A membrane fusion protein mutant PSI-7M induced under weak acidic conditions, its encoding gene and application

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to a membrane fusion protein mutant PSI-7M induced under weakly acidic conditions, its encoding gene and application. The present invention provides an induced membrane fusion protein mutant PSI-7M, and the amino acid sequence of the induced membrane fusion protein mutant PSI-7M is shown in SEQ ID NO.1. The present invention introduces 7 amino acid mutations into the wild-type PSI protein to change the isoelectric point of the protein, and then can achieve the activity of mediating membrane fusion in the range of 4.5≤pH<7 under weakly acidic conditions through the distribution of its own surface charge. The membrane fusion-inducing protein mutant PSI-7M of the present invention breaks through the characteristic that the original protein can only perform membrane fusion under relatively strong acidic conditions, and significantly improves the scope of application of PSI protein cell membrane fusion.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a membrane fusion protein mutant PSI-7M induced under weakly acidic conditions, a coding gene thereof and an application thereof. Background Art

[0002] Aspartic protease (AP) is an important class of proteolytic enzymes that participate in the body's metabolism and biological regulation. It is widely present in animals, plants, and pathogenic bacteria cells. APs are found in plant organs and tissues such as flowers, leaves, and stems. In plants, APs are related to physiological processes such as protein precursor processing, protein digestion, and programmed cell apoptosis. Plant APs are mainly composed of three parts: the N-terminal domain, the plant-specific insertion sequence (PSI), and the C-terminal structure. The N-terminal region and the C-terminal region have a high degree of homology with the N-terminal and C-terminal domains of AP proteins in mammals and microorganisms. The PSI sequence is unique to plant APs. APs in mammalian cells do not contain the PSI domain and are unique to plant cells.

[0003] In plant cells, PSI exists in the form of zymogen as part of AP enzyme and has antibacterial defense function of plant cells. After infection by exogenous pathogens, AP zymogen is transported to the vacuole and processed into mature AP hydrolase in the acidic environment of the vacuole. In this process, PSI separates from APs and interacts with the biological membrane of the vacuole to induce the fusion of the vacuole membrane and the cell membrane. Using genetic engineering, recombinantly expressed PSI protein also has antibacterial activity and shows resistance to a variety of pathogens that infect plants or human cells. However, natural PSI protein has the activity of inducing membrane fusion and exerting antibacterial effect only when the pH is lower than 4.5. In vivo, the microenvironment of most cells is a neutral solution environment with a pH between 7.0-7.4; or in some local infection sites, the cells are in a weak acid environment, that is, the pH is between 6.0-7.0. Therefore, in this environment, the natural PSI protein does not have the ability to induce membrane fusion. Therefore, it is particularly urgent to modify PSI protein so that it has membrane fusion activity under near-neutral conditions. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a membrane fusion protein mutant PSI-7M induced under weakly acidic conditions and its encoding gene and application, which can efficiently induce membrane fusion under conditions where the pH value is higher than 4.5.

[0005] The present invention provides a membrane fusion inducing protein mutant PSI-7M, and the amino acid sequence of the membrane fusion inducing protein mutant PSI-7M is shown in SEQ ID NO.1.

[0006] The present invention also provides a gene encoding the protein mutant PSI-7M described in the above technical solution.

[0007] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector encoding the protein mutant PSI-7M described in the above technical solution.

[0009] Preferably, the recombinant vector includes a starting vector and a gene encoding the protein mutant PSI-7M described in the above technical solution.

[0010] The present invention also provides a method for preparing the protein mutant PSI-7M described in the above technical solution, comprising the following steps:

[0011] Using pET-32a(+) as a starting vector, inserting the coding gene described in the above technical solution between the Msc I and Xho I restriction sites of the pET-32a(+), deleting the His tag carried by pET-32a(+), and keeping the other sequences of pET-32a(+) unchanged, to obtain a recombinant vector;

[0012] Transforming host cells with the recombinant vector to obtain recombinant bacteria;

[0013] Cultivating the recombinant bacteria to obtain a culture solution containing the expressed protein;

[0014] The culture fluid containing the expressed protein is recovered and purified to obtain the protein mutant PSI-7M.

[0015] The present invention also provides a recombinant bacterium expressing the protein mutant PSI-7M described in the above technical solution.

[0016] The present invention also provides the use of the protein mutant PSI-7M or the gene or the protein mutant PSI-7M obtained by the preparation method described in the above technical scheme in products that induce membrane fusion.

[0017] Preferably, when the product is used to induce membrane fusion, 4.5≤pH<7.

[0018] The present invention discloses an induced membrane fusion protein mutant PSI-7M, and the amino acid sequence of the induced membrane fusion protein mutant PSI-7M is shown in SEQ ID NO.1. According to the different dynamic properties of different amino acids in the process of binding to the membrane of the wild-type PSI protein, the present invention introduces mutations into the wild-type PSI protein, and mutates some glutamic acid and aspartic acid residues in the wild-type PSI protein into histidine and glutamine. Specifically, the D at position 40 of the wild-type PSI protein is mutated to H, the E at position 54 is mutated to H, the E at position 56 is mutated to H, the E at position 58 is mutated to H, the E at position 64 is mutated to H, the E at position 89 is mutated to Q, and the E at position 93 is mutated to Q. The mutations of the present invention increase the number of positive charges on the surface of the PSI protein, making it easier for the PSI protein to bind to the cell membrane, and can still have a strong activity of mediating membrane fusion under the condition of 4.5≤pH<7. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 The results of SDS-PAGE experiments were performed in Example 2.

[0021] Figure 2 The results of SDS-PAGE experiment in step (5) of Comparative Example 1;

[0022] Figure 3 The results of SDS-PAGE experiment in step (7) of Comparative Example 1;

[0023] Figure 4 is the efficiency of wild-type protein PSI in mediating membrane fusion under different pH conditions;

[0024] Figure 5 The figure shows the efficiency of the protein mutant PSI-7M in mediating membrane fusion under different pH conditions. DETAILED DESCRIPTION

[0025] The present invention provides a membrane fusion inducing protein mutant PSI-7M, and the amino acid sequence of the membrane fusion inducing protein mutant PSI-7M is shown in SEQ ID NO.1.

[0026] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is specifically: IVSMECKTIVSQYGEMIWDLLVSGVRPDQVCSQAGLCFVHGAQHVSSNIKTVVHRHTHGSSVGHAPLCTACEMAVVWMQNQLKQEGTKQKVLQYVNQLCEKIP. The induced membrane fusion protein mutant PSI-7M of the present invention is a natural protein PSI in which the D at position 40 is mutated to H, the E at position 54 is mutated to H, the E at position 56 is mutated to H, the E at position 58 is mutated to H, the E at position 64 is mutated to H, the E at position 89 is mutated to Q, and the E at position 93 is mutated to Q. The amino acid sequence of the natural protein PSI of the present invention is shown in SEQ ID No. 3, specifically: IVSMECKTIVSQYGEMIWDLLVSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0027] The present invention also provides a gene encoding the protein mutant PSI-7M described in the above technical solution. In the present invention, the nucleotide sequence of the gene is preferably as shown in SEQ ID NO.2, specifically 5'-ATCGTTTCCATGGAATGCAAGACTATTGTTTCTCAGTATGGTGAGATGATCTGGGACCTGCTGGTGTCTGGTGTGCGTCCGGACCAGGTGTGTTCTCAGGCAGGTCTGTGCTTCGTGCACGGCGCGCAGCACGTATCCTCCAACATCAAAACCGTTGTTCATCGCCACACTCACGGTTCCAGCGTTGGCCACGCCCCGCTGTGCACCGCATGCGAGATGGCCGTGGTTTGGATGCAGAACCAGCTGAAACAGGAGGGTACTAAGCAGAAAGTCCTGCAGTACGTTAACCAGCTGTGCGAAAAAATTCCG-3'.

[0028] The present invention also provides a recombinant vector comprising the gene described in the above technical solution. In the present invention, the recombinant vector preferably includes a starting vector and the gene described in the above technical solution. The starting vector of the present invention preferably includes pET-32a(+). In the specific implementation of the present invention, it is preferred to insert the gene described in the above technical solution between the MscI and Xho I restriction sites of pET-32a(+), delete the His tag carried by the original plasmid vector itself, and keep the other sequences of pET-32a(+) unchanged to obtain the recombinant vector. The plasmid pET-32a(+) described in the present invention preferably carries ampicillin resistance. The present invention has no strict requirements on the method for constructing the recombinant vector, and a method well known in the art can be used.

[0029] The present invention also provides a method for preparing the induced membrane fusion protein mutant PSI-7M described in the above technical solution, characterized in that it comprises the following steps:

[0030] Using pET-32a(+) as a vector, inserting the coding gene described in the above technical solution between the Msc I and Xho I restriction sites of the pET-32a(+), deleting the His tag carried by pET-32a(+), and keeping the other sequences of pET-32a(+) unchanged, to obtain a recombinant vector;

[0031] Transforming the recombinant vector into Escherichia coli host cells to obtain recombinant bacteria;

[0032] Cultivating the recombinant bacteria to obtain a culture solution containing the expressed protein;

[0033] The culture fluid containing the expressed protein is recovered and purified to obtain the protein mutant PSI-7M.

[0034] The present invention transforms the recombinant vector described in the above technical solution into a host cell to obtain a recombinant bacterium. In the present invention, the host cell preferably includes an Escherichia coli competent cell, and more preferably Rosetta-gamiB (DE3) pLysS. The Rosetta-gamiB (DE3) pLysS described in the present invention is purchased from Shanghai Jingkang Biological Co., Ltd. The present invention has no strict requirements on the method of transformation, and a method well known in the art can be used.

[0035] After obtaining the recombinant bacteria, the present invention cultures the recombinant bacteria to obtain a culture solution containing the expressed protein. The present invention preferably uses an LB culture medium containing antibiotics to culture the recombinant bacteria. The antibiotics described in the present invention preferably include kanamycin (Kana), chloramphenicol (Chl), tetracycline (Tet) and ampicillin (Amp); the concentration of kanamycin in the LB culture medium is preferably 15 μg / mL, the concentration of chloramphenicol is preferably 34 μg / mL, the concentration of tetracycline is preferably 12.5 μg / mL, and the concentration of ampicillin is preferably 50 μg / mL. The temperature of the culture described in the present invention is preferably 37°C, the time of the culture is preferably 16 hours, and the rotation speed of the culture is preferably 250rpm. The present invention preferably uses an incubator for the culture.

[0036] After obtaining the culture fluid containing the expressed protein, the present invention preferably recovers and purifies the culture fluid containing the expressed protein to obtain the protein mutant PSI-7M. In the present invention, the recovery and purification preferably use a Ni-NTA column. The recovery and purification steps of the present invention preferably include: combining the cultured protein with the balanced Ni-NTA column, washing with buffer 1, buffer 2 and buffer 3 in sequence, collecting the eluate after the buffer 3 column; dialyzing the eluate after the buffer 3 column for the first time to obtain a dialysate; after enzymatic digestion of the dialysate, washing with buffer 1 and buffer 3 in sequence, collecting the eluate after the buffer 3 column; dialyzing the eluate after the buffer 3 column for the second time to obtain a purified protein. The buffer 1 of the present invention includes Tris and NaCl; the concentration of Tris in the buffer 1 is preferably 20mM, and the concentration of NaCl is preferably 300mM; the pH of the buffer 1 is preferably 7.42. The buffer 2 of the present invention includes Tris, NaCl and imidazole; the concentration of Tris in the buffer 2 is preferably 20mM, the concentration of NaCl is preferably 300mM, and the concentration of imidazole is preferably 20mM; the pH of the buffer 2 is preferably 7.42. The buffer 3 of the present invention includes Tris, NaCl and imidazole; the concentration of Tris in the buffer 3 is preferably 20mM, the concentration of NaCl is preferably 300mM, and the concentration of imidazole is preferably 300mM; the pH of the buffer 3 is preferably 7.42.

[0037] In the present invention, the first dialysis preferably uses a dialysis bag with a molecular weight cutoff of 7 kDa; the second dialysis preferably uses a dialysis bag with a molecular weight cutoff of 1 kDa.

[0038] In the present invention, when the dialysate obtained from the first dialysis is subjected to enzymatic cleavage, thrombin is preferably used; the activity of the thrombin is preferably 0.5U / μL; and the amount of the thrombin is preferably 1μL / mg protein. The present invention uses thrombin for enzymatic cleavage to remove the TrxA solubilizing tag connected to the protein, thereby avoiding affecting the properties of the protein itself.

[0039] The present invention also provides a recombinant bacterium expressing the protein mutant PSI-7M described in the above technical solution.

[0040] The present invention also provides the use of the membrane fusion inducing protein mutant PSI-7M or the encoding gene or the membrane fusion inducing protein mutant PSI-7M obtained by the preparation method described in the above technical solution in the preparation of a product that induces membrane fusion. In the present invention, when the product is used to induce membrane fusion, 4.5≤pH<7, more preferably 4.8-6.0, and more preferably 5.4.

[0041] The present invention introduces mutations at key positions in the natural protein PSI to affect the charge carried by the protein, so that the isoelectric point pI changes from 4.63 to 7.65, so that the mutated protein can still have a strong activity of mediating membrane fusion under the condition of 4.5≤pH<7. The results of the embodiment show that the protein mutant PSI-7M provided by the present invention has a liposome fusion efficiency of 31.68% after induction for 1200s at pH=3.4; the liposome fusion efficiency of 45.00% after induction for 1200s at pH=4.8; the liposome fusion efficiency of 69.27% ​​after induction for 1200s at pH=5.4; and the liposome fusion efficiency of 49.00% after induction for 1200s at pH=6.0.

[0042] To further illustrate the present invention, a membrane fusion protein mutant PSI-7M induced under weakly acidic conditions, its encoding gene and application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] Construction of recombinant vector pET-32a(+)-PSI-7M

[0045] (1) Site-directed mutagenesis

[0046] Using a plasmid containing a coding gene of a natural protein PSI (specific nucleotide sequence: SEQ ID NO.4: 5'-ATCGTTTCCATGGAATGCAAGACTATTGTTTCTCAGTATGGTGAGATGATCTGGGACCTGCTGGTGTCTGGTGTGCGTCCGGACCAGGTGTGTTCTCAGGCAGGTCTGTGCTTCGTGGACGGCGCGCAGCACGTATCCTCCAACATCAAAACCGTTGTTGAGCGCGAGACTGAAGGTTCCAGCGTTGGCGAGGCCCCGCTGTGCACCGCATGCGAGATGGCCGTGGTTTGGATGCAGAACCAGCTGAAACAGGAGGGTACTAAGGAGAAAGTCCTGGAGTACGTTAACCAGCTGTGCGAAAAAATTCCG-3') as a template, site-directed mutagenesis was performed to obtain a coding gene shown in SEQ ID NO.2;

[0047] (2) Insert the gene shown in SEQ ID NO.2 between the Msc I and Xho I restriction sites of PET-32a(+), delete the His tag carried by the original plasmid vector itself, and obtain the recombinant vector pET-32a(+)-PSI-7M. The specific steps are as follows.

[0048] The above steps were entrusted to Nanjing GenScript Biotech Co., Ltd.

[0049] Example 2

[0050] (1) Preparation of competent cells: 500 μL of Rosetta-gami B (DE3) pLysS strain was inoculated into 50 mL LB liquid medium and cultured at 37°C, 220 rpm for 4 h. 600 Between 0.4 and 0.5, transfer the bacterial solution to a 50mL centrifuge tube, place on ice for 20 minutes, centrifuge at 4°C, 4000rpm for 10 minutes, and discard the supernatant. Add 10mL of pre-cooled 0.1M CaCl2 to the precipitate, resuspend the cell precipitate, centrifuge at 4°C, 4000rpm for 10 minutes, discard the supernatant, and repeat twice. Finally, add 3mL of pre-cooled 0.1M CaCl2, resuspend the cells again, use a pipette to distribute the competent cells, add 100μL to each 1.5mL EP tube, add an equal volume of 50% glycerol, and store at -80°C.

[0051] (2) Transformation: Take out 4 μg of the recombinant vector pET-32a(+)-PSI-7M dry powder obtained in Example 1 and add 80 μL of ddH2O to dissolve. Take 10 μL of the plasmid aqueous solution and add it to the competent cells prepared in step (1), gently shake and mix, and ice bath for 25 minutes. Then place the bacterial solution in a 42°C water bath, heat shock for 45 seconds, and ice bath the bacterial solution for 2 minutes after heat shock. Then add 2 mL of LB liquid culture medium to the bacterial solution, place it in a shaker at 37°C and 220 rpm for 60 minutes. Take 50 μL of the bacterial solution and spread it on the LB solid culture medium to which 15 μg / mL Kana, 34 μg / mL Chl, 12.5 μg / mL Tet and 50 μg / mL Amp have been added, and culture overnight.

[0052] (3) Sequencing and preservation: Take a single colony after overnight culture, extract the plasmid, and sequence it. The sequencing results show that there is a nucleotide sequence consistent with SEQ ID NO.2, and the recombinant bacteria are obtained; the recombinant bacteria are inoculated into LB liquid culture medium to which 15μg / mL Kana, 34μg / mL Chl, 12.5μg / mL Tet and 50μg / mL Amp have been added, and cultured in a shaking incubator at 250rpm and 37℃ for 16h. Take 700μL of the bacterial solution and inject it into a cryopreservation tube, add 50% glycerol at a volume ratio of 1:1, and the glycerol bacteria are prepared.

[0053] (4) Cultivation of recombinant bacteria: Take 1 mL of glycerol bacteria and add it to 50 mL of LB liquid culture medium (containing 15 μg / mL Kana, 34 μg / mL Chl, 12.5 μg / mL Tet and 50 μg / mL Amp) supplemented with antibiotics, and culture it in a shaker at 37°C and 220 rpm for 8 h. Then, add 50 mL of the bacterial solution to 900 mL of LB liquid culture medium (containing 15 μg / mL Kana, 34 μg / mL Chl, 12.5 μg / mL Tet and 50 μg / mL Amp) supplemented with antibiotics, and culture it under shaking conditions at 37°C and 220 rpm for 3 h. Use a UV spectrophotometer to measure the OD value of the bacterial solution. 600 At 0.6-0.8 (recorded as the pre-induction bacterial solution). Add 500 μL of 1M IPTG solution to the pre-induction bacterial solution, ice bath for 10 minutes, transfer to a 30°C, 200rpm shaker for 14 hours to obtain the post-induction bacterial solution. The post-induction bacterial solution was centrifuged at 4°C, 4000rpm for 10 minutes to collect the cell precipitate. Use 50mL of 20mM Tris buffer (pH 7.42) to resuspend the cell precipitate. Add the resuspended bacterial solution to a high-pressure homogenizer precooled at 4°C, and break the bacteria for 3 minutes at 820bar and 860bar. After the high-pressure homogenizer breaks the bacteria, recover the bacterial solution, centrifuge it at 4°C, 12000rpm for 30 minutes, and collect the supernatant.

[0054] (5) Add the supernatant after centrifugation in step (4) to the Ni-NTA column (which has been rinsed with 20 mM Tris buffer at pH = 7.4), place it in a shaker at 4°C and 200 rpm for 3 hours. After the binding is completed, use 50 mL of buffer 1 (composed of an aqueous solution of Tris and NaCl, pH = 7.42, wherein the Tris concentration is 20 mM and the NaCl concentration is 300 mM), buffer 2 (composed of an aqueous solution of Tris, NaCl and imidazole, pH = 7.42, wherein the Tris concentration is 20 mM, the NaCl concentration is 300 mM, and the imidazole concentration is 20 mM), buffer 3 (composed of an aqueous solution of Tris, NaCl and imidazole, pH = 7.42, wherein the Tris concentration is 20 mM, the NaCl concentration is 300 mM, and the imidazole concentration is 20 mM), and buffer 4 (composed of an aqueous solution of Tris, NaCl and imidazole, pH = 7.42). .42, wherein the Tris concentration is 20 mM, the NaCl concentration is 300 mM, and the imidazole concentration is 300 mM) for washing, and the eluates after the column are collected respectively. The bacterial solution before induction in step (4) (referred to as before induction), the bacterial solution after induction in step (4) (referred to as before after induction), the eluate after washing with buffer 1 (referred to as buffer 1), the eluate after washing with buffer 2 (referred to as buffer 2), and the eluate after washing with buffer 3 (referred to as buffer 3) are used as samples for SDS-PAGE experiment. The results are shown in FIG. Figure 1 shown.

[0055] according to Figure 1 It can be seen that after washing with buffer 3, a 26.7 kDa protein band appeared, confirming that the target protein was in the eluate after washing with buffer 3.

[0056] (6) The eluate containing the target protein (i.e., the eluate after washing with buffer 3) was transferred to a 7kDa dialysis bag and dialyzed overnight in 3L of dialysis solution (20mM Tris, 100mM NaCl) at pH=8.0. The dialyzed protein was then transferred to a 50mL centrifuge tube, and 1μL of thrombin (0.5U / μL) was added per 1mg of protein. The protein solution was digested overnight to obtain the digested protein solution;

[0057] (7) The protein solution after enzymatic digestion and the washed Ni-NTA column were shaken in a shaker at 16°C and 200 rpm for 2 h, and the flow-through was collected. The Ni-NTA column after binding was washed with buffer 1 and buffer 3 in step (5) in sequence, and the eluate after washing with buffer 1 and the eluate after washing with buffer 3 were collected respectively.

[0058] The protein solution after enzyme cleavage obtained in step (6) (referred to as after enzyme cleavage), the flow-through solution in step (7) (referred to as flow-through after cleavage), the eluate after washing with buffer 1 in step (7) (referred to as buffer 1 after cleavage), and the eluate after washing with buffer 3 in step (7) (referred to as buffer 3 after cleavage) were used as samples for SDS-PAGE experiments. The results are as follows: Figure 1shown.

[0059] according to Figure 1 It can be seen that in the eluate after washing with buffer 3, PSI-7M mutant protein appeared with a band size of 11.7 kDa, confirming that the target protein was in the eluate after washing with buffer 3.

[0060] (8) The eluate after washing with buffer 3 in step (7) was transferred to a 1 kDa dialysis bag and dialyzed in 3 L of pure water for 24 h to obtain an aqueous solution of the protein, which was recorded as an aqueous solution of the PSI-7M protein.

[0061] (9) Isoelectric point detection

[0062] The online prediction software ExPASy Compute pI / Mw was used to input the amino acid sequence corresponding to the coding gene shown in SEQ ID NO.2, and the theoretical isoelectric point was calculated, i.e., 7.65, indicating that the isoelectric point of the PSI-7M protein obtained in the present invention is 7.65.

[0063] Comparative Example 1

[0064] 1. Construction of the recombinant vector pET-32a(+)-PSI

[0065] The same as Example 1, except that a plasmid containing a gene encoding a natural protein PSI (specific nucleotide sequence as shown in SEQ ID NO.4) is used as a template, site-directed mutagenesis is not performed, and the His tag of the original pET-32a (+) plasmid vector is not deleted. The nucleotide sequence encoding the natural protein PSI is inserted between the Msc I and Xho I restriction sites of the PET-32a (+) plasmid to obtain a recombinant vector pET-32a (+)-PSI.

[0066] 2. Preparation of PSI protein aqueous solution

[0067] (1) preparing competent cells according to the method of step (1) of Example 2;

[0068] (2) Transformation was performed according to the method of step (2) of Example 2, except that the recombinant vector pET-32a(+)-PSI-7M powder in step (2) of Example 2 was replaced with the recombinant vector pET-32a(+)-PSI powder;

[0069] (3) sequencing and storing according to the method of step (3) of Example 2;

[0070] (4) Cultivating the recombinant bacteria according to the method of step (4) of Example 2;

[0071] (5) The same as step (5) of Example 2, SDS-PAGE experiment was performed with the bacterial solution after induction in step (4) (referred to as after induction), the supernatant after centrifugation in step (4) combined with the flow-through after Ni-NTA column (referred to as flow-through), the eluate after washing with buffer 1 (referred to as buffer 1), the eluate after washing with buffer 2 (referred to as buffer 2), and the eluate after washing with buffer 3 (referred to as buffer 3) as samples. The results are shown in FIG. Figure 2 shown.

[0072] according to Figure 2 It can be seen that after washing with buffer 3, a 26.7 kDa protein band appeared, confirming that the target protein was in the eluate after washing with buffer 3;

[0073] (6) The eluate containing the target protein (i.e., the eluate after washing with buffer 3) was transferred to a 7kDa dialysis bag and dialyzed overnight in 3L of dialysis solution (20mM Tris, 100mM NaCl) at pH=8.0. The dialyzed protein was then transferred to a 50mL centrifuge tube, and 1μL of thrombin (0.5U / μL) was added per 1mg of protein. The protein was digested overnight to obtain the digested protein;

[0074] (7) The protein solution after enzyme digestion and the cleaned Ni-NTA column were shaken in a shaker at 16°C and 200 rpm for 2 hours, and the flow-through was collected. The Ni-NTA column after the combination was washed with buffer 1 and buffer 3 in step (5) in turn, and the eluate after the wash with buffer 1 and the eluate after the wash with buffer 3 were collected respectively. The eluate after the wash with buffer 3 in step (5) (before enzyme digestion), the protein solution after enzyme digestion obtained in step (6) (recorded as post-enzyme digestion), the flow-through in step (7) (recorded as post-cut flow-through), the eluate after the wash with buffer 1 (recorded as buffer 1), and the eluate after the wash with buffer 3 (recorded as buffer 3) were used as samples for SD-PAGE experiment. The results are as follows: Figure 3 shown.

[0075] according to Figure 3 It can be seen that a PSI protein band with a band size of 11.7 kDa appears in the flow-through of step (7), confirming that the target protein is in the flow-through of step (7).

[0076] (8) The flow-through liquid from step (7) was transferred to a 1 kDa dialysis bag and dialyzed in 3 L of pure water for 24 h to obtain an aqueous solution of the protein, i.e., an aqueous solution of the PSI protein.

[0077] Application Example 1

[0078] 1. Construct non-fluorescent labeled liposomes: Dissolve non-fluorescent labeled phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in chloroform at a molar ratio of 1:1:1, and mix and stir for 30 minutes. Use N2 to blow dry, dissolve in different pH phosphate buffers, ultrasonically oscillate for 5 minutes to form liposomes, freeze and thaw repeatedly in liquid nitrogen and 37 degrees Celsius water bath ten times to form single-layer liposomes, and repeatedly squeeze through a polycarbonate membrane with a pore size of 100nm ten times to form liposomes with a diameter distribution within 100nm;

[0079] 2. Construct fluorescently labeled liposomes: Dissolve non-fluorescently labeled phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, rhodamine-labeled ethanolamine, and NBD-labeled ethanolamine in chloroform at a molar ratio of 1:1:1:0.6:0.6, and mix and stir for 30 minutes. Use N2 to blow dry, dissolve in different pH phosphate buffers, ultrasonically oscillate for 5 minutes to form liposomes, freeze and thaw repeatedly in liquid nitrogen and a 37 degree Celsius water bath ten times to form monolayer liposomes, and repeatedly squeeze through a polycarbonate membrane with a pore size of 100 nm ten times to form liposomes with a diameter distribution within 100 nm (this step is performed under light-proof conditions);

[0080] 3. The constructed fluorescently labeled liposomes and non-fluorescently labeled liposomes were mixed at a molar ratio of 1:9, and diluted to 300 μM and randomly divided into test group 1, test group 2, test group 3 and test group 4. The pH of the mixed solution of test group 1 was adjusted to 3.4, the pH of the mixed solution of test group 2 was adjusted to 4.8, the pH of the mixed solution of test group 3 was adjusted to 5.4, the pH of the mixed solution of test group 4 was adjusted to 6.0, and the pH of the mixed solution of test group 5 was adjusted to 7.0;

[0081] The test groups 1 to 5 were randomly divided into a blank group, a control group, a PSI group, and a PSI-7M group, and the following operations were performed respectively:

[0082] Blank group: no ingredients were added to the mixture;

[0083] Control group: Triton X-100 was added to the mixture to make its volume concentration 0.2%;

[0084] PSI group: the PSI protein aqueous solution obtained in Comparative Example 1 was added to the mixed solution to make its volume concentration 20 μM;

[0085] PSI-7M group: the PSI-7M protein aqueous solution obtained in Example 2 was added to the mixed solution to make the final volume concentration 20 μM;

[0086] 4. Monitor the fluorescence signal intensity of each treatment group in step 3 after 1200s of reaction, and calculate the fusion efficiency according to the following formula. The results are shown in Tables 1 to 2 and Figures 4-5 .

[0087] Fusion efficiency (%) = (f t -f0) / (f 100 -f0)

[0088] Among them, f t represents the fluorescence intensity of the PSI group or PSI-7M group at time t, f0 represents the fluorescence intensity of the blank group at time t, and f 100 represents the fluorescence intensity of the control group at time t.

[0089] Table 1 Fluorescence signal intensity of each treatment group at 1200s reaction time

[0090]

[0091] Table 2 Fusion efficiency of PSI group and PSI-7M group at 1200s reaction time (%)

[0092]

[0093] Note: When the fusion efficiency is negative, it means that the protein does not show the activity of inducing membrane fusion, and the efficiency of promoting membrane fusion is treated as 0.

[0094] according to Figures 4-5 As can be seen from Tables 1 and 2, the protein mutant PSI-7M obtained by the present invention still has a strong activity of mediating membrane fusion at pH = 4.8 and higher pH conditions compared to the natural PSI protein.

[0095] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A protein mutant PSI-7M that induces membrane fusion, characterized in that: The amino acid sequence of the induced membrane fusion protein mutant PSI-7M is shown in SEQ ID NO.

1.

2. A gene encoding the protein mutant PSI-7M according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A recombinant vector comprising the gene according to claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector comprises a starting vector and the gene according to claim 2 or 3.

6. The method for preparing the inducible membrane fusion protein mutant PSI-7M according to claim 1, characterized in that: The following steps are involved: Using pET-32a(+) as a starting vector, inserting the gene described in claim 2 or 3 between the MscI and XhoI restriction sites of the pET-32a(+), deleting the His tag carried by pET-32a(+), and keeping the other sequences of pET-32a(+) unchanged, to obtain a recombinant vector; Transforming host cells with the recombinant vector to obtain recombinant bacteria; Cultivating the recombinant bacteria to obtain a culture solution containing the expressed protein; The culture fluid containing the expressed protein is recovered and purified to obtain the induced membrane fusion protein mutant PSI-7M.

7. A recombinant bacterium expressing the protein mutant PSI-7M according to claim 1.

8. Use of the membrane fusion inducing protein mutant PSI-7M according to claim 1, or the gene according to claim 2 or 3, or the membrane fusion inducing protein mutant PSI-7M obtained by the preparation method according to claim 6 in the preparation of a product that induces membrane fusion; When the product is used to induce membrane fusion, 4.8≤pH≤6.

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