DDX42 fusion protein and preparation method thereof

Abstract

The invention belongs to the field of biological medicines, and relates to a DDX42 fusion protein and a preparation method thereof. The amino acid sequence of the DDX42 fusion protein is as shown in SEQ ID NO: 1. The method comprises the following steps: 1) amplifying a nucleic acid sequence for coding the DDX42 fusion protein; 2) cloning the nucleic acid sequence to a prokaryotic expression vector; (3) transforming the prokaryotic expression vector into an E.coli strain; 4) inducing expression of the DDX42 fusion protein; and 5) purifying the DDX42 fusion protein. The DDX42 fusion protein has ATP hydrolase activity and helicase activity and plays an important role in inhibiting HIV-1 and various normal-chain RNA viruses, the method is easy for large-scale production and can be used for high-throughput screening of a DDX42 helicase inhibitor, and an expression system of the DDX42 fusion protein is simple.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a DDX42 fusion protein and a preparation method and application thereof. Background Art

[0002] DDX42 (DEAH-Box Helicase42) is a member of the DEAD-box protein family and a type of ATP-dependent RNA helicase that plays a key role in many processes of RNA metabolism, such as transcription, translation initiation, and ribosome biogenesis. Its main function is to unwind the double-stranded structure of RNA, promote the single-stranded state of RNA molecules by unwinding RNA, and also provide the necessary conditions for the subsequent modification and processing of RNA. At the same time, relevant studies have shown that DDX42 is an inhibitor of HIV-1 and can inhibit various positive-strand RNA viruses. The depletion of DDX42 increases retroviral infection. In addition, DDX42 is also involved in the assembly of U2snRNP, which is part of the early spliceosome assembly.

[0003] Structurally, the 3'UTR of DDX42 is very long, almost extending to the second poly(A) site, with a low GC content and multiple consecutive U sequences, which are generally considered to be signs of RNA degradation. This structural property may affect its translational regulation. DDX42 plays an important role in gene expression regulation, spliceosome assembly, and antiviral immune response. The RNA helicase DDX42 was identified as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increased HIV-1 DNA accumulation and infection in cell lines and primary cells. Overexpression of DDX42 inhibited HIV-1 infection, while expression of a dominant negative mutant increased infection. In addition, DDX42 also plays an important role in antiviral immunity, for example, it can inhibit HIV-1 replication, independent of interferon. At the same time, DDX42 also shows inhibitory effects on other viruses such as Zika virus and SARS-CoV-2.

[0004] In recent years, with the rapid development of molecular biology technology, the research on DDX42 has also made significant progress. Scientists have revealed the expression pattern and functional characteristics of DDX42 in various cell types through gene knockout, RNA interference and other technologies. Studies have found that abnormal expression of DDX42 in tumor cells may be closely related to the occurrence and development of tumors. The role of DDX42 in neurodegenerative diseases has also attracted widespread attention. Studies have shown that abnormal expression or dysfunction of DDX42 may lead to neuronal damage and death, thereby causing neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. At present, there are few research methods for obtaining DDX42 recombinant protein, and most of them are extracted from full-length DDX42 recombinant proteins obtained in cell culture. Because the molecular weight of DDX42 protein is as high as 120kD or more, the vector construction is complex, the purification process is difficult, the cost is high, and the yield is low, these factors limit the development of high-throughput DDX42 protein inhibitor screening. Summary of the invention

[0005] The present invention expresses a recombinant human RNA helicase DDX42 fusion protein through an E. coli expression system. The growth cycle of E. coli is fast and efficient. A large amount of soluble fusion proteins with enzymatic activity can be prepared in a short time, which is suitable for high-throughput screening of DDX42 inhibitors. Therefore, the purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a method for cloning a truncated DDX42 gene and a method for preparing a fusion protein that is easily obtained by using a human skin fibroblast HSF sequence as a library template. The prepared fusion protein has good solubility in E. coli, and has good ATP hydrolase activity and unwinding activity.

[0006] In one aspect, the present invention provides a DDX42 fusion protein having both ATP hydrolase and helicase activities, wherein the amino acid sequence of the DDX42 fusion protein is shown in SEQ ID NO:1.

[0007] Compared with the full-length human RNA helicase DDX42 protein, the N-terminus of the DDX42 fusion protein lacks 489 amino acids and the C-terminus lacks 802 amino acids. At the same time, a fusion tag, thioredoxin (TrxA), is added to the N-terminus, and a purification tag, histidine tag (His-Tag), is added to the C-terminus.

[0008] Therefore, in an embodiment of the present invention, the N-terminus of the DDX42 fusion protein has a fusion tag, namely, thioredoxin A (TrxA).

[0009] In a second aspect, the present invention provides a nucleic acid encoding a DDX42 fusion protein.

[0010] In a specific embodiment, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:2.

[0011] In a third aspect, the present invention provides an expression vector comprising the nucleic acid of the second aspect.

[0012] In a preferred embodiment of the present invention, the expression vector is a bacterial expression vector, such as pET.32m.3c.

[0013] In a fourth aspect, the present invention provides a strain containing the expression vector according to the third aspect of claim.

[0014] In a preferred embodiment, the strain may be an E. coli strain, such as Rosetta (DE3), BL21 (DE3) or BL21 pLysS.

[0015] In a fifth aspect, the present invention provides a method for preparing a DDX42 fusion protein, comprising the following steps:

[0016] 1) Amplifying the nucleic acid sequence encoding the DDX42 fusion protein;

[0017] 2) Cloning the nucleic acid sequence into a prokaryotic expression vector;

[0018] 3) Transforming the prokaryotic expression vector into the E. coli strain;

[0019] 4) Induce the expression of DDX42 fusion protein, and

[0020] 5) Purification of DDX42 fusion protein.

[0021] In a specific embodiment of the present invention, the prokaryotic expression vector may be a bacterial expression vector, such as pET.32m.3c.

[0022] In a specific embodiment of the present invention, the E. coli strain may be Rosetta (DE3), BL21 (DE3) or BL21pLysS.

[0023] In a specific embodiment of the invention, purification can be performed, for example, by affinity chromatography.

[0024] In a sixth aspect, the present invention provides the use of DDX42 fusion protein in:

[0025] 1) Preparation of preparations for unwinding double-stranded oligonucleic acid structures

[0026] 2) Preparation of drugs for inhibiting HIV-1 virus

[0027] 3) High-throughput screening of inhibitors of DDX42 helicase or ATP hydrolase activity.

[0028] Compared with the prior art, the present invention has the following beneficial effects. Specifically, the present invention expresses the recombinant human RNA helicase DDX42 protein through the E. coli expression system, can obtain a large amount of target protein in a short time, greatly improves the yield, and overcomes the disadvantages of large molecular weight, complex spatial structure and low purification efficiency of natural DDX42. The present invention intercepts the fragment of the natural human RNA helicase DDX42 gene through genetic engineering fusion protein technology, and expresses the fusion protein in a prokaryotic system, with high operability and expression amount, stable purification process, low cost, and is conducive to the large-scale production of DDX42 and high-throughput screening of inhibitors in the later stage. Compared with the expression systems of various eukaryotic cells used in the existing inventions, it overcomes the shortcomings of complex construction, difficult process, high cost and low yield. The E. coli expression system used in the present invention is easy to culture, has a shorter growth cycle, fast passage speed, and the expressed recombinant human RNA helicase DDX42 protein has a higher yield, stronger activity, and is easier to achieve large-scale and expanded protein purification (see Table 1). The recombinant human RNA helicase DDX42 protein prepared by the present invention also has good biological activity. It is determined by in vitro detection that it has good ATP hydrolase and helicase activities at the same time. The DDX42 fusion protein with ATP hydrolase and helicase activities of the present invention can be used for DDX42 activity analysis, high-throughput screening of DDX42 inhibitors, intermolecular interactions, antibody and detection kit preparation and other physiological and biochemical operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the DDX42 gene truncation map;

[0030] Figure 2 and Figure 3 This is the construction map of the truncated version of the E. coli recombinant plasmid pET32m.3C-DDX42;

[0031] Figure 4 This is the double enzyme digestion verification diagram of the recombinant plasmid pET.32m.3c-DDX42;

[0032] Figure 5 The induced expression of DDX42 fusion protein in E. coli BL21 (DE3), where + indicates induction; - indicates no induction;

[0033] Figure 6 This is the nickel column purification of DDX42 fusion protein;

[0034] Figure 7 The results of the ATP helicase activity assay of DDX42 fusion protein;

[0035] Figure 8The results of RNA helicase activity assay of DDX42 fusion protein;

[0036] Fig. 9 Comparison of protein sequences of native DDX42 and DDX42 fusion protein. DETAILED DESCRIPTION

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all performed according to conventional experimental conditions, such as those described in Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2012), or according to the conditions recommended by the manufacturer's instructions; the materials, reagents, etc. used, unless otherwise specified, can all be obtained through commercial channels.

[0038] Example 1: Amplification of the gene fragment of the truncated DDX42 protein

[0039] The gene library (GenBank) was searched to obtain the wild-type nucleotide and amino acid sequences of human DDX42. The GenBank accession numbers were NM_203499.3 and NP_987095.1, respectively. The full-length human wild-type DDX42 target gene was amplified using the cDNA library of normal human cells Human Skin Fibroblast, HSF (Shanghai Bolson Biotechnology Co., Ltd.) as a template using the Snap Gene software for online primer design. Referring to the protein sequence of DDX42, 489 amino acids were truncated at the N-terminus of the DDX42 protein and 802 amino acids were truncated at the C-terminus, retaining the core enzyme activity region. The truncated protein was as follows Figure 1 As shown, the truncated DDX42 primers were designed. The primers are shown in Table 1:

[0040] Table 1: Primer sequences

[0041]

[0042]

[0043] The target gene fragment was obtained by PCR polymerase chain reaction, and a high concentration fragment solution was obtained by rapid purification kit (Beijing Quanshijin Biotechnology Co., Ltd., #S20513). The specific PCR reaction conditions are shown in Table 2.

[0044] Table 2: PCR polymerase chain reaction

[0045]

[0046] The PCR amplification program was as follows: pre-denaturation at 94°C for 1 min; denaturation at 98°C for 10 s, annealing at 55-65°C for 15 s, extension at 68°C for 3 min, 33 cycles; extension at 68°C for 10 min, and termination at 4°C.

[0047] Example 2: Construction and identification of expression plasmid

[0048] The restriction endonucleases BamHI / XhoI (New England Biotechnology (Beijing) Co., Ltd.) were used to double-digest the DDX42 truncated type and the vector pET.32m.3c (purchased from the BioVector plasmid vector strain cell protein antibody gene storage center). The double-digestion systems are shown in Table 3 and Table 4 respectively:

[0049] Table 3: Truncated DDX42 double enzyme digestion system

[0050] cDNA (1 μg) 7μL B H 1μL XOt 1μL rcutsmart 10μL ddH2O 81μL

[0051] Table 4: Double restriction enzyme digestion system of vector pET32M.3C

[0052] cDNA (1 μg) 3.7μL B H 1μL XOt 1μL rcutsmart 10μL <![CDATA[ddH 2 The]]> 34.3μL

[0053] Reagents were added according to the system, mixed and heated at 37°C for 2 h, and 1% nucleic acid gel was prepared for running and cutting. After recovery using gel DNA recovery kit (Hangzhou Xinjing Biological Reagent Development Co., Ltd., 2001050), T4 ligase (New England Biotechnology (Beijing) Co., Ltd., M0202V) was used for ligation reaction. The ligation system is shown in Table 5:

[0054] Table 5: Ligation reaction system

[0055] Total volume 20μl T4 DNA Ligation Buffer (10X) 2μl Vector DNA (5.8kb) 50ng Insert DNA (1.6kb) 68.97ng Enzyme-free water to20μl T4 DNA ligase 1μl

[0056] After adding and mixing according to the ligation reaction system in Table 5, the ligation was carried out overnight at room temperature, and the ligation product was transformed into E. coli DH5α (Shanghai Angyu Biotechnology Co., Ltd., G6016). The specific steps of the transformation are as follows:

[0057] 1) Take out E. coli DH5α competent cells (Shanghai Angyu Biotechnology Co., Ltd., G6016) from the -80℃ refrigerator and place them on ice until they are completely thawed;

[0058] 2) Add 5 μL of the obtained ligation product to the competent cells, gently pipette to mix, and continue to place on ice for 30 minutes;

[0059] 3) Place the mixture after ice bath in a 42°C water bath for heat shock for 1 min;

[0060] 4) After heat shock, quickly place the competent cells back on ice and place them in an ice bath for 3 minutes;

[0061] 5) Add 700 μL of LB liquid culture medium to the competent cells after ice bath, and place them in a 37°C constant temperature shaker for shaking culture and recovery for 45 to 60 minutes;

[0062] 6) The revived bacterial solution was centrifuged at 12000 rpm for 1 min, 100 μL of the supernatant was collected and the precipitate was evenly blown and spread on a plate containing 100 mg / mL ampicillin (BBI Life Sciences

[0063] ,#[69-52-3]) on LB plates and cultured inverted at 37°C overnight.

[0064] 5-10 monoclonal colonies were picked and placed in 3 mL LB liquid medium supplemented with ampicillin, shaken to the logarithmic phase, centrifuged at 8000 rpm for 2 min to obtain strains, and plasmids were extracted using a plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., A0717A). The obtained plasmids were digested again with restriction endonucleases BamH1 / Not1 (New England Biotechnology (Beijing) Co., Ltd.) for verification. The digestion identification results were as follows: Figure 4 As shown in (42-6 to 42-10 all carry the correct gene fragment (1.6K)), the correct DDX42 truncated plasmid can be sequenced, namely the plasmid pET32M.3C-DDX42, and its construction map is shown in Figure 2 , Figure 3 shown.

[0065] Example 3: Protein expression and purification

[0066] Small amount of inducible expression

[0067] Transformation: Take 1 μL of DDX42 recombinant plasmid and 30 μL E.coliBL21 (DE3) pLysS competent cells (Shenzhen Sai Ensi Biotechnology Co., Ltd., G6030-10) and mix them for transformation (30 min on ice-42 °C, 1 min-3 min on ice); mix with 700 μL LB without antibiotics, shake at 37 °C 200 rpm for 1 h, centrifuge at 4000 rpm for 1 min to collect bacteria, keep 100 μL for re-suspension, apply to the plate, and invert the plate in a 37 °C incubator for overnight culture;

[0068] Induction by picking: Pick 4 monoclones with good growth and easy picking and put them into 3mL LB medium tubes containing 100μg / mL ampicillin and 34μg / mL chloramphenicol, shake at 290rpm and 37℃ until the logarithmic phase, take 1mL and add it to 250μL glycerol, and store it at -80℃; 1mL is used for the control group; 1mL is added with 10μL of 100mM IPTG (Shanghai Aladdin Biochemical Technology Co., Ltd., isopropyl-β-D-thiogalactoside, I104812), and continue to induce protein expression; 18℃, induce for 4h;

[0069] Protein extraction: The bacterial solution was collected by centrifugation, and 250 μL of PBS (Shenzhen Ruixin Biotechnology Co., Ltd.) containing 1% protease inhibitor (Kailian Gene Technology Co., Ltd., HY-K0010) was added and mixed; ultrasonic disruption was performed until a translucent bacterial solution was formed; and the supernatant was collected by centrifugation at 4°C for 10 min;

[0070] Take 20 μL of sample and mix with 4 μL 5X loading buffer (250 mM Tris-HCl PH = 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol to make 5 mL of 5X loading buffer) and boil at 95℃ for 5 min before loading on gel and staining with Coomassie brilliant blue for 1 hour before decolorization. Small amount induction. The results are shown in Figure 5 As shown, it was shown that the DDX42 fusion protein was expressed in a soluble form.

[0071] Example 4: Large-scale protein expression

[0072] After obtaining the induced target protein, the E. coli BL21 (DE3) strain with higher induction efficiency was expanded to 500 mL LB medium. After the logarithmic phase, IPTG with a final concentration of 1 mM was added, and protein expression was continued at 18°C. After 5 hours, the bacterial liquid was collected by centrifugation.

[0073] The expressed truncated DDX42 was purified using a nickel affinity chromatography column (Shanghai Biotech Co., Ltd.). E. coli cells were resuspended in lysis buffer (composition see Table 6), disrupted with 15% ultrasonic power for 15-25 minutes until the bacterial solution was transparent, centrifuged at 10000rpm and 4℃ for 30 minutes, and the supernatant was collected. The supernatant was mixed with the His-tagged protein agarose purification resin (Yisheng Biotechnology (Shanghai) Co., Ltd., 20502ES50) activated for 1 hour by inversion for 1 hour to allow the resin and protein to fully bind. The beads were washed upside down 5 times with cleaning solution 1 (composition see Table 6) and cleaning solution 2 (composition see Table 6), respectively, and then the washed solution was passed through the nickel column, and the elution solution (composition see Table 6) was used to elute the fusogenic DDX42 target protein. The purified protein was verified for purity by running a gel again, and then 10% glycerol was added and mixed, and then stored at -80℃ for a long time. The eluted protein sample was identified by SDS-PAGE, and the results are as follows. Figure 6 As shown: A specific target protein band was observed at around 72 kDa. The concentration of the purified DDX42 fusion protein reached 2.5 mg / mL by using a protein quantitative reagent. That is, in a 0.5 L bacterial culture system, the total yield of the DDX42 fusion protein that can be extracted reaches 2-5 mg (purity greater than 95%).

[0074] Table 6: Solutions required for nickel column affinity chromatography

[0075] name Phosphate buffer (mM) Sodium chloride (mM) Imidazole (mM) Triton100 β-ME(mM) Lysis buffer 50 150 25 1％ 2 Lotion 1 50 150 25 0.1％ 1 Lotion 2 50 500 25 0.1％ 1 Eluent 50 / 250 / 1

[0076] Example 5: ATPase activity detection

[0077] The ATPase activity of the DDX42 fusion protein was detected as follows:

[0078] 1. Preparation of DNA substrates. Design and synthesize two DNA primers containing different substrate labels, namely biotin-labeled DNA: 5'-biotin-GCTGACCCTGCTCCCAATCGTAATCTATAGTGTCACCTA-3'; and digoxigenin-labeled DNA: 5'-DIG-CGATTGGGAGCAGGGTCAGC-3'. The two single-stranded DNA oligomers containing different labels were resuspended at a molar ratio of 1:1 and mixed in the annealing reaction solution (2mM HEPES, pH7.0, 0.05MNaCl, 0.1mM EDTA and 0.01% (w / v) SDS). The reaction system was heated to 100℃ for 5min, and then cooled to 65℃ for 30min. The DNA annealing reaction was set at 22℃ and reacted for 4h. The final DNA double-stranded molecule was diluted with deionized water to a DNA substrate solution with a final concentration of 2.5ng / μL, and stored in aliquots at -20℃ for later use.

[0079] 2. Sample preparation: Prepare enzyme reaction solution (25mM 4-mops, 5μM ATP, 2mM DTT, 3mM MnCl 2 , 100 μg / mL BSA). Add 90 μL of reaction solution, 2 ng DNA and 4 μL of DDX42 fusion protein to each well. Place in a 37°C incubator for 1 hour.

[0080] 3. Preparation of detection reagents: ATP detection reagent (Shanghai Biotech Co., Ltd., S0026-1) and ATP diluent (Shanghai Biotech Co., Ltd., S0026-2) were diluted at a ratio of 1:9 and added to a new 96-well plate, 100 μL per well.

[0081] 4. ATP detection: 40 μL of the solution with complete ATPase reaction was added to the ATP detection reagent, and the mixture was shaken and mixed using a multifunctional microplate reader (Victor Nivo 3S, Ruifudi Biomedical (Shanghai) Co., Ltd.), and the ATP concentration was detected after shaking for 1 min.

[0082] 5. The plate was read with a chemiluminescent microplate reader (VictorNivo 3S, Ruifudi Biomedical (Shanghai) Co., Ltd.) to analyze the signal. The original data and analysis are shown in Table 7. In Table 7, the values ​​of the wells with fusion protein DDX42 added were significantly reduced compared with those without fusion protein DDX42, and there was a significant difference (P<0.01), indicating that the DDX42 fusion protein has ATPase activity.

[0083] The results showed that the DDX42 fusion protein had significant ATPase activity, such as Figure 7 As shown. Figure 7 It can be seen that the ATPase activity of the DDX42 fusion protein is significantly higher than that of the control group, and has obvious inhibitory properties.

[0084] Table 7: Raw data of ATPase activity of DDX42 fusion protein

[0085]

[0086] Example 6: Helicase activity detection

[0087] The biotin-labeled oligo DNA molecule is annealed and paired with another digoxigenin-labeled oligo DNA molecule to form a double-stranded DNA molecule with a 3'-protruding part. This double-stranded DNA molecule is spread on a 96-well plate with a neutral avidin link at the bottom. After the helicase reaction, the single-stranded DNA molecule containing the digoxigenin label will dissociate from the plate. Then, by adding an anti-digoxigenin secondary antibody with an alkaline phosphatase label and the alkaline phosphatase substrate CSPD to the 96 plate, the chemiluminescent signal generated by each well on the plate is analyzed to identify the activity of the helicase. Generally, the more the chemiluminescent signal decreases, the stronger the helicase activity. The helicase activity of the DDX42 fusion protein is detected based on this mechanism as follows:

[0088] 1. Avidin (Thermo Fisher Scientific (China) Co., Ltd., 31000) was diluted with 0.5M Na 2 CO 3 (pH = 9.3) was diluted to 15 μg / mL, 100 μL per well, and incubated at 4°C overnight.

[0089] 2. Pour out the avidin incubated overnight, wash with PBS buffer three times, pat dry the residual buffer on a paper towel, and dry at room temperature for 15 minutes.

[0090] 1.3. Incubate each well with 100 μL 0.1% BSA (bovine serum albumin, dissolved in PBS) at room temperature for 1 hour.

[0091] 4. After washing three times with PBS buffer, pat dry the residual buffer on a paper towel and dry at room temperature for 15 minutes.

[0092] 5. The annealed DNA (DNA substrate prepared in Example 5) was dissolved in 1M PBS 1M NaCl, and 4 ng DNA (75 μL) was added to each well of a 96-well plate and incubated at room temperature for 2 h.

[0093] 6. After washing twice with PBS buffer, wash once with 200 μL of 50 mM Tris-HCl (pH 7.5) containing 50 mM NaCl.

[0094] 7. Prepare the reaction solution (25mM 4-MOPS (pH 7.0), 5mM ATP, 2mM DTT, 3mM MnCl 2 , and 100 μg / mL BSA), 95 μL of reaction solution and 5 μL of helicase were added to each well (no enzyme was added to the control group); the reaction was carried out at 37°C for 60 min.

[0095] 8. After the enzyme reaction is completed, wash twice with 150 mM NaCl (200 μL / time), and then dry at room temperature for 15 minutes.

[0096] 9. Wash the 96-well plate with washing solution (0.1 M maleic acid, 0.15 M NaCl and 0.3% Tween 20 (pH 7.5)) for another 5 min, 200 μL per well.

[0097] 10. Blocking: Add 300 μL of 10% BSA (dissolved in a mixture of 0.1 M maleic acid and 0.15 M NaCl, pH 7.5) to each well and block for 30 min.

[0098] 11. Remove the blocking solution, add 20 μL of anti-digoxigenin secondary antibody-AP (Beckman Coulter Life Sciences (Shanghai) Co., Ltd., Ms mAb to Digoxigenin [21H8] (AP), ab119345 diluted 1:5000 in blocking solution) to each well, and incubate at room temperature for 30 minutes.

[0099] 12. Wash twice with buffer (0.1 M Tris-HCl, 0.1 M NaCl (pH 9.5)), 100 μL each time.

[0100] 13. Add 20 μL of detection solution (containing 0.1 M Tris-HCl, 0.1 M NaCl (pH 9.5)) and chemiluminescent substrate CSPD (Thermo Fisher Scientific, T2141) (25 mM (20:1)) to each well and let stand for 5 min.

[0101] 14. Use a multifunctional microplate reader with chemiluminescence function to detect RLU value, analyze the signal, and read the well for 10s / well. The original data and analysis are shown in Table 8. In Table 8, the numerical comparison of DDX42 without fusion protein and fusion protein DDX42 shows that the numerical value of the well with fusion protein DDX42 is significantly reduced, and there is a significant difference (P<0.01), indicating that DDX42 fusion protein has helicase activity.

[0102] The results showed that the DDX42 fusion protein had significant helicase activity, such as Figure 8 As shown. Figure 8 It can be seen that the luminescent signal of the experimental group with DDX42 fusion protein is significantly lower than that of the control group (without enzyme), and the reading value difference is obvious (see Table 8 for specific data). It can be judged that the DDX42 fusion protein catalyzes a large number of aoligo-DNA depolymerization reactions, proving that the DDX42 fusion protein has obvious helicase activity.

[0103] Table 8: Helicase activity data of DDX42 fusion protein

[0104]

[0105] Based on the above data, compared with the mammalian cell expression system, it can be concluded that the purification of DDX42 recombinant protein through the E. coli expression system has more prominent advantages, as shown in Table 9:

[0106] Table 9: Comparison of DDX42 protein production systems

[0107]

Claims

1. DDX42 fusion protein, characterized in that The amino acid sequence of the DDX42 fusion protein is shown in SEQ ID NO:

1.

2. The DDX42 fusion protein according to claim 1, characterized in that The N segment of the DDX42 fusion protein has a fusion expression tag, namely, thioredoxin A (TrxA).

3. A nucleic acid, characterized in that The nucleic acid encodes the DDX42 fusion protein of claim 1.

4. The nucleic acid according to claim 3, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

2.

5. An expression vector, characterized in that The expression vector contains the nucleic acid of claim 3, and preferably the expression vector is a bacterial expression vector, such as pET.32m.3c.

6. A strain characterized in that The strain contains the expression vector according to claim 5, and preferably the strain is an E. coli strain, such as Rosetta (DE3), BL21 (DE3) or BL21 pLysS.

7. A method for preparing a DDX42 fusion protein, characterized in that: The method comprises the following steps: 1) Amplifying the nucleic acid sequence encoding the DDX42 fusion protein; 2) cloning the nucleic acid sequence into a prokaryotic expression vector; 3) transforming the prokaryotic expression vector into an E. coli strain; 4) inducing the expression of the DDX42 fusion protein, and 5) Purifying the DDX42 fusion protein.

8. The method according to claim 7, characterized in that The prokaryotic expression vector is a bacterial expression vector, such as pET.32m.3c.

9. The method according to claim 7, characterized in that: The E. coli strain is Rosetta (DE3), BL21 (DE3) or BL21 pLysS.

10. Use of the DDX42 fusion protein of claim 1 in: 1) preparing a preparation for unwinding the double-stranded structure of oligonucleic acid, 2) Preparation of drugs for inhibiting HIV-1 virus, or 3) High-throughput screening of inhibitors of DDX42 helicase or ATP hydrolase activity.

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