Method for preparing human metapneumovirus nucleocapsid protein based on insect-baculovirus system

Abstract

The invention discloses a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system. Comprising the following steps: 1) constructing a recombinant vector with a human metapneumovirus nucleocapsid protein coding sequence: sequentially connecting a sequence for promoting secretory expression, the human metapneumovirus nucleocapsid protein coding sequence and a tag sequence from 5'to 3 'ends in the recombinant vector; 2) converting the recombinant vector with the human metapneumovirus nucleocapsid protein coding sequence constructed in the step 1) into a competent cell containing a baculovirus genome plasmid Bacmid to obtain a recombinant baculovirus genome plasmid; and 3) transfecting insect cells with the recombinant baculovirus genome plasmid obtained in the step 2), culturing and purifying to obtain the human metapneumovirus nucleocapsid protein. The invention provides the preparation method of the human metapneumovirus nucleocapsid protein, and a material support is provided for subsequent further research.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and specifically relates to a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system. Background Art

[0002] Human metapneumovirus is a single-stranded negative-sense RNA virus that can cause respiratory diseases. Since it was first discovered in 2001, there have been reports of infections all over the world. Especially in winter and spring, the elderly, children, and people with low immunity are at risk of infection. However, currently, no company on the market produces and sells human metapneumovirus nucleocapsid protein, and there is no patent application related to the expression of human metapneumovirus nucleocapsid protein in China. Summary of the Invention

[0003] The purpose of this method is to provide a preparation method and its application for stably expressing human metapneumovirus nucleocapsid protein.

[0004] The present invention provides a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system, including:

[0005] 1) Constructing a recombinant vector carrying the coding sequence of human metapneumovirus nucleocapsid protein: In the recombinant vector, a sequence promoting secretory expression, the coding sequence of human metapneumovirus nucleocapsid protein, and a tag sequence are sequentially connected from the 5' end to the 3' end; the coding sequence of human metapneumovirus nucleocapsid protein is as shown in Sequence 2;

[0006] 2) Transforming the recombinant vector carrying the coding sequence of human metapneumovirus nucleocapsid protein constructed in step 1) into competent cells containing the baculovirus genomic plasmid Bacmid to obtain a recombinant baculovirus genomic plasmid;

[0007] 3) Transfecting the recombinant baculovirus genomic plasmid obtained in step 2) into insect cells, continuously transfecting the P0 generation virus in the culture supernatant into insect cells at least once, and purifying the recombinant baculovirus particles in the culture supernatant to obtain human metapneumovirus nucleocapsid protein.

[0008] Further, the tag sequence is the Flag and His tags as shown in Sequence 3.

[0009] Further, the recombinant vector is the pFastBac-N-Flag-His vector.

[0010] Further, the competent cells of the baculovirus genomic plasmid Bacmid are DH10Bac competent cells.

[0011] Further, the insect cells are ExpiSf9 TM Insect cells.

[0012] The present invention also claims to protect the human metapneumovirus nucleocapsid protein prepared by the above method using an insect-baculovirus system.

[0013] The applications of the above method or the above protein in any of the following aspects should also be within the protection scope of the present invention:

[0014] 1) The application of the human metapneumovirus nucleocapsid protein in the preparation of products for detecting human metapneumovirus infection;

[0015] 2) The application of the human metapneumovirus nucleocapsid protein in the preparation or screening of anti-human metapneumovirus drugs;

[0016] 3) The application of the human metapneumovirus nucleocapsid protein in the preparation or screening of anti-human metapneumovirus vaccines.

[0017] The beneficial effects of the present invention are as follows: The present invention provides a preparation method of the human metapneumovirus nucleocapsid protein, providing material support for subsequent further research. As the most abundant protein in human metapneumovirus, the nucleocapsid protein can play a role in the clinical detection of human metapneumovirus infection in children, the elderly, and immunocompromised populations; the nucleocapsid protein gene is very conservative and can also be used to screen and obtain antiviral drugs or vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 For insect cells ExpiSf9 TM The western-blot result of the N protein in the cell culture medium 72 hours after the insect cells ExpiSf9 were infected with the P1 generation virus carrying the baculovirus genome fused with the human metapneumovirus nucleocapsid (N) protein. Since the N protein is fused with the detection tag flag, mouse anti-flag primary antibody and goat anti-mouse secondary antibody were used to detect whether the target protein was contained in the culture medium.

[0019] Figure 2 For the SDS-PAGE diagram of the human metapneumovirus nucleocapsid protein finally obtained by using the preparation and purification method described in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0021] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0022] The experimental materials in the following examples: pFastBac-I plasmid, DH5α competent cells (purchased from TransGenBiotech), DH10Bac competent cells, kanamycin, gentamicin, tetracycline (all purchased from Solarbio), yeast extract, tryptone (both purchased from Oxoid), NaCl (purchased from Solarbio), ExpiFectamine Sf TM Transfection reagent (Thermo, product number A38915), ExpiSf TM CD medium (Thermo, product number A3767802), Ni Sepharose 6 Fast Flow packing material (purchased from Cytiva).

[0023] Example 1 Preparation method of human metapneumovirus nucleocapsid protein (N protein)

[0024] The gene sequence of human metapneumovirus (hMPV) is shown as ncbi reference sequence YP_009513265.1. The coding nucleotide sequence of the nucleocapsid (N) protein of human metapneumovirus (hMPV) prepared in the present invention is shown as Sequence 1, and the amino acid sequence of the encoded protein is shown as Sequence 2. Its preparation method includes the following steps:

[0025] 1. Preparation of recombinant plasmid:

[0026] Add Flag (shown as Sequence 5, and the encoded protein is shown as Sequence 6) and His tag (shown as Sequence 3, and the encoded protein is shown as Sequence 4) to the C-terminus of the coding sequence of the nucleocapsid protein of human metapneumovirus (i.e., the sequence shown as Sequence 1) to obtain a fusion sequence; use the fusion sequence to replace the sequence between the BamHⅠ and XhoⅠ restriction enzyme sites on the pFastBac-I vector, and keep other sequences unchanged to obtain the recombinant plasmid pFastBac-N-Flag-His. The plasmid sequencing result is correct.

[0027] 2. Transformation of recombinant plasmid

[0028] The recombinant plasmid pFastBac-N-Flag-His was transformed into DH10Bac competent cells, and the cells were spread on an LB solid plate containing antibiotics kanamycin + gentamicin + tetracycline + IPTG + X-GAL for blue-white screening (working concentration of gentamicin: 10 μg / ml; working concentration of kanamycin: 50 μg / ml; working concentration of tetracycline: 10 μg / ml. IPTG and X-GAL can be spread directly after the solid medium solidifies at the bottom of the bacterial culture dish. IPTG was dissolved in H2O to prepare a stock solution with a concentration of 50 mg / ml. After filtration, 40 μl was added to every 10 ml of the screening medium; X-Gal was dissolved in DMF to prepare a stock solution with a concentration of 20 mg / ml. After filtration, 40 μl was added to every 10 ml of the screening medium), and cultured inverted in the dark at 37 °C for more than 48 hours.

[0029] 3. Pick white colonies, perform colony PCR using pUC / M13 primers (as shown in Sequence 7 and Sequence 8), and select the positive colonies for amplification.

[0030] 4. Use an LB liquid medium containing antibiotics kanamycin + gentamicin + tetracycline for amplification and obtain Bacmid (the Bacmid refers to the plasmid pFastBac-N-Flag-His with a baculovirus genome that has successfully ligated the coding sequence of the nucleocapsid (N) protein of human metapneumovirus (hMPV)).

[0031] 5. Transfect the Bacmid into 25 ml of ExpiSf9 6 insect cells at a density of 2.5 × 10 TM cells / ml (cell viability ≥ 90%), place them in a shaker at 27 °C and 125 rpm, and after 3 - 4 days, centrifuge at 350 g for 5 min to collect the supernatant.

[0032] 6. The product obtained in step (5) is the 0th generation virus (P0). Take 2 ml to infect ExpiSf9 6 cells at a density of 5 × 10 TM cells / ml, place them in a shaker at 27 °C and 125 rpm, and the cell supernatant can be taken for western-blot detection 5 - 6 days after infection.

[0033] 7. The 1st generation virus (P1) obtained in step (6) can be used for small-scale protein preparation. If a relatively large amount of protein is needed, the 2nd generation virus (P2) needs to be prepared, and the procedure is the same as (6).

[0034] 8. After centrifuging the cell culture in step (7) at 4000 rpm for 5 min, the supernatant is taken and passed through a nickel column, and eluted with 50 mM Tris, 150 mM NaCl, and 300 mM imidazole to obtain the N protein.

[0035] 9. Result verification:

[0036] 1) Perform WB detection on the P1 generation virus supernatant in step 7) (run SDS gel on the supernatant, transfer the membrane, block with 10% skim milk for 1 h, incubate with flag primary antibody overnight at 4 °C, wash three times with TBST, incubate with secondary antibody at room temperature for 1 h, wash three times with TBST and then expose), and the results are as Figure 1 shown. It can be seen from Figure 1 that the target protein with a size of 54 kDa can be detected in the supernatant of ExpiSf9 TM insect cells infected with the P1 generation baculovirus containing the human metapneumovirus N protein gene sequence.

[0037] 2) The SDS-PAGE result of the purified human metapneumovirus N protein is as Figure 2 shown. Figure 2 In Figure 2 , the first lane is the marker band, from top to bottom are 170 kDa, 130 kDa, 100 kDa, 70 kDa (red), 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, and 10 kDa (green). Since 10% polyacrylamide gel is used, the 15 kDa band and the 10 kDa band cannot be separated. The picture can be obtained after staining with Coomassie Brilliant Blue and decolorization.

[0038] In

[0039] , by using this method, a relatively pure human metapneumovirus N protein with a size of 54 kDa can be obtained.

[0040] The sequences involved in the present invention are as follows:

[0041] hMPV N base sequence: (Sequence 1)

[0042] ACAACCACCGCCGTGACCCCTAGCTCCCTGCAACAGGAGATCACACTGCTTTGTGGCGAGATCCTGTACGCCAAACATGCAGATTACAAGTACGCCGCTGAGATCGGCATCCAATACATCAGCACAGCCCTGGGCAGCGAGAGAGTGCAGCAGATCCTGCGCAACAGCGGCAGCGAGGTGCAGGTGGTGCTCACCCGGACCTACAGCCTGGGAAAAATTAAGAACAACAAGGGCGAAGACCTGCAGATGCTGGACATCCACGGCGTGGAAAAGTCCTGGGTCGAGGAAATCGACAAGGAAGCCAGAAAGACAATGGCCACCCTGCTGAAGGAAAGCTCAGGCAACATCCCTCAGAACCAGAGACCTTCTGCCCCTGATACACCTATCATTCTGCTGTGCGTGGGCGCTCTGATCTTCACCAAGCTGGCTAGCACCATCGAGGTGGGCCTGGAAACAACCGTGCGGAGAGCAAATAGAGTGCTGAGCGATGCCCTGAAAAGATACCCCAGAATGGACATCCCCAAGATCGCCCGGAGCTTCTACGACCTCTTCGAGCAGAAGGTGTACCACCGGAGCCTGTTCATCGAGTACGGCAAAGCCCTGGGCTCGTCTTCTACCGGCTCTAAGGCCGAATCCCTGTTCGTGAACATCTTTATGCAGGCCTACGGCGCCGGACAGACCATGCTGAGATGGGGCGTGATCGCCAGAAGCAGCAACAACATCATGCTGGGCCACGTGTCCGTGCAAGCTGAACTGAAGCAGGTTACAGAGGTTTACGACCTGGTGCGGGAAATGGGCCCAGAGAGCGGCCTGCTGCACCTGAGGCAGAGCCCAAAGGCTGGACTGCTCAGCCTGGCCAACTGCCCCAATTTCGCCAGCGTGGTGCTGGGAAATGCCAGCGGCCTGGGTATTATCGGCATGTACCGTGGAAGAGTGCCTAATACCGAGCTGTTTAGCGCCGCTGAGAGCTATGCCAAGAGCCTGAAAGAGAGCAAC

[0043] hMPV N amino acid sequence: (Sequence 2)

[0044] TTTAVTPSSLQQEITLLCGEILYAKHADYKYAAEIGIQYISTALGSERVQQILRNSGSEVQVVLTRTYSLGKIKNNKGE

[0045] DLQMLDIHGVEKSWVEEIDKEARKTMATLLKESSGNIPQNQRPSAPDTPIILLCVGALIFTKLASTIEVGLETTVRRAN

[0046] RVLSDALKRYPRMDIPKIARSFYDLFEQKVYHRSLFIEYGKALGSSSTGSKAESLFVNIFMQAYGAGQTMLRWGVIARS

[0047] SNNIMLGHVSVQAELKQVTEVYDLVREMGPESGLLHLRQSPKAGLLSLANCPNFASVVLGNASGLGIIGMYRGRVPNTE

[0048] LFSAAESYAKSLKESN

[0049] His tag sequence: (Sequence 3)

[0050] CACCACCACCACCACCAC,

[0051] The corresponding amino acid sequence is: (Sequence 4)

[0052] HHHHHH

[0053] Flag tag sequence: (Sequence 5)

[0054] GATTACAAGGATCATGATGGAGACTACAAGGACCACGACATCGATTACAAGGATGACGACGATAAG, the corresponding amino acid sequence is: (Sequence 6)

[0055] DYKDHDGDYKDHDIDYKDDDDK pUC / M13 primer forward primer(Sequence 7)

[0056] (5'-CCCAGTCACGACGTTGTAAAACG-3'),

[0057] and pUC / M13 reverse primer(Sequence 8)

[0058] (5'-AGCGGATAACAATTTCACACAGG-3')

Claims

1. A method for preparing human metapneumovirus nucleocapsid protein based on the insect-baculovirus system, characterized in that, Comprising: 1) Constructing a recombinant vector carrying the coding sequence of the human metapneumovirus nucleocapsid protein: In the recombinant vector, a sequence promoting secretory expression, the coding sequence of the human metapneumovirus nucleocapsid protein, and a tag sequence are sequentially linked from the 5'-end to the 3'-end; the coding sequence of the human metapneumovirus nucleocapsid protein is as shown in Sequence 2; 2) Transforming the recombinant vector carrying the coding sequence of the human metapneumovirus nucleocapsid protein constructed in step 1) into competent cells containing the baculovirus genomic plasmid Bacmid to obtain a recombinant plasmid carrying the baculovirus genome; 3) Transfecting the recombinant plasmid carrying the baculovirus genome obtained in step 2) into insect cells, for the P0 generation virus in the culture supernatant, continuously transfecting insect cells at least once, and purifying the culture supernatant to obtain the human metapneumovirus nucleocapsid protein.

2. The method according to claim 1, characterized in that, The tag sequence is the Flag and His tag sequences.

3. The method according to claim 1, wherein The recombinant vector is the pFastBac-N-Flag-His vector. The pFastBac-N-Flag-His vector adds Flag and His tags to the C-terminus of the coding sequence of the nucleocapsid protein of the human metapneumovirus to obtain a fusion sequence; the sequence between the BamHⅠ and XhoⅠ restriction enzyme sites on the pFastBac-Ⅰ vector is replaced with the fusion sequence, and other sequences remain unchanged to obtain a recombinant plasmid.

4. The method according to claim 1, wherein The competent cells of the baculovirus genomic plasmid Bacmid are DH10Bac competent cells.

5. The method according to claim 1, characterized in that, The insect cells are ExpiSf9TM insect cells.

6. Preparing the human metapneumovirus nucleocapsid protein using the insect-baculovirus system prepared by the method according to any one of claims 1-5.

7. Use of the method according to any one of claims 1-5 or the protein according to claim 6 in the preparation of a product for detecting human metapneumovirus infection.

8. Use of the method according to any one of claims 1-5 or the protein according to claim 6 in the preparation or screening of anti-human metapneumovirus drugs.

9. Use of the method according to any one of claims 1-5 or the protein according to claim 6 in the preparation or screening of anti-human metapneumovirus vaccines.

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