CN112326963B - Application of eukaryotic expression type A influenza virus PB2cap protein - Google Patents
Application of eukaryotic expression type A influenza virus PB2cap protein Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering, and particularly relates to eukaryotic expression and application of a PB2cap protein of an influenza A virus. The invention discloses application of eukaryotic expression of an influenza A virus PB2cap protein in detection of RNA cap binding activity of the influenza A virus. The invention constructs eukaryotic expression plasmid pCAGGS-PB2cap (aa: 318-483) based on eukaryotic expression vector pCAGGS, and carries out eukaryotic expression on PB2cap by transient transfection, and the expression protein and m 7 The binding capacity of GTP in vitro is used for evaluating the RNA cap binding activity of influenza virus, and a novel detection index can be provided for the differential research of the PB2 protein function of influenza A virus.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to eukaryotic expression and application of a PB2cap protein of an influenza A virus.
Background
Influenza viruses belong to the orthomyxoviridae family, including A, B, C, D type four; the host spectrum of the A-type influenza virus (Influenza A virus, IAV) is the widest, and the A-type influenza virus can infect various animals such as human beings, birds, pigs, horses, dogs and the like and can cause influenza pandemic, and has important public health significance (Zheng Teng, chen Zhihua. History of avian influenza and biological report of public health significance [ J ], 2002, 37 (4): 13-15). IAV is a single-stranded, negative-strand RNA virus whose genome is divided into 8 segments, and encodes more than 10 viral proteins, such as PB2, PB1-F2, PA-X, HA, NP, NA, M1, M2, NS1, NS2, etc. (Mostafa A, abdelwhab EM, mettenleiter TC, et al, zoonotic Potential of Influenza A Viruses: A Comprehensive Overview [ J ]. Viruses,2018,10 (9): 497.).
In the IAV lifecycle, polymerase proteins (PB 2, PB1, PA) together with Nucleoprotein (NP) catalyze transcription and replication of host cell nuclear viral RNA (Reich S, guilligay D, pflug A, et al structural light into cap-snatching and RNA synthesis by influenza polymerase [ J ]. Nature,2014,516 (7531):361-366.). During transcription, the PB2 subunit is first bound to the cap (cap) structure at the 5 'end of the host mRNA (Fechter P, mingay L, sharps J, et al, two aromatic residues in the PB [ J ]. J BiolChem,2003,278 (22): 20381-20388.; ulmanen I, broni BA, krug RM, et al, role of two of the influenza virus core P proteins in recognizing cap 1structures (M7 GpppNm) on RNAs and in initiating viral RNA transcription [ J ]. Proc Natl AcadSci USA,1981,78 (12): 7355-7359.), and then cleaved by a PA subunit having endonuclease activity at a position 8-14nt downstream of the 5' cap structure (Dias A, bouper D, crespin T, et al, the cap snatching endonuclease of influenza virus polymerase resides in the PA subunit [ J ]. Nature,2009,458 (7240):914-918.; fodor E, crow M, mingaLJ et al, A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs [ J ]. J ] Virol 89, sch7, 5:18:67 ]; vir [ J ]. Proc Natl AcadSci USA, 8239:, for the synthesis of viral self mRNA (Biswas SK, nayak DP, et al, identification of four conserved motifs among the RNA-dependent polymerase encoding elements [ J ] EMBO J,1989,8 (12): 3867-3874); poch O, sauvget I, delaue M, et al, identification analysis of the conserved motifs of influenza A virus polymerase basic protein [ J ] J Virol,1994,68 (3): 1819-1826.). Therefore, the cap-binding domain of PB2 subunit (PB 2 cap) initiates transcription and replication of the virus by its unique "cap-grasping" mechanism, which plays a vital role.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for constructing a eukaryotic expression plasmid of pCAGGS-PB2cap, eukaryotic expression, identification and purification of PB2cap protein, and application of the eukaryotic expression of PB2cap protein.
The invention discloses application of eukaryotic expression of an influenza A virus PB2cap protein in detection of RNA cap binding activity of the influenza A virus.
In the invention, a gene fragment (namely PB2 cap) of a 318-483 th amino acid cap-binding domain (aa: 318-483) of PB2 protein is amplified by PCR, a histidine tag (6 XHis: CATCATCACCACACCAC) is added at the 3' end, and the fragment is connected with a linear eukaryotic expression vector pCAGGS after double digestion by EcoRI and XhoI. And further transfecting 293T cells with the successfully constructed eukaryotic expression plasmid pCAGGS-PB2cap, collecting cell lysates after 48 hours, and separating and purifying by using a nickel (Ni+) affinity chromatographic column to obtain the eukaryotic expression fusion protein of PB2cap and histidine tag.
The invention also discloses the PB2cap protein expressed by eukaryotic cells and m in vitro 7 The binding capacity of GTP is used for evaluating the RNA cap binding activity of influenza virus, and provides a new detection index for the differential research of the PB2 protein function of influenza A virus.
The specific RNA cap binding experimental method comprises the following steps:
1) Suction 100 mu L m 7 GTP agar magnetic beads (Jena Bioscience: EDA-m) 7 GTP-ATTO 488) to a new 1.5mL finger tube;
2) Centrifuging at 500rpm for 30s to pellet the agar beads, carefully discarding the supernatant;
3) Adding 500 mu L TBS (pH 8.0) to resuspend the agar magnetic beads, repeating the step 2), washing the agar magnetic bead preservation solution for balancing, and repeating the washing for 2 times;
4) At well balanced m 7 50 mug eukaryotic expression PB2cap protein is added into GTP agar magnetic beads, and a shaking table at 4 ℃ is slowly shaken to be combined for 3 hours;
5) Centrifuging at 500rpm for 30s to pellet the agar beads, carefully discarding the supernatant;
6) Adding 500 μl of washing solution (50 mM Tris-HCl pH8.0, 200mM NaCl, 2mM DTT) to resuspend the agar magnetic beads, repeating step 5) to wash off unbound proteins, repeating washing 5 times in total;
7) To remove non-specific binding, 600. Mu.L of a solution containing 1mM m was added 7 Washing solution of GTP (Sigma: 7-methyl guanosine5' -triphosphite) was slowly shaken and combined for 1 hour at 4℃in a shaker;
8) Centrifuging at 500rpm for 30s to pellet the agar beads, carefully discarding the supernatant; washing again, and discarding the supernatant;
9) 50. Mu.L of 2x loading buffer was added to the agar beads, and after boiling, SDS-PAGE Coomassie Brilliant blue staining or Western-Blot analysis was performed.
The invention constructs eukaryotic expression plasmid pCAGGS-PB2cap (aa: 318-483) based on eukaryotic expression vector pCAGGS, and carries out eukaryotic expression on PB2cap by transient transfection, and the expression protein and m 7 The binding capacity of GTP in vitro is used for evaluating the RNA cap binding activity of influenza virus, and a novel detection index can be provided for the differential research of the PB2 protein function of influenza A virus.
Drawings
FIG. 1 PCR identification of the bacterial solutions of eukaryotic expression plasmids pCAGGS-MZ34-PB2cap and pCAGGS-SH14-PB2 cap.
FIG. 2 shows the sequencing result of the region of the foreign gene MZ34-PB2cap inserted into the pCAGGS-MZ34-PB2cap eukaryotic expression plasmid. And (3) injection: the pCAGGS vector sequences at both ends of the inserted exogenous gene MZ34-PB2cap are underlined.
FIG. 3 shows the sequencing result of the SH14-PB2cap region of the exogenous gene inserted into the pCAGGS-SH14-PB2cap eukaryotic expression plasmid. And (3) injection: the pCAGGS vector sequences at both ends of the inserted exogenous gene SH14-PB2cap are underlined.
FIG. 4 plasmid maps of pCAGGS-MZ34-PB2cap (A) and pCAGGS-SH14-PB2cap (B) eukaryotic expression plasmids. And (3) injection: the grey shaded portion in the lower right corner of the circular plasmid map is the region in which the exogenous gene MZ34-PB2cap or SH14-PB2cap is inserted.
FIG. 5 Western-Blot identification of MZ34-PB2cap and SH14-PB2cap eukaryotic protein expression.
FIG. 6 cap binding ability of eukaryotic expressed PB2cap proteins from different strains.
FIG. 7MZ34 and SH14 strain PB2 gene-induced polymerase activity.
FIG. 8 A.sub. rMZ34 of a recombinant virus containing MZ34-PB2 gene and a recombinant virus rMZ-SH 14 containing SH14-PB2 gene PB2 Its PB2 protein expression level.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials and reagents used, unless otherwise specified, are commercially available. The materials and reagents used were as follows:
high fidelity enzyme (2× TransStart FastPfu Fly PCR SuperMix), homologous recombination kit (pEASY-Uni Seamless Cloning and Assembly Kit) were purchased from Beijing full gold company; transfection reagents were purchased from Qiagen; western cell lysate, his tag protein purification kit and BCA protein concentration determination kit are purchased from Biyundian corporation; m7GTP-immobilized agarose (EDA-m 7GTP-ATTO 488) was purchased from Jena Bioscience; eukaryotic expression vector pCAGGS was purchased from the BioVector plasmid vector strain cell protein antibody gene collection-NTCC classical culture collection.
1. Eukaryotic expression plasmids for constructing PB2 caps of different strains (MZ 34 and SH 14) by homologous recombination method
According to the Cap-binding domain ranges reported in the relevant literature (Schrauwen EJ, de Graaf M, herfst S, et al Determins of virulence of influenza A virus [ J ]. Eur J ClinMicrobiol Infect Dis,2014,33 (4): 479-490.) the PCR products were amplified using the PbW 2000 recombinant plasmid of the PB 14 (A/chicken/Shanghai/14/2001 (H9N 2), the PB2 gene GenBank accession number was KY 005860.1), the PB2 gene GenBank accession number was EU 753319.1) as template (Liu Jiao, hao Xiaoli, li Xiuli, et al G1-like and PB2, M genes of the F/98-like evolutionary lineages in the H5N6 subtype avian influenza virus reassortment [ J ]. Chinese preventive Protect school of PB, 2019,41 (11): 987-993), the cloning of DNA polymerase and the corresponding to the BrH 5:6 (CAT 2) gene and the CAT 2-binding domain of the PCR products were amplified using the high-DNA polymerase and the PbB gene of the CAT 1:6:6:. According to the specification of the homologous recombination kit, the recovered target fragment is connected with a linear eukaryotic expression vector pCAGGS subjected to double digestion by EcoRI and XhoI, after transformation, suspected positive plasmids are respectively extracted for bacterial liquid PCR identification and sequencing verification, and finally 2 recombinant eukaryotic expression plasmids pCAGGS-MZ34-PB2cap and pCAGGS-SH14-PB2cap are respectively constructed.
TABLE 1 primer for constructing MZ34 and SH14 strain PB2cap eukaryotic expression plasmid by homologous recombination method
Note that: homology arms are underlined; the added His tag is shown in italics.
The bacterial liquid PCR identification result (figure 1) shows that the eukaryotic expression recombinant plasmids pCAGGS-MZ34-PB2cap and pCAGGS-SH14-PB2cap can both amplify exogenous gene fragments of about 500bp by using construction primers, and further sequencing verification (or figure 2 (SEQ ID NO. 5) and figure 3 (SEQ ID NO. 6) show that the exogenous genes have no mutation, the insertion positions are accurate, the construction of the eukaryotic expression recombinant plasmids pCAGGS-MZ34-PB2cap and pCAGGS-SH14-PB2cap is successful, and the plasmid map is shown in figure 4.
2. Eukaryotic expression purification MZ34-PB2cap and SH14-PB2cap proteins
The eukaryotic expression plasmids pCAGGS-MZ34-PB2cap and pCAGGS-SH14-PB2cap which were successfully constructed were each transfected at 24. Mu.g into 293T cells in a cell culture dish (diameter: 10 cm). After 48h of transfection, the supernatant was discarded and the cell lysate was prepared using Western cell lysate. According to the specification of the His tag protein purification kit, mixing supernatant after lysate centrifugation with a Ni+ column at 4 ℃ for 1h, adding the mixed solution into a purification tube, washing the mixed solution for 8 to 10 times, eluting protein for 3 to 5 times by using an eluent, dialyzing the eluted protein by using PBS (pH 8.0), detecting the protein concentration by using a BCA kit, and finally carrying out protein verification by using a first antibody of the His tag.
Western-blot results show that eukaryotic expression PB2cap proteins of MZ34 and SH14 strains are successfully expressed (shown in figure 5), and the post-purification effect is good, and the strain has no impurity protein.
3. In vitro cap combination experiment
Suction 100 mu L m 7 GTP was followed by 2 washes with TBS (pH 8.0) at equilibrium m 7 50. Mu.g of eukaryotic expression pCAGGS-MZ34-PB2cap and pCAGGS-SH14-PB2cap proteins were added to GTP, and after slow shaking and binding at 4℃for 3h, 500. Mu.L of washing solution (50 mM Tris-HCl pH8.0, 200mM NaCl, 2mM DTT) was repeatedly washed 5 times, and 600. Mu.L of a solution containing 1mM m was added to remove non-specific binding 7 After the washing solution of GTP is slowly shaken and combined for 1h by a shaking table at 4 ℃, washing is carried out again, the supernatant is discarded, 50 mu L of 2x loading buffer is added into the agar magnetic beads, and SDS-PAGE coomassie brilliant blue staining or Western-Blot analysis is carried out after boiling.
Western-Blot and SDS-PAGE Coomassie brilliant blue staining both indicate that the binding capacity of MZ34-PB2cap protein, which is eukaryotic expressed by using pCAGGS as a vector, to m7GTP in vitro is significantly higher than that of SH14-PB2cap protein (FIG. 6).
Notably, this result is also consistent with other index results for determining differences in MZ34, SH14 strain PB2 protein function: as shown in FIG. 7, under the same conditions that other RNP plasmids (PB 1, PA and NP) are all derived from MZ34 strain, the polymerase activity induced by MZ34-PB2 on 293T cells for 24h is significantly stronger than that of SH14-PB2; as shown in FIG. 8, in the case where the other 7 gene fragments (PB 1 and PA, HA, NP, NA, M, NS) of the recombinant virus are both MZ34 strain-derived, the recombinant virus rMZ containing MZ34-PB2 gene induces a higher level of PB2 protein expression in CEF cells than in the recombinant virus rMZ-SH 14 containing SH14-PB2 gene in 3h and 6h at the early stage of infection PB2 . Thus, we believe that the invention employs a eukaryotic expressed PB2cap protein with the 5' end cap structure of mRNA (m 7 GTP) to evaluate RNA cap binding activity of influenza virus in vitro, and can provide a new detection index for differential studies of PB2 protein function.
Finally, it should be noted that the above description is only of the preferred embodiments of the invention and is not intended to limit the invention. While the invention has been described in detail with reference to the preferred embodiments thereof, it will be apparent to one skilled in the art that modifications may be made to the techniques described above, or equivalents may be substituted for elements thereof. Modifications, equivalents, and the like, which are within the spirit and principles of the present invention, are intended to be included within the scope of the present invention.
SEQUENCE LISTING
<110> university of Yangzhou
<120> eukaryotic expression of influenza A virus PB2cap protein and uses thereof
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 43
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
catcattttg gcaaagaatt atgaggatca gttcatcctt cag 43
<210> 2
<211> 66
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
ggaaaaagat ctgctagctc gattagtggt gatggtgatg atgcatttta ctaactctca 60
ctcctc 66
<210> 3
<211> 44
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
catcattttg gcaaagaatt atgaggatca gttcatcttt tagc 44
<210> 4
<211> 66
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
ggaaaaagat ctgctagctc gattagtggt gatggtgatg atgcatttta ctaactctca 60
ctcctc 66
<210> 5
<211> 557
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
catcattttg gcaaagaatt atgaggatca gttcatcctt cagcttcgga ggtttcactt 60
ttaaaaggac aagtgggtca tctgtcaaaa gggaagaaga agtgctcaca ggcaacctcc 120
aaacattgaa aataagagta catgaaggat atgaggaatt cacaatggtt gggagaagag 180
caacagccat tctaaggaaa gcaaccagaa gactgattca actgatagtg agtgggaaag 240
acgagcaatc aatcgccgag gcaatcatag tggcaatggt gttctcacaa gaggattgta 300
tgataaaggc agtgagaggt gatttgaact ttgtcaacag agcgaaccag cggctaaatc 360
ccatgcatca actcctgagg catttccaaa aggatgcaaa ggtcctgttc caaaactggg 420
gaattgagcc cattgacaat gtaatgggga tgatcggaat attgcctgac atgaccccca 480
gcacagagat gtccttgaga ggagtgagag ttagtaaaat gcatcatcac catcaccact 540
aatcgagcta gcagatc 557
<210> 6
<211> 557
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
catcattttg gcaaagaatt atgaggatca gttcatcttt tagctttgga ggattcactt 60
tcaaaagaac aagtgggtca tctgtcaaga aggaagagga agtgcttacg ggcaacctcc 120
aaacactgaa aataagagta catgaggggt atgaggaatt cacaatggtt gggcgaagag 180
caacagctat cctgaggaaa gcaaccagaa ggctgattca gctgatagta agtggaagag 240
atgaacaatc aatcgctgaa gcgatcattg tagcaatggt gttctcacag gaagattgca 300
tgataaaagc agtcagaggt gatctgaatt tcgtaaatag agcaaatcaa aggttaaacc 360
ccatgcatca actccttagg cacttccaaa aagatgcaaa ggtgctattt cagaactggg 420
gaattgaacc tattgacgat gtcatgggga tgatcggaat attacctgac atgactccaa 480
gcacagagat gtcactgaga ggagtaagag ttagtaaaat gcatcatcac catcaccact 540
aatcgagcta gcagatc 557
Claims (4)
1. Application of eukaryotic expressed PB2cap protein of influenza A virus in detecting RNA cap binding activity of influenza A virus.
2. The use according to claim 1, wherein said eukaryotic expressed influenza a virus PB2cap protein is obtained by the following method: and adding a histidine tag at the 3' -end of the cap-binding domain to construct eukaryotic expression plasmid, transfecting 293T cells for 48 hours by using the recombinant plasmid, collecting cell lysates, and separating and purifying by using a nickel affinity chromatography column to obtain PB2cap and histidine-tagged fusion protein.
3. The use according to claim 2, wherein in constructing the eukaryotic expression plasmid, the eukaryotic expression vector is pCAGGS and the cleavage sites are ecori, xhoi.
4. The use according to claim 1, wherein the eukaryotic expressed PB2cap protein is associated with m in vitro 7 GTP was subjected to cap binding experiments as follows:
1) Suction 100 mu L m 7 GTP agar beads were transferred to a new 1.5mL finger tube;
2) Centrifuging at 500rpm for 30s to pellet the agar beads, carefully discarding the supernatant;
3) Adding 500 mu LTBS, re-suspending the agar magnetic beads at pH of 8.0, repeating the step 2), washing the agar magnetic bead preservation solution for balancing, and repeating balancing for 2 times;
4) At well balanced m 7 50 mug eukaryotic expression PB2cap protein is added into GTP agar magnetic beads, and a shaking table at 4 ℃ is slowly shaken to be combined for 3 hours;
5) Centrifuging at 500rpm for 30s to pellet the agar beads, carefully discarding the supernatant;
6) Adding 500 mu L of washing solution to resuspend the agar magnetic beads, repeating the step 5) to wash unbound proteins, and repeating the washing for 5 times;
7) To remove non-specific binding, 600. Mu.L of a solution containing 1mM m was added 7 Washing liquid of GTP, and shaking the shaking table at 4 ℃ slowly for combination for 1h;
8) Centrifuging at 500rpm for 30s to pellet the agar beads, carefully discarding the supernatant;
9) 50. Mu.L of 2 Xloadingbuffer was added to the agar beads, and after boiling, SDS-PAGE Coomassie Brilliant blue staining or Western-Blot analysis was performed.
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