to a strep tag, a His tag, a GST tag, a pelB signal tag or an ompA signal tag; and the protein tag facilitating detection of the polypeptide ligand includes but is not limited to a horseradish peroxidase (HRP) tag, a beta-galactosidase tag, a luciferase tag, a green fluorescent protein (GFP) tag, an HcRed tag, a DsRed tag or a cyan fluorescent protein (CFP) tag.

[0014] The method for preparing the polypeptide ligand according to the first aspect of the embodiments of the second aspect of the present application comprises the following steps: Introducing the coding gene of the polypeptide ligand into a biological cell to express the coding gene.

[0015] The enzyme complex according to the third aspect of the embodiments of the present application comprises the polypeptide ligand according to the first aspect of the embodiments and MMLV reverse transcriptase.

[0016] According to some embodiments of the present application, the molar ratio of the polypeptide ligand to MMLV reverse transcriptase is (1-6): 1. For example, it can be 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1 or 6: 1.

[0017] According to some embodiments of the present application, the polypeptide ligand is electrostatically complementary to the MMLV reverse transcriptase.

[0018] The method of preparing the enzyme complex according to the third aspect of the fourth aspect of the present application comprises a step of preparing a mixture of the polypeptide ligand and the MMLV reverse transcriptase.

[0019] According to some embodiments of the present application, the method of preparing further comprises incubating the mixture.

[0020] According to some embodiments of the present application, the molar ratio of the polypeptide ligand to the MMLV reverse transcriptase is (1-6): 1.

[0021] According to some embodiments of the present application, the incubation is for a time period of 10 min-90 min. For example, it can be for 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min or 90 min.

[0022] According to some embodiments of the present application, the incubation is at a temperature of 4°C-37°C. For example, it can be at a temperature of 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C or 37°C.

[0023] The biological material related to the polypeptide ligand according to the first aspect of the fifth aspect of the present application comprises at least one of: b1) a nucleic acid molecule encoding the polypeptide ligand according to the first aspect of the present application; b2) an expression cassette comprising the nucleic acid molecule of b1); b3) a vector comprising the nucleic acid molecule of b1) or the expression cassette of b2); b4) a transgenic cell line comprising the nucleic acid molecule of b1), the expression cassette of b2) or the vector of b3); b5) a recombinant microorganism comprising the nucleic acid molecule of b1), the expression cassette of b2) or the vector of b3).

[0024] According to some embodiments of the present application, the nucleic acid molecule of b1) has any one of b1-1) to b1-2): b1-1) a DNA molecule having the nucleotide sequence as set forth in SEQ ID NO: 2; b1-2) has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% or more identity to the nucleotide sequence shown in SEQ ID NO: 2, and encodes the polypeptide ligand.

[0025] In some embodiments of the present application, the expression cassette refers to a DNA capable of expressing the polypeptide ligand in a host cell. The DNA can include not only a promoter initiating the transcription of the polypeptide ligand-encoding gene, but also a terminator terminating the transcription of the polypeptide ligand-encoding gene. Further, the expression cassette can also include an enhancer sequence.

[0026] In some embodiments of the present application, the vector can be a plasmid, a cosmid, a bacteriophage, or a viral vector. For example, it can be a PET-32a vector.

[0027] In some embodiments of the present application, the vector can be a vector obtained by inserting a coding gene encoding the polypeptide ligand into a multiple cloning site.

[0028] In some embodiments of the present application, the transgenic cell line does not contain reproductive material.

[0029] The polypeptide ligand described in the first aspect of the sixth aspect of the embodiments of the present application or the biological material described in the fifth aspect of the embodiments of the present application is used for improving the stability of MMLV reverse transcriptase or preparing a product containing MMLV reverse transcriptase.

[0030] According to some embodiments of the present application, the product includes at least one of a reagent, a detection plate, a detection chip, and a kit.

[0031] The enzyme complex described in the third aspect of the seventh aspect of the embodiments of the present application is used in any one of c1) to c4): c1) detecting an RNA sample; c2) preparing a product for detecting an RNA sample; c3) reverse transcribing an RNA to synthesize a cDNA; c4) preparing a product for reverse transcribing an RNA to synthesize a cDNA.

[0032] According to some embodiments of the present application, the product includes at least one of a reagent, a detection plate, a detection chip, and a kit.

[0033] According to some embodiments of the present application, the product comprises at least one of a reverse transcription reaction product, an RT-PCR amplification product, an RT-qPCR product, a cDNA library construction product, a cDNA end rapid amplification product, and an RNA sequencing product.

[0034] In some embodiments of the present application, the product is a reverse transcription reaction product; the reverse transcription reaction product further comprises at least one of a reverse transcription reaction buffer, dNTPs, an RNase inhibitor, and a reverse transcription reaction primer.

[0035] In some embodiments of the present application, the product is an RT-PCR amplification product; the RT-PCR amplification product further comprises at least one of PCR water, an RT-PCR amplification buffer, dNTPs, a DNA polymerase, and a PCR amplification primer.

[0036] In some embodiments of the present application, the product is an RT-qPCR product; the RT-qPCR product further comprises at least one of PCR water, an RT-qPCR amplification buffer, dNTPs, a DNA polymerase, a qPCR amplification primer, and a probe.

[0037] In some embodiments of the present application, the product is a cDNA library construction product; the cDNA library construction product further comprises an adaptor.

[0038] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Schematic diagram of the structural site of the ligand of the present application binding to MMLV RT; wherein the green part is MMLV reverse transcriptase, and the blue part is the polypeptide ligand of the present application; Figure 2 Purification result of the ligand of the present application; wherein lane M: protein marker, lane 1: polypeptide ligand; Figure 3 Differential scanning fluorometry (DSF) detection result of Example 2; Figure 4 Agarose gel electrophoresis result of Example 3; wherein lane M: protein marker, lane 1: MMLV RT; lane 2: MMLV RT + binder 1; Figure 5 Ct difference value curve diagram of MMLV reverse transcriptase placed for different days under different treatments. DETAILED DESCRIPTION

[0040] The concept and technical effects of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0041] The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by market purchase.

[0042] Unless otherwise specified, the MMLV RT of the embodiments of the present application is purchased from Hunan Aikeerui Biological Engineering Co., Ltd., and the item number is CM0267.

[0043] Example 1 1. Design of ligand: According to artificial intelligence machine learning and sex design, combined with the MMLV RT binding pocket position, the polypeptide ligand (binder1) with the amino acid sequence as shown in SEQ ID NO: 1 is designed, which can be combined with MMLV RT as a ligand. The corresponding structure site map is shown in Figure 1 .

[0044] MAAEKEKLLEELDIIAGLALLGKAPVASISRLYDIIEELKKKGYNKEALDVALEEIDAVLRQDDVTEEQLIAVVVAAVTAVKEIIEEA (SEQ ID NO: 1).

[0045] 2. Synthesis of ligand: (1) The coding gene (nucleotide sequence as shown in SEQ ID NO: 2) of the ligand is synthesized by whole gene synthesis, and the coding gene is homologously recombined into the vector PET-32a to obtain a recombinant expression vector.

[0046] ATGGCGGCAGAAAAAGAAAAACTGCTGGAAGAGCTGGATATTATCGCCGGTCTGGCGCTGCTGGGTAAAGCGCCGGTTGCCTCAATCAGCCGTCTGTATGATATTATTGAAGAGCTGAAAAAAAAAGGTTATAACAAAGAAGCGCTGGATGTTGCGCTGGAAGAAATTGATGCCGTACTGCGTCAGGATGATGTTACCGAAGAGCAGCTGATTGCTGTGGTGGTTGCTGCGGTAACCGCGGTAAAAGAAATTATTGAAGAAGCGTAA (SEQ ID NO: 2).

[0047] (2) Transform the sequenced recombinant expression vector into host cells E. coli In BL21(DE3), after culture, a single colony was picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin, and cultured to OD 600 0.6-0.8, IPTG was added to a final concentration of 0.4 mmol / L-0.6 mmol / L, and the culture was induced at 37°C for 6 h. The culture was centrifuged at 4200 rpm at 4°C, the bacterial cells were collected, and lysis buffer was added and sonicated. The expression of the target protein was detected by SDS-PAGE electrophoresis, and the recombinant expression vector contained in the strain was extracted for sequencing verification, and a strain that could successfully express the target protein and was correctly sequenced was selected.

[0048] (3) The recombinant bacteria obtained in step (2) capable of expressing polypeptide ligands were inoculated into 100 mL of LB medium containing 100 μg / mL ampicillin and placed in a 37°C shaker for overnight culture to obtain a seed solution.

[0049] (4) The seed solution obtained in step (3) was inoculated into 2 L of LB medium containing 100 μg / mL ampicillin at a volume ratio of 1:100, and cultured at 37°C on a shaker until the OD 600 0.6-0.8; IPTG was added to a final concentration of 0.4 mmol / L-0.6 mmol / L, and the shaking induction was continued at 37°C for 6 h; the induced bacterial cells were collected by centrifugation, weighed, and the wet weight was recorded, and stored at -20°C.

[0050] (5) According to the recorded wet weight of the bacterial cells, the lysing buffer (50 mM Tris-HCl, 300 mM NaCl, 0.15% Triton X-100, pH 7.8) was added to the frozen bacterial cells at 10 mL / g of bacterial cells to resuspend the bacterial cells, and the bacterial cells were lysed by a high-pressure disrupter. The lysed bacterial cells were centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was taken; the supernatant was further centrifuged at 12,000 rpm for 30 min at 4°C, and then filtered through a 0.22 μm microporous filter to obtain a filtrate.

[0051] (6) Ni affinity chromatography column purification: The filtrate obtained in step (5) was loaded into a chromatography column that had been equilibrated with buffer A (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 5(v / v)% glycerol, pH 7.8). After loading was completed, the column was first washed with buffer A, and then eluted with a gradient of 0%-100% of buffer B (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, 5(v / v)% glycerol, pH 7.8), and the eluted components were detected by SDS-PAGE protein electrophoresis, and the eluate was collected according to the detection results and dialyzed into buffer C (20 mM Tris-HCl, 200 mM NaCl, 5(v / v)% glycerol, 1 mM DTT, 1 mM EDTA, pH 7.8) for standby. In the components collected from the nickel ion affinity chromatography containing the target protein elution buffer, ULP1 protease (concentration of 1 wt% of the target protein) was added, and nickel ion affinity chromatography purification separation was performed again.

[0052] (7) Anion exchange chromatography purification: The dialyzed sample obtained by Ni affinity chromatography column purification was loaded into a chromatography column that had been equilibrated with buffer C. After loading was completed, the column was first washed with buffer C, and then eluted with a gradient of 0%-100% of buffer D (20 mM Tris-HCl, 600 mM NaCl, 5(v / v)% glycerol, 1 mM DTT, 1 mM EDTA, pH 7.8), and the eluted components were detected by Tricine-SDS-PAGE protein electrophoresis, and the eluate was collected according to the detection results, dialyzed using a dialysis bag with a molecular weight of 10 KDa, and then stored in a storage solution (20 mM Tris-HCl, 600 mM NaCl, 1 mM DTT, 50(v / v)% glycerol, 1 mM EDTA, pH 7.8) for standby, and the concentration of the purified mutant was determined by the Coomassie brilliant blue method.

[0053] The purification results are shown in Table 1. Figure 2

[0054] ​Example 2 (1) The ligand prepared in Example 1 was mixed with MMLV reverse transcriptase (MMLV RT) at a molar ratio of 6:1, incubated at 16°C for 30 min, diluted to 500 ng / μL (in terms of protein concentration), and a protein sample (MMLV RT+binder1) was obtained. An equal amount of MMLV reverse transcriptase was used as a control protein sample (MMLV RT).

[0055] (2) After the fluorescent dye Sypro Orange was diluted to 20x with dimethyl sulfoxide (DMSO), the DSF reaction system shown in Table 1 was prepared, and the system was placed in a real-time fluorescence quantitative PCR instrument. The excitation and emission wavelengths were adjusted to the corresponding wavelengths of the fluorescent dye, and the DSF reaction was performed (DSF reaction program: 4°C for 2 min; then enter the temperature program: temperature range from 25°C to 99°C, 0.05°C / 10 s, measure the fluorescence intensity once; select the X1M3 channel). The fluorescence intensity-temperature curve obtained was used to calculate the Tm value and related parameters of the protein sample on the software.

[0056] Table 1

[0057] The fluorescence intensity-temperature curve is shown in Figure 3 .

[0058] After the ligand of the present application was added, the thermal stability of MMLV reverse transcriptase was improved, and the Tm value was increased from 50.90°C to 67.34°C, about 16.44°C.

[0059] Example 3 (1) The ligand prepared in Example 1 was mixed with MMLV reverse transcriptase at a molar ratio of 6:1, incubated at 16°C for 30 min, and an enzyme complex was obtained.

[0060] (2) The RNA template pre-reaction annealing system (final concentration of dNTP Mix 1 mmol / L, final concentration of Oligo dT Primer 0.25 μmol / L, 500 ng template (293T RNA), and water to 10 μL) was prepared and placed in a PCR instrument for pre-reaction annealing reaction (reaction program: 65°C, 5 min; 4°C, hold), and an annealing system reaction solution was obtained.

[0061] (3) Prepare 20 μL reverse transcription system (final concentration of 1 x RTase Reaction Buffer, MMLV reverse transcriptase or enzyme complex obtained in step (1) (final concentration of 20 U / μL in terms of MMLV reverse transcriptase enzyme activity), final concentration of 1 U / μL RNase Inhibitor, and 10 μL annealing system reaction solution obtained in step (2), and the rest is water), and place it in a PCR instrument for reverse transcription reaction (reaction program: 60°C for 30 min; 70°C for 15 min; 4°C hold), to obtain the reverse transcription product.

[0062] (4) Prepare the PCR amplification system (final concentration of 1 x Exp Taq Master Mix, final concentration of 1 μmol / L Test 1, final concentration of 1 μmol / L Test 2, 2 μL reverse transcription product obtained in step (3), and water to make up to 50 μL), and place it in a PCR instrument for amplification (reaction program: 94°C for 1 min; 94°C for 30 sec; 56°C for 30 sec, 72°C for 10 min for 30 cycles; 72°C for 10 min; 4°C hold), and take 5 μL of the amplification product for electrophoresis detection of the results using 1% agarose gel.

[0063] Test 1: 5'-CAGGTGACCAAGCTGGTGTTC-3' (SEQ ID NO: 3); Test 2: 5'-GCTGCAGGATCCACTGTTGTT-3' (SEQ ID NO: 4).

[0064] The results are shown in Figure 4 .

[0065] The enzyme complex obtained by incubating the ligand binder 1 with the MMLV reverse transcriptase at 16°C for 30 min can reverse transcribe to obtain a cDNA product, and the amount of product obtained by subsequent PCR amplification is consistent with that of the MMLV reverse transcriptase. This indicates that the ligand binder 1 has no effect on the normal application of the MMLV reverse transcriptase.

[0066] Example 4 (1) Mix the ligand prepared in Example 1 with the MMLV reverse transcriptase according to a molar ratio of 6:1, and incubate at 16°C for 30 min. Continue to place at 37°C for 1, 3, 5, 7, 10, 13, and 15 days, respectively. Dilute the enzyme complex to a concentration of 0.01 μM, and use it as the experimental group enzyme sample. The same amount of MMLV reverse transcriptase without the addition of ligand is also treated in the same way, and used as the control group enzyme sample.

[0067] (2) Prepare the reverse transcription system (final concentration of 1xRTase Reaction Buffer Mix II, final concentration of 0.1 μmol / L Test 3, final concentration of 1 U / μL RNase Inhibitor, 0.4 μg MS2 RNA, 5 μL enzyme sample, and water to make up to 20 μL), and place it in a PCR instrument for reverse transcription reaction (reaction procedure: 37℃, 30 sec; 85℃, 5 sec; 4℃, hold), after the reaction, the reverse transcription reaction product can be placed at -20℃ for standby.

[0068] (3) Prepare the qPCR reaction system (2 μL of the reverse transcription reaction product obtained in step (2), final concentration of 1x SYBRGreen Pro Taq HS Premix, final concentration of 0.2 μmol / L Test 3, final concentration of 0.2 μmol / L Test 4, and water to make up to 25 μL), and place it in a quantitative qPCR instrument for qPCR reaction (reaction procedure: 95℃ 30 sec; 95℃ 5 sec; 60℃, 30 sec, 40 cycles), n=2. Export the Ct value data obtained by the qPCR instrument, calculate the Ct difference value of the control group and the experimental group, and draw a curve graph of the Ct difference value under different storage days.

[0069] Test 3: 5'-TTCAGCGAACTTCTTGTAA-3' (SEQ ID NO: 5); Test 4: 5'-CATCCGTAGCCTTATTGG -3' (SEQ ID NO: 6).

[0070] The results are shown in Table 1. Figure 5

[0071] After the MMLV reverse transcriptase was incubated with binder 1 and placed at 37℃ for 15 days, the Ct difference value was very small. This indicates that binder 1 can effectively improve the stability of MMLV reverse transcriptase.

[0072] The above embodiments of the present application are described in detail in combination with the examples, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application.​

Claims

1. A polypeptide ligand, characterized in that, The polypeptide ligand is any one of a1) to a3): a1) a polypeptide ligand having an amino acid sequence as set forth in SEQ ID NO: 1; a2) a polypeptide ligand having 90% or more identity to the polypeptide ligand as set forth in a1) and the same activity, obtained by substitution and / or deletion and / or addition of amino acid residues of the polypeptide ligand of a1); a3) a fusion polypeptide ligand obtained by linking a protein tag to the N-terminus or / and C-terminus of a1) or a2).

2. A method of producing a polypeptide ligand according to claim 1, characterized in that, comprising the following steps: introducing a gene encoding the polypeptide ligand into a biological cell, so that the encoding gene is expressed.

3. An enzyme complex, characterized in that, The polypeptide ligand and the MMLV reverse transcriptase of claim 1.

4. The enzyme complex of claim 3, wherein, The molar ratio of the polypeptide ligand and the MMLV reverse transcriptase is (1-6):

1.

5. The method of producing an enzyme complex according to claim 3 or 4, characterized in that, comprising the following steps:

6. The production method according to claim 5, wherein The preparation method further comprises: incubating the mixture; Preferably, the incubation time is 10 min-90 min; Preferably, the incubation temperature is 4°C-37°C.

7. Biomaterials associated with the polypeptide ligand of claim 1, characterized in that, The biological material comprises at least one of b1) to b5): b1) a nucleic acid molecule encoding the polypeptide ligand; b2) an expression cassette comprising the nucleic acid molecule of b1); b3) a vector comprising the nucleic acid molecule of b1) or the expression cassette of b2); b4) a transgenic cell line comprising the nucleic acid molecule of b1), the expression cassette of b2) or the vector of b3); b5) a recombinant microorganism comprising the nucleic acid molecule of b1), the expression cassette of b2) or the vector of b3).

8. The biomaterial of claim 7, wherein, The nucleic acid molecule of b1) has any one of b1-1) to b1-2): b1-1) a DNA molecule having a nucleotide sequence as set forth in SEQ ID NO: 2; b1-2) a DNA molecule having 70% or more identity to the nucleotide sequence as set forth in SEQ ID NO: 2, and encoding the polypeptide ligand.

9. Use of the polypeptide ligand of claim 1 or the biological material of any one of claims 7 to 8 in improving the stability of MMLV reverse transcriptase or preparing a product comprising MMLV reverse transcriptase.

10. Use of the enzyme complex of claim 3 or 4 in any one of c1) to c4): c1) detecting an RNA sample; c2) preparing a product for detecting an RNA sample; c3) reverse transcribing an RNA to synthesize a cDNA; c4) preparing a product for reverse transcribing an RNA to synthesize a cDNA.

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