CN113121665A - Two modified high-efficiency low-immunogenicity sealwort agglutinin proteins - Google Patents
Two modified high-efficiency low-immunogenicity sealwort agglutinin proteins Download PDFInfo
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Abstract
The invention discloses two mutant polygonatum agglutinin proteins with high activity and low immunogenicity. Belongs to the field of biotechnology. The invention designs a mutant protein mrPCL2 (mutant site G19Q) and a mutant protein mrPCL5 (mutant site P100R) with low immunogenicity by combining bioinformatics analysis and an immunoinformatics method based on a wild type polygonatum sibiricum lectin sequence,the two mutant sequences are respectively connected with pET21a vector by using recombination technology, thus constructing fusion expression recombinant plasmid. Transferring the plasmid into a prokaryotic expression system Rosettatm-gami B (DE 3) plys host bacteria, followed by Ni utilization2+And a large amount of mutant protein is obtained by means of Sepharose column affinity chromatography and the like. Experiments prove that the mutant polygonatum agglutinin obtained by the invention has the characteristics of high purity, high activity, low toxicity and low immunogenicity, and has good inhibition effect on tumor cells. The invention lays a foundation for the development and application of the polygonatum agglutinin.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to preparation and application of two high-efficiency low-immunogenicity polygonatum agglutinin recombinant mutants.
Background
Polygonatum cyrtonema agglutinin (A), (B), (C), (Polygonatum cyrtonemalectin, PCL) is a class of carbohydrate-binding proteins extracted from rhizomes of polygonatum sibiricum and has typical lectin activity (hemagglutination and carbohydrate-binding activity). PCL has binding specificity for mannose and is also effective in binding sialic acid. And gp120 on the surface of HIV virions has a large amount of mannose and sialic acid, and the surface of tumor cells also abnormally expresses a plurality of sialylated T antigens and Tn antigens, so that PCL can be combined with gp120 and mannose and sialic acid on the surface of tumor cells, thereby effectively blocking and blocking recognition and combination of gp120 and CD4 on the surface of host cells, and having the effects of efficiently inhibiting tumor activity and resisting HIV infection.
PCL has low toxicity to normal cells, low toxic and side effects and good druggability. However, as the isohexin of human body, the polygonatum agglutinin has stronger immunogenicity, and is easy to activate the autoimmune reaction of the body when entering the body, and generates neutralizing antibodies to influence the curative effect. At present, natural extracts and macromolecular drugs with antitumor and antiviral effects are applied to future clinical treatment, and the important problem that the unnecessary immunogenicity needs to be reduced like PCL is solved.
The specificity of an antigen depends on the nature, number and spatial configuration of the antigenic epitopes that underlie the antigenicity of the protein. The deep research of protein epitope has important significance for the design of polypeptide and novel vaccine molecules and the development of diagnostic reagents. The prediction of antigen epitope by adopting an immune informatics method and a bioinformatics tool has become a powerful tool for epitope determination.
Therefore, the invention uses a bioinformatics tool to predict the PCL epitope, selects a target peptide segment to carry out amino acid mutation and carries out molecular docking simulation with a main histocompatibility compound with higher distribution frequency in Chinese population, determines two single-point mutations with obviously reduced binding tightness, and carries out experimental verification. Constructing a recombinant PCL mutant, purifying and detecting the physicochemical property and the anti-tumor activity of the PCL mutant after prokaryotic expression, and finally obtaining two high-efficiency low-immunogenicity rhizoma polygonati agglutinin recombinant mutants.
At present, no report related to PCL recombinant protein for reducing immunogenicity is available, the invention provides a new thought for reducing immunogenicity and researching and developing of novel medicaments with stronger immunogenicity, such as immunotoxin, phytohemagglutinin and the like, provides theoretical and technical support for creating a new generation of innovative medicaments with antiviral and antitumor activities and low immunogenicity, and provides basic research support for reducing immunogenicity of natural medicaments, reducing toxic and side effects of macromolecular medicaments and avoiding the technical problem that a large amount of neutralizing antibodies are generated in vivo.
Disclosure of Invention
In order to reduce the immunogenicity of polygonatum agglutinin PCL under the condition of ensuring the activity, the invention obtains the high-efficiency low-immunogenicity polygonatum agglutinin mutants mrPCL2 and mrPCL5 which are successfully expressed by modifying the gene of the polygonatum agglutinin PCL.
In the first aspect of the invention, two modified high-efficiency low-immunogenicity sealwort lectins are provided, and the modified mutant sealwort lectin mrPCL2 is prepared by converting the amino acid sequence of PCL (SEQ ID NO: 1 glycine at position 19 is replaced with glutamine; the mutant polygonatum agglutinin mrPCL5 is prepared by mixing the amino acid sequence SEQ ID NO: 1 proline to 100 to arginine; protein abbreviations are shown in table 1 below.
The second aspect of the invention provides two preparation methods for modifying the agglutinin of polygonatum sibiricum with high efficiency and low immunogenicity, which comprises the following steps:
(1) performing single mutation on 19 th and 100 th amino acids of the amino acid sequence of PCL, wherein the replaced sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 4; designing and synthesizing RFP-g4s-mrPCL2 gene, and amino acid sequence SEQ ID NO: 6, the nucleic acid sequence is shown in SEQ ID NO: 7. RFP-g4s-mrPCL5 gene, amino acid sequence SEQ ID NO: 8, the nucleic acid sequence is shown in SEQ ID NO: 9;
(2) constructing a modified polygonatum agglutinin recombinant plasmid: the RFP-g4s-mrPCL2 gene and the RFP-g4s-mrPCL5 gene are respectively inserted into a carrier pET-21aNdeI andHind III, restriction enzyme cutting sites;
(3) constructing a recombinant expression strain containing RFP-g4s-mrPCL2 gene and RFP-g4s-mrPCL5 gene: the recombinant plasmid is transformed into a clonic bacterium DH5 alpha to extract the plasmid, and finally transformed into an expression bacterium Rosetta-gami B (DE 3) plys;
(4) preparation of modified polygonatum cyrtonema agglutinin protein mrPCL2 and mrPCL 5: a single clone was picked and inoculated into 5ml of LB medium containing 15. mu.g/ml Kan, 100. mu.g/ml Amp, 34. mu.g/ml Cam, 12.5. mu.g/ml Tet, and cultured overnight at 37 ℃ with shaking at 200 rpm. The following day is as follows: transferring 100 percent of the strain into an LB culture medium containing Kan, carrying out shaking culture at 37 ℃ and 200rpm to enable the OD value to reach 0.5-0.6, taking part of the strain liquid as a control before induction, adding IPTG to enable the final concentration to be 0.5 mmol/L, carrying out shaking induction culture at 37 ℃ and 200rpm for 12 h, and inducing the expression of the target protein.
The cells were centrifuged at 8000rpm for 10min, and after induction expression, the cells were collected, suspended in a nickel column Binding Buffer (20 mM PB, 500mM NaCl, 10mM imidazole, pH = 7.4), and subjected to sonication with 1mM PMSF. The ultrasonic power is 52W, the ultrasonic time is 3s, the interval is 7s, 90 times, two ultrasonic cycles are carried out, and the total time is 30min. The sonicated total protein sample was centrifuged at 8000rpm at 4 ℃ for 10min, and the supernatant protein sample was collected and passed through a 0.22 μm filter to remove insoluble material. Starting to load Ni which has been previously balanced by Binding Buffer2+-Sepharose Fast Flow affinity chromatography, after loading, washing with Wash BufferWashing the column to remove the non-hanging column protein, wherein about 20 column volumes are needed, finally eluting with 250mM imidazole concentration, and collecting the eluent; then using enterokinase and molecular sieve chromatography to carry out enzyme digestion and separation on the fusion protein to obtain mutant protein, and using ddH2Ultrafiltration with O followed by lyophilization.
In the third aspect of the invention, the application of the modified high-efficiency low-immunogenicity sealwort lectin in the antitumor cell proliferation activity is provided.
Compared with the prior art, the invention has the following beneficial effects:
1. the recombinant mutant PCL is an expression protein, and no report related to PCL recombinant protein for reducing immunogenicity exists at present.
2. The invention provides a new idea for reducing the immunogenicity of novel medicaments with stronger immunogenicity, such as immunotoxin, phytohemagglutinin and the like, and research and development.
3. The invention provides theoretical and technical support for creating a new generation of innovative medicine with antiviral and antitumor activities and low immunogenicity, and provides basic research support for reducing the immunogenicity of natural medicine, reducing toxic and side effects of macromolecular medicine and avoiding the technical problem that a large amount of neutralizing antibodies are generated in vivo.
TABLE 1 description of protein abbreviations.
Drawings
FIG. 1 shows the results of affinity chromatography after induction of expression of the fusion protein. FIG. A, lanes 1-4 are the results of purification after affinity chromatography of mrPCL 2; 5 is a protein Marker; FIG. B, lanes 1, 3-4, shows the results of purification after affinity chromatography of mrPCL 5; 2 is a protein Marker; 5 is mrPCL5 sample before affinity chromatography.
FIG. 2 shows enterokinase cleavage of mutant Polygonatum sibiricum lectin fusion protein. Lanes 1 and 2 are rfp-g4s-mrPCL2, rfp-g4s-mrPCL5, respectively; lane 3 is protein Marker; lane is uncleaved rfp-g4s-mrPCL 5.
FIG. 3 shows the purification results of mutant Polygonatum sibiricum lectin after enterokinase digestion. Panel A, molecular sieve chromatography after cleavage of mrPCL2, with two distinct peaks. FIG. B shows the sample mrPCL2 after the enterokinase enzyme digestion and the molecular sieve treatment; 7 is a protein Marker; panel C, enterokinase digested sample after sieving through molecular sieves. Lanes 1-4: mrPCL 5; lane 5: a protein Marker; lane 6: samples before separation.
FIG. 4 shows the sugar inhibitory activity of wild-type, recombinant and mutant lectins.
FIG. 5 is a CCK-8 method for determining toxicity of wild-type, recombinant and mutant lectins to LO2 in normal cells
FIG. 6 shows the results of CCK-8 assay for the inhibition of Hela and HepG2 by wild-type, recombinant and mutant lectins.
FIG. 7 shows the morphological changes of Hela and HepG2 cells after 24h treatment with wild-type, recombinant and mutant lectins. A) Inhibition of Hela and HepG2 cells by nclk; B) inhibition of Hela and HepG2 cells by wrPCL; C) the inhibitory effect of mrPCL5 on Hela and HepG2 cells; D) inhibitory effect of mrPCL2 on Hela and HepG2 cells.
FIG. 8 shows the morphological changes of Hela and HepG2 cells after 48h treatment with wild type, recombinant and mutant lectins after DAPI staining.
FIG. 9 shows SDS-PAGE analysis of purified antibody. M is protein molecular mass standard; PCL purified antibody; 2: wrPCL purified antibody; B) m is protein molecular mass standard; 3: mrPCL2 purified antibody; 4: mrPCL5 purified antibody.
FIG. 10 shows the results of antibody titer detection by ELISA.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
The molecular biological methods involved in this experiment are conventional and well known to those skilled in the art. For details not described in the present invention, please refer to the molecular cloning guidelines, edited by Sum Brooks, D.W. Lassel, etc.
Example 1 design of high Performance Low immunogenic mutant mrPCL2 (G19Q) and cloning of expression vector
The method is characterized in that the wild type polygonatum sibiricum agglutinin gene sequence SEQ ID NO: 1 is a template, a mutation site G19Q (namely the glycine at the 19 th site of the protein is changed into glutamine) is designed, the amino acid sequence after mutation is shown as SEQ ID NO. 2, and the nucleic acid sequence is shown as SEQ ID NO. 3. Further, a fusion protein sequence RFP-G4s-mrPCL2 (G19Q) was designed. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 6, the nucleotide sequence of the fusion protein is shown in SEQ ID NO: 7, synthesized by Beijing Ongchongke Biometrics Ltd.
Example 2 design of high Performance Low immunogenic mutant mrPCL5 (P100R) and cloning of expression vector
Mutant protein mrPCL5 is expressed by a wild type polygonatum agglutinin gene sequence SEQ ID NO: 1 is taken as a template, a mutation site P20H (namely proline of the 100 th site of the protein is changed into arginine) is designed, the amino acid sequence after mutation is shown as SEQ ID NO. 4, and the nucleic acid sequence is shown as SEQ ID NO. 5. And then designing a fusion protein sequence RFP-g4s-mrPCL4, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: and 8, the nucleotide sequence of the fusion protein is shown in SEQ ID NO: 9, synthesized by Beijing Ongchongke Biometrics Ltd.
The cloning, expression and purification and immunization processes of the mutant protein of the embodiment 1 and the embodiment 2 are the same, and the specific embodiment is as follows:
example 3 construction of recombinant mutant Polygonatum sibiricum lectin expression vector
The fusion genes RFP-g4s-mrPCL2 and RFP-g4s-mrPCL5 in example 1 and example 2 were passed throughNdeI andHind III, inserting the restriction enzyme cutting site into a vector pET-21 a; the recombinant plasmid is transformed into a clonality bacterium DH5 alpha, then the plasmid is extracted, and finally the plasmid is transformed into an expression bacterium Rosetta-gami B (DE 3) plys. The single clone is selected and the correctness of the recombinant plasmid is identified by using a sequencing technology, and the sequencing work is finished by Beijing Optimalaceae biology, Inc.
Example 4 expression and purification of mutant Polygonatum sibiricum lectin fusion proteins
The clones verified to be correct in example 3 were picked and inoculated into 5ml of LB medium containing 15. mu.g/ml Kan, 100. mu.g/ml Amp, 34. mu.g/ml Cam, 12.5. mu.g/ml Tet, and cultured overnight at 37 ℃ with shaking at 200 rpm. The following day is as follows: transferring 100 proportion into LB culture medium containing Kan, shaking culturing at 37 deg.C and 200rpm to make OD value reach 0.5-0.6, adding IPTG to make final concentration be 0.5 mmol/L, shaking inducing culturing at 37 deg.C and 200rpm for 12 h, and inducing expression of target protein.
After induction expression, centrifugation was carried out at 8000rpm for 10min, and the cells were collected, suspended in a nickel column Binding Buffer (20 mM PB, 500mM NaCl, 10mM imidazole, pH = 7.4), and PMSF was added to a final concentration of 1 mM. Then, ultrasound is carried out, wherein the ultrasound power is 52W and is 8 percent of the total power 650W, the ultrasound time is 3s, the interval is 7s, 90 times, and the ultrasound is carried out for two cycles, and the total time is 30min. The sonicated total protein sample was centrifuged at 8000rpm at 4 ℃ for 10min, and the supernatant protein sample was collected and passed through a 0.22 μm filter to remove insoluble material. Starting to load Ni which has been previously balanced by Binding Buffer2+-Sepharose Fast Flow affinity column, after loading, washing the column with Wash Buffer to Wash the unbound column protein, approximately 20 column volumes, and finally eluting with 250mM imidazole concentration and collecting the eluate. ddH for chromatographic column2O washing and storing with 20% ethanol. The purified sample was identified by 15% SDS-PAGE to obtain a relatively pure fusion protein, see FIG. 1.
Example 5 enzymatic cleavage and purification of mutant Polygonatum sibiricum lectin fusion proteins
The mutant fusion proteins rfp-g4s-mrPCL2 and rfp-g4s-mrPCL5 purified in example 4 were subjected to enzyme digestion experiment by using enterokinase to obtain cleaved mrPCL2 and mrPCL5 proteins, the enzyme digestion results are shown in FIG. 2, and the enzyme digestion reaction system is shown in Table 2. After the enzyme digestion, the redundant protein sequences (including rfp, histidine tag, g4s, etc.) of about 31kDa are obtained, and the target proteins mrPCL2 and mrPCL5 of 11.9kDa are bands.
TABLE 2 Enterokinase digestion reaction System
Selecting Sephacryl S-100 for molecular sieve (gel filtration) chromatography; simultaneously, normal saline (0.9% sodium chloride solution) is selected asEluting the buffer solution; loading the gel column material into a chromatographic column (the inner diameter: the height =1: 20-1: 100), compacting and keeping the gel column material wet; and (3) closing the outflow port after the buffer solution is balanced, adding the sample solution after enzyme digestion by using an injector, opening the outflow port, and continuously adding a large amount of eluent for elution by using a mechanical pump after the sample permeates into the gel bed. OD280And detecting the peak of the eluted protein by ultraviolet, and collecting the eluted protein with the peak value. The purification results were checked for protein using 15% SDS-PAGE and are shown in FIG. 3. As a result, it was found that the target proteins mrPCL2 and mrPCL5, which were purified by subjecting the mixture of the fusion proteins rfp-g4s-mrPCL2 and rfp-g4s-mrPCL5, which were digested with enzymes, to dialysis concentration and then to molecular sieve chromatography, had molecular weights of approximately 11.9 kDa.
Example 6 detection of coagulant Activity of mutant Polygonatum sibiricum lectin
The blood coagulation activity detection of the recombinant mutant sealwort lectins mrPCL2 and mrPCL5 obtained in example 5 was performed using fresh rabbit red blood cells. Healthy rabbit erythrocytes are taken and made into 2% (v/v) erythrocyte suspension by using physiological saline. Weighing freeze-dried products of recombinant mutant type sealwort agglutinin mrPCL2 and mrPCL5, preparing the freeze-dried products into 1mg/ml by using normal saline, and performing multiple dilution of all protein samples on a V-shaped 96-hole micro serum dilution plate. Firstly, 25 mul of physiological saline is added into each hole, 25 mul of protein sample solution is added into a first hole, a micropipette is used for mixing evenly, 25 mul of protein sample solution is taken out from the first hole and mixed evenly into a second hole, 25 mul of protein sample solution is taken out from the second hole and added into a third hole, the dilution is continued by the analogy, after all samples are diluted, 25 mul of 2% (V/V) rabbit blood erythrocyte suspension is added into each hole, and the mixture is vibrated for 5min on a micro mixer. All mixtures were incubated at room temperature for 2h and the agglutination results of the protein samples were observed microscopically to indicate the agglutination activity as the lowest sample concentration that produced the coagulation reaction, the results of which are given in table 3 below. Wild type polygonatum agglutinin PCL, recombinant polygonatum agglutinin wrPCL as control samples were provided by the applicant's laboratory.
It can be seen that the activity intensity of the recombinant wild type polygonatum agglutinin wrPCL is relatively close compared with that of the plant wild type nPCL. The mutants mrPCL2 and mrPCL5 maintained activity, although slightly reduced compared to the wild type, but still maintained useful activity intensity.
TABLE 3 clotting activity of wild type, recombinant and mutant Polygonatum sibiricum lectin
Note: "+" indicates agglutination, "-" indicates no agglutination. "+" Agluting, "-" Non-agluting.
Example 7 sugar inhibition assay of mutant Polygonatum sibiricum lectin
After recombinant mutant Polygonatum sibiricum lectins mrPCL2 and mrPCL5 (1 mg/ml) obtained in example 5 were diluted in duplicate with reference to the agglutination activity assay, thyroglobulin, mannan, mannose, galactose, fructose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid were prepared in physiological saline to a concentration of 160mmol/L (monosaccharide) and 4mg/ml (glycan and glycoprotein), and 25. mu.L of each prepared sugar solution was added to each well. After the addition of the sugar solution, the sample was incubated with a shaker for 10min in the same manner as in example 6, and the concentration of the lowest protein sample in the agglutination reaction was measured, and the activity of the protein samples treated with different carbohydrates was measured to express the agglutination activity as the lowest sample concentration for the coagulation reaction, and the measurement results are shown in FIG. 4. The results of the tests show that although the sugar inhibition experiment results of the mutant and the wild type polygonatum agglutinin have partial differences, the sugar binding specificity is similar, and the mutant and the wild type polygonatum agglutinin are both specifically bound with mannose (mannan). Secondly, Thyroglobulin (Thyroglobulin) and Sialic acid (Sialic acid) have strong inhibitory effects on Thyroglobulin and Sialic acid.
Example 8 determination of antitumor cell proliferation Activity of mutant Polygonatum sibiricum lectin
Cytotoxicity of mrPCL2 and mrPCL5 obtained in example 5 on LO2 cells was measured by the CCK-8 method. HeLa, HepG2 and LO2 cells were cultured in 96-well tissue culture plates at a density of 5X 10 cells per well with 100. mu.l of cells added3Individual cells/well. After incubation for 24h at 37 ℃, mrPCL2 and mrPCL5 with different concentrations are added into the cultured cells, and CO is added at 37 DEG C2Respectively in 5% concentration cell culture boxAfter 24h and 48h of incubation, 10. mu.l of CCK-8 solution was added to each well. Wells with 100 μ l of cell culture medium and 10 μ l of CCK-8 solution (no cells) were blank. A well to which the same amount of cells and 10. mu.l of CCK-8 solution (no drug) were added in the experimental group was used as a negative control. The cell culture plate is further incubated in the cell culture box for 1-4h, and the absorbance is measured at a wavelength of 450 nm.
The rate of viable cells of normal cells and the rate of inhibition of tumor cells were calculated according to the following formula:
cell viability ratio(%) =(A490,treated-A490, blank)/(A490,control-A490, blank)×100%；
cell inhibitory ratio(%) =1-(A490,treated -A490, blank)/ (A490, control-A490, blank)×100%
the results are shown in FIG. 5. nPCL, wrPCL, mrPCL2, mrPCL5 were less toxic to normal hepatocytes LO 2. mrPCL2, mrPCL5 are less toxic to normal cells than wild type lectin.
For tumor cells, as seen in fig. 6, mrPCL2 and mrPCL5 showed substantially the same tumor cell inhibiting effect as wild-type polygonatum agglutinin under the treatment time condition of 24h and 48 h. The two mutants obtained in the experiment keep the original tumor cell inhibition activity compared with the wild type polygonatum agglutinin.
As can be seen from FIG. 7, the mutant still has better ability to inhibit tumor cells compared to the wild-type protein. Meanwhile, wild type and mutant PCL treated 24h HepG2 were stained with DAPI, and apoptosis was observed as shown in FIG. 8, and the results were found to be consistent with those observed directly.
Example 9 preliminary testing of immunogenicity of mutant Polygonatum sibiricum lectin
Polyclonal antibody preparation and antibody purification by using mrPCL2 and mrPCL5 proteins with purity of more than 90% obtained in example 5, the total protein amount of each protein is about 5mg-6mg respectively, and the purity meets more than 85% required by antibody preparation through detection. Animals prepared for each antibody were 2 new zealand white rabbits, three immunizations in total. And (3) respectively detecting the titer of the antiserum after one week of each immunization, killing the immunized animals after the titer of the antiserum reaches the standard after the third immunization, collecting the antiserum, and purifying the antibody by adopting a Protein A affinity chromatography method. The antibody purification results are shown in lanes 3 and 4 of FIG. 9, respectively, and the antibody was obtained at a purity of 85% or more, about 3.52 mg and 3.82 mg.
The titer of the prepared antiserum and Antibody is detected by ELISA, Rabbit antibodies of Anti-mrPCL 2 and mrPCL5 are used as primary antibodies, and Goat Anti-Rabbit Antibody Goat Anti-Rabbit IgG (H & L) Antibody [ IRDye 800cw ] is used as secondary antibodies. The optimal coating concentration (4 mug/ml) and optimal secondary antibody dilution concentration (1: 120000) are obtained by a matrix method, and the titer of the prepared antibody is detected by ELISA. Setting 2.1 times of OD reading of the negative control hole as a threshold value, judging that the OD value is greater than the threshold value is positive, judging that the OD value is less than the threshold value is negative, and determining the result. The titer of the antibody was determined as the maximum antibody dilution concentration at which the OD value was positive, and the results are shown in tables 4 to 7 below and FIG. 10. The antibody titer of mrPCL2 was found to be about 256K, the antibody titer of mrPCL5 was found to be about 256K, and the purity was found to be 85% or more. The nPCL2 antibody titer is about 512K, the wrPCL antibody titer is about 512K, and compared with nPCL and wrPCL, the immunogenicity of the two mutants is obviously reduced.
TABLE 4 detection results of the mrPCL2 purified antibody
Initial dilution: 1: 500; the titer, i.e. the highest dilution of the sample OD/blank OD being not less than 2.1
TABLE 5 detection results of the purified antibody of mrPCL5
Initial dilution: 1: 500; the titer, i.e. the highest dilution of the sample OD/blank OD being not less than 2.1
TABLE 6 detection results of PCL purified antibody
Initial dilution: 1: 500; the titer, i.e. the highest dilution of the sample OD/blank OD being not less than 2.1
TABLE 7 detection results of PCL purified antibody
Initial dilution: 1: 500; the titer is the highest dilution that the OD/blank OD of the sample is more than or equal to 2.1.
Sequence listing
<110> Sichuan university
<120> two modified high-efficiency low-immunogenicity sealwort lectin proteins
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 110
<212> PRT
<213> Polygonatum cyrtonema)
<400> 1
Val Asn Ser Leu Ser Ser Pro Asn Ser Leu Phe Thr Gly His Ser Leu
1 5 10 15
Glu Val Gly Pro Ser Tyr Arg Leu Ile Met Pro Gly Asp Cys Asn Phe
20 25 30
Val Leu Tyr Asp Ser Gly Lys Pro Val Trp Ala Ser Asn Thr Gly Gly
35 40 45
Leu Gly Ser Gly Cys Arg Leu Thr Leu His Asn Asn Gly Asn Leu Val
50 55 60
Ile Tyr Asp Gln Ser Asn Arg Val Ile Trp Gln Thr Lys Thr Asn Gly
65 70 75 80
Lys Glu Asp His Tyr Val Leu Val Leu Gln Gln Asp Arg Asn Val Val
85 90 95
Ile Tyr Gly Pro Val Val Trp Ala Thr Gly Ser Gly Pro Ala
100 105 110
<210> 2
<211> 110
<212> PRT
<213> Polygonatum cyrtonema)
<400> 2
Val Asn Ser Leu Ser Ser Pro Asn Ser Leu Phe Thr Gly His Ser Leu
1 5 10 15
Glu Val Gln Pro Ser Tyr Arg Leu Ile Met Pro Gly Asp Cys Asn Phe
20 25 30
Val Leu Tyr Asp Ser Gly Lys Pro Val Trp Ala Ser Asn Thr Gly Gly
35 40 45
Leu Gly Ser Gly Cys Arg Leu Thr Leu His Asn Asn Gly Asn Leu Val
50 55 60
Ile Tyr Asp Gln Ser Asn Arg Val Ile Trp Gln Thr Lys Thr Asn Gly
65 70 75 80
Lys Glu Asp His Tyr Val Leu Val Leu Gln Gln Asp Arg Asn Val Val
85 90 95
Ile Tyr Gly Pro Val Val Trp Ala Thr Gly Ser Gly Pro Ala
100 105 110
<210> 3
<211> 330
<212> DNA
<213> Polygonatum cyrtonema)
<400> 3
gtcaattctc tgtcttcccc caacagcctc ttcaccggcc attccctcga ggtgcagccc 60
tcttaccgtc tcattatgcc gggagactgc aactttgtgt tgtacgactc aggcaaacct 120
gtttgggcgt ccaacaccgg cgggctcggc agtggctgcc gcttgacgtt gcacaacaac 180
gggaacctcg tcatctacga tcagagcaac cgtgtgattt ggcagaccaa gacgaacggg 240
aaggaggacc attacgtgct ggtgctgcag caagaccgca atgtggtcat ctacggccct 300
gtagtttggg ccacaggctc tggaccggcc 330
<210> 4
<211> 110
<212> PRT
<213> Polygonatum cyrtonema)
<400> 4
Val Asn Ser Leu Ser Ser Pro Asn Ser Leu Phe Thr Gly His Ser Leu
1 5 10 15
Glu Val Gly Pro Ser Tyr Arg Leu Ile Met Pro Gly Asp Cys Asn Phe
20 25 30
Val Leu Tyr Asp Ser Gly Lys Pro Val Trp Ala Ser Asn Thr Gly Gly
35 40 45
Leu Gly Ser Gly Cys Arg Leu Thr Leu His Asn Asn Gly Asn Leu Val
50 55 60
Ile Tyr Asp Gln Ser Asn Arg Val Ile Trp Gln Thr Lys Thr Asn Gly
65 70 75 80
Lys Glu Asp His Tyr Val Leu Val Leu Gln Gln Asp Arg Asn Val Val
85 90 95
Ile Tyr Gly Arg Val Val Trp Ala Thr Gly Ser Gly Pro Ala
100 105 110
<210> 5
<211> 330
<212> DNA
<213> Polygonatum cyrtonema)
<400> 5
gtcaattctc tgtcttcccc caacagcctc ttcaccggcc attccctcga ggtggggccc 60
tcttaccgtc tcattatgcc gggagactgc aactttgtgt tgtacgactc aggcaaacct 120
gtttgggcgt ccaacaccgg cgggctcggc agtggctgcc gcttgacgtt gcacaacaac 180
gggaacctcg tcatctacga tcagagcaac cgtgtgattt ggcagaccaa gacgaacggg 240
aaggaggacc attacgtgct ggtgctgcag caagaccgca atgtggtcat ctacggccgt 300
gtagtttggg ccacaggctc tggaccggcc 330
<210> 6
<211> 371
<212> PRT
<213> Polygonatum cyrtonema)
<400> 6
His His His His His His Val Ser Lys Gly Glu Glu Asp Asn Met Ala
1 5 10 15
Ile Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val
20 25 30
Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr
35 40 45
Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu
50 55 60
Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys
65 70 75 80
Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser
85 90 95
Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly
100 105 110
Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe
115 120 125
Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro
130 135 140
Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met
145 150 155 160
Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys
165 170 175
Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys
180 185 190
Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys
195 200 205
Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr
210 215 220
Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr
225 230 235 240
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Asp Asp Asp Asp Lys Val Asn Ser Leu Ser Ser Pro Asn Ser Leu Phe
260 265 270
Thr Gly His Ser Leu Glu Val Gln Pro Ser Tyr Arg Leu Ile Met Pro
275 280 285
Gly Asp Cys Asn Phe Val Leu Tyr Asp Ser Gly Lys Pro Val Trp Ala
290 295 300
Ser Asn Thr Gly Gly Leu Gly Ser Gly Cys Arg Leu Thr Leu His Asn
305 310 315 320
Asn Gly Asn Leu Val Ile Tyr Asp Gln Ser Asn Arg Val Ile Trp Gln
325 330 335
Thr Lys Thr Asn Gly Lys Glu Asp His Tyr Val Leu Val Leu Gln Gln
340 345 350
Asp Arg Asn Val Val Ile Tyr Gly Pro Val Val Trp Ala Thr Gly Ser
355 360 365
Gly Pro Ala
370
<210> 7
<211> 1141
<212> DNA
<213> Polygonatum cyrtonema)
<400> 7
gggaattcca tatgcaccac caccaccacc acgtgagcaa gggcgaggag gataacatgg 60
ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg 120
agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc 180
tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca 240
tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt 300
ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga 360
ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg 420
gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct 480
cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga 540
agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc 600
ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg 660
aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca 720
tggacgagct gtacaagggt ggtggtggta gcggcggcgg cggcagtggt ggcggtggct 780
ctgacgacga cgacaaggtc aattctctgt cttcccccaa cagcctcttc accggccatt 840
ccctcgaggt gcagccctct taccgtctca ttatgccggg agactgcaac tttgtgttgt 900
acgactcagg caaacctgtt tgggcgtcca acaccggcgg gctcggcagt ggctgccgct 960
tgacgttgca caacaacggg aacctcgtca tctacgatca gagcaaccgt gtgatttggc 1020
agaccaagac gaacgggaag gaggaccatt acgtgctggt gctgcagcaa gaccgcaatg 1080
tggtcatcta cggccctgta gtttgggcca caggctctgg accggcctaa taaaagcttg 1140
g 1141
<210> 8
<211> 371
<212> PRT
<213> Polygonatum cyrtonema)
<400> 8
His His His His His His Val Ser Lys Gly Glu Glu Asp Asn Met Ala
1 5 10 15
Ile Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val
20 25 30
Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr
35 40 45
Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu
50 55 60
Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys
65 70 75 80
Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser
85 90 95
Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly
100 105 110
Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe
115 120 125
Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro
130 135 140
Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met
145 150 155 160
Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys
165 170 175
Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys
180 185 190
Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys
195 200 205
Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr
210 215 220
Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr
225 230 235 240
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Asp Asp Asp Asp Lys Val Asn Ser Leu Ser Ser Pro Asn Ser Leu Phe
260 265 270
Thr Gly His Ser Leu Glu Val Gly Pro Ser Tyr Arg Leu Ile Met Pro
275 280 285
Gly Asp Cys Asn Phe Val Leu Tyr Asp Ser Gly Lys Pro Val Trp Ala
290 295 300
Ser Asn Thr Gly Gly Leu Gly Ser Gly Cys Arg Leu Thr Leu His Asn
305 310 315 320
Asn Gly Asn Leu Val Ile Tyr Asp Gln Ser Asn Arg Val Ile Trp Gln
325 330 335
Thr Lys Thr Asn Gly Lys Glu Asp His Tyr Val Leu Val Leu Gln Gln
340 345 350
Asp Arg Asn Val Val Ile Tyr Gly Arg Val Val Trp Ala Thr Gly Ser
355 360 365
Gly Pro Ala
370
<210> 9
<211> 1141
<212> DNA
<213> Polygonatum cyrtonema)
<400> 9
gggaattcca tatgcaccac caccaccacc acgtgagcaa gggcgaggag gataacatgg 60
ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg 120
agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc 180
tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca 240
tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt 300
ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga 360
ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg 420
gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct 480
cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga 540
agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc 600
ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg 660
aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca 720
tggacgagct gtacaagggt ggtggtggta gcggcggcgg cggcagtggt ggcggtggct 780
ctgacgacga cgacaaggtc aattctctgt cttcccccaa cagcctcttc accggccatt 840
ccctcgaggt ggggccctct taccgtctca ttatgccggg agactgcaac tttgtgttgt 900
acgactcagg caaacctgtt tgggcgtcca acaccggcgg gctcggcagt ggctgccgct 960
tgacgttgca caacaacggg aacctcgtca tctacgatca gagcaaccgt gtgatttggc 1020
agaccaagac gaacgggaag gaggaccatt acgtgctggt gctgcagcaa gaccgcaatg 1080
tggtcatcta cggccgtgta gtttgggcca caggctctgg accggcctaa taaaagcttg 1140
g 1141
Claims (6)
1. A modified protein of a high-efficiency and low-immunogenicity rhizoma polygonati lectin is characterized in that the modified rhizoma polygonati lectin is mrPCL2, and the amino acid sequence of the modified rhizoma polygonati lectin is shown as SEQ ID NO. 2.
2. A modified nucleotide sequence of polygonatum agglutinin is characterized in that the nucleotide sequence is shown as SEQ ID NO 3.
3. A modified protein of a high-efficiency and low-immunogenicity rhizoma polygonati lectin is characterized in that the modified rhizoma polygonati lectin is mrPCL5, and the amino acid sequence of the modified rhizoma polygonati lectin is shown as SEQ ID NO. 4.
4. A modified nucleotide sequence of polygonatum agglutinin is characterized in that the nucleotide sequence is shown as SEQ ID NO. 5.
5. A method for preparing modified Polygonatum sibiricum lectin described in claims 1 and 3, comprising the following steps:
(1) performing single mutation on 19 th and 100 th amino acids of the amino acid sequence of PCL, wherein the replaced sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 4; designing and synthesizing RFP-g4s-mrPCL2 gene, and amino acid sequence SEQ ID NO: 6, the nucleic acid sequence is shown in SEQ ID NO: 7. RFP-g4s-mrPCL5 gene, amino acid sequence SEQ ID NO: 8, the nucleic acid sequence is shown in SEQ ID NO: 9;
(2) constructing a modified polygonatum agglutinin recombinant plasmid: inserting RFP-g4s-mrPCL2 gene and RFP-g4s-mrPCL5 gene into Nde I and Hind III restriction enzyme cutting sites of a vector p ET-21a respectively;
(3) constructing a recombinant expression strain containing RFP-g4s-mrPCL2 gene and RFP-g4s-mrPCL5 gene: the recombinant plasmid is transformed into a clonic bacterium DH5 alpha to extract the plasmid, and finally transformed into an expression bacterium Rosetta-gami B (DE 3) plys;
(4) preparing modified sealwort lectin proteins mrPCL2 and mrPCL 5: selecting a single clone, inoculating the single clone into 5ml LB culture solution containing 15 mug/ml Kan, 100 mug/ml Amp, 34 mug/ml Cam and 12.5 mug/ml Tet, carrying out shake culture at 37 ℃ and 200rpm for overnight, and carrying out shake culture on the next day according to a ratio of 1: transferring 100 proportion into LB culture medium containing Kan, culturing at 37 deg.C and 200rpm under shaking to make OD value reach 0.5-0.6, taking part of the bacterial liquid as control before induction, adding IPTG to make final concentration 0.5 mmol/L, culturing at 37 deg.C and 200rpm under shaking for 12 h, inducing expression of target protein, centrifuging at 8000rpm for 10min, collecting thallus after induction expression, suspending thallus with nickel column combined with Buffer Binding Buffer (20 mMPB, 500mMNaCl, 10mM imidazole, pH = 7.4), adding 1mM PMSF, performing ultrasonic treatment at ultrasonic power of 52W, ultrasonic treatment for 3s, interval of 7s, 90 times, ultrasonic treatment for two cycles, total time of 30min, centrifuging total protein sample at 8000rpm, 4 deg.C for 10min after ultrasonic treatment, collecting supernatant protein sample, removing insoluble substance with 0.22 μm filter membrane, and starting to load onto Ni balanced with Binding Buffer in advance2+-Sepharose Fast Flow affinity chromatography, after loading, washing the column with Wash Buffer to Wash the non-suspended proteins, about 20 column volumes, and finally eluting with 250mM imidazole concentration, collecting the eluate; the fusion protein was then cleaved and separated by enzyme using enterokinase and molecular sieve chromatography to obtain a mutant protein, which was ultrafiltered with dd H2O, followed by lyophilization.
6. The use of the modified Polygonatum cyrtonema lectin of claims 1,3 for anti-tumor cell proliferation activity.
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| WO2023245415A1 (en) * | 2022-06-21 | 2023-12-28 | 四川大学 | Use of polygonatum cyrtonema hua. lectin in blocking invasion and infection of novel coronavirus |
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| CN114446392A (en) * | 2022-01-21 | 2022-05-06 | 华东理工大学 | Method for identifying key arginine amino acid residue position when protein is combined with nucleic acid aptamer |
| CN114446392B (en) * | 2022-01-21 | 2024-01-30 | 华东理工大学 | Method for confirming key essential amino acid residue site of protein combined with aptamer |
| WO2023245415A1 (en) * | 2022-06-21 | 2023-12-28 | 四川大学 | Use of polygonatum cyrtonema hua. lectin in blocking invasion and infection of novel coronavirus |
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