CN113121665B

CN113121665B - Two modified low-immunogenicity sealwort agglutinin proteins

Info

Publication number: CN113121665B
Application number: CN202110323835.0A
Authority: CN
Inventors: 鲍锦库; 吴传芳; 郑茹潇; 曹东炜; 王意达; 陈宣铭; 李洋均; 邓杰; 周洵羽
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2023-03-10
Anticipated expiration: 2041-03-26
Also published as: CN113121665A

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 on the basis of a wild type polygonatum sibiricum lectin sequence, and connects the two mutant sequences with a pET21a vector by using a recombination technology to construct a fusion expression recombinant plasmid. Transferring the plasmid into prokaryotic expression system Rosetta ^tm -gami B (DE 3) plys host bacteria, followed by Ni ²⁺ 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

Two modified low-immunogenicity sealwort agglutinin proteins

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation and application of two low-immunogenicity polygonatum agglutinin recombinant mutants.

Background

Rhizome of Siberian solomonseal lectin (C)Polygonatumcyrtonemalectin, 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 virion has a large amount of mannose and sialic acid, and the surface of tumor cell also abnormally expresses a large amount of sialylated T antigen and Tn antigen, so that PCL can be combined with gp120 and mannose and sialic acid on the surface of tumor cell, thereby effectively blocking and blocking recognition and combination of gp120 and CD4 on the surface of host cell, 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 the polygonatum sibiricum lectin PCL under the condition of ensuring the activity, the invention obtains the high-efficiency low-immunogenicity polygonatum sibiricum lectin mutants mrPCL2 and mrPCL5 which are successfully expressed by modifying the gene of the polygonatum sibiricum lectin PCL.

The first aspect of the invention provides two modified high-efficiency low-immunogenicity polygonatum agglutinin, wherein the modified mutant polygonatum agglutinin mrPCL2 is prepared by mixing 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. (ii) proline at position 100 is replaced with 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 the 19 th amino acid and the 100 th amino acid of the amino acid sequence of the PCL, wherein the replaced sequences are shown as SEQ ID NO. 2 and SEQ ID NO; designing and synthesizing RFP-g4s-mrPCL2 gene, 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: RFP-g4s-mrPCL2 geneRFP-g4s-mrPCL5 gene is inserted into a vector pET-21a respectivelyNdeI andHind III, restriction enzyme cutting sites;

(3) Constructing a recombinant expression strain containing RFP-g4s-mrPCL2 gene and RFP-g4s-mrPCL5 gene: transforming the recombinant plasmid into a clonal bacterium DH5 alpha to extract the plasmid, and finally transforming into an expression bacterium Rosetta-gami B (DE 3) plys;

(4) Preparing modified polygonatum cyrtonema agglutinin proteins 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 (20mMPB, 500mM NaCl,10mM imidazole, pH = 7.4), added to 1mM PMSF, and subjected to sonication. The ultrasonic power is 52W, the ultrasonic time is 3s, the interval is 7s,90 times, and the ultrasonic time is 30min. The sonicated total protein sample was centrifuged at 8000rpm,4 ℃ for 10min, and the supernatant protein sample was collected and filtered through a 0.22 μm filter to remove insoluble materials. Starting to load Ni which has been previously balanced by Binding Buffer ²⁺ -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; then using enterokinase and molecular sieve chromatography to carry out enzyme digestion and separation on the fusion protein to obtain mutant protein, and using ddH ₂ O ultrafiltration 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 in three aspects:

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 and researching and developing novel medicaments with stronger immunogenicity, such as immunotoxin, phytohemagglutinin and the like.

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 mrPCL2; 5 is a protein Marker; FIG. B, lanes 1,3-4 show the results of purification after affinity chromatography of mrPCL5; 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-mrPCL5.

FIG. 3 shows the purification results of mutant Polygonatum sibiricum lectin after enterokinase digestion. Panel A, molecular sieve chromatography after cleavage of mrPCL2, has two distinct peaks. FIG. B is the mrPCL2 sample 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: mrPCL5; lane 5: a protein Marker; lane 6: the pre-separation sample.

FIG. 4 shows the sugar inhibitory activity of wild-type, recombinant and mutant lectins.

FIG. 5 is a CCK-8 method for determining the toxicity of wild-type, recombinant and mutant lectins to LO2 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) The inhibition effect of nPCL on Hela and HepG2 cells; b) The inhibition effect of wrPCL on Hela and HepG2 cells; c) The inhibition 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 treated with wild-type, recombinant and mutant lectins 48h after DAPI staining.

FIG. 9 shows SDS-PAGE analysis of purified antibodies. M is protein molecular mass standard; 1, pcl purification 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

Further features and advantages of the present invention will be understood by reference to the following detailed description when considered 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-potency and Low-immunogenicity mutant mrPCL2 (G19Q) and cloning of expression vector

Wild type polygonatum sibiricum lectin gene sequence SEQ ID NO:1 is a template, a mutation site G19Q (namely that the 19 th glycine of the protein is changed into glutamine) is designed, the mutated amino acid sequence 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 Ongchooks Biotechnology Ltd.

Example 2 design of high Performance Low immunogenic mutant mrPCL5 (P100R) and cloning of expression vector

The mutant protein mrPCL5 is expressed by a wild type polygonatum sibiricum agglutinin gene sequence SEQ ID NO:1 is a template, a mutation site P20H (namely proline at the 100 th site of the protein is changed into arginine) is designed, the mutated amino acid sequence 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 introducedNdeI andHind III, inserting restriction enzyme cutting sites 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 picked and the correctness of the recombinant plasmid is identified by using a sequencing technology, and the sequencing work is finished by Beijing Optimala biology Limited.

Example 4 expression and purification of mutant Polygonatum sibiricum lectin fusion proteins

The correct clones verified 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 of expression, centrifugation was carried out at 8000rpm for 10min, and the cells were collected, suspended in a nickel column Binding Buffer (20mMPB, 500mMNaCl,10mM imidazole, pH = 7.4), and PMSF was added to a final concentration of 1mM. Then, ultrasonic treatment was carried out at a power of 52W, which is 8% of the total power of 650W, for 3s ultrasonic treatment and 7s interval90 times, two ultrasonic cycles, and 30min total time. The sonicated total protein sample was centrifuged at 8000rpm,4 ℃ for 10min, and the supernatant protein sample was collected and filtered through a 0.22 μm filter to remove insoluble materials. Starting to load Ni which has been previously balanced with Binding Buffer ²⁺ -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 column ₂ O washing and storing with 20% ethanol. The purified samples were subjected to 15% SDS-PAGE identification to yield the purer fusion proteins, see FIG. 1.

Example 5 digestion 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 experiments using enterokinase to obtain cleaved mrPCL2 and mrPCL5 proteins, the enzyme digestion results are shown in FIG. 2, and the enzyme digestion reaction systems are shown in Table 2. After the enzyme digestion, bands of target proteins mrPCL2 and mrPCL5 with 11.9kDa of redundant protein sequences (including rfp, histidine tag, g4s and the like) about 31kDa are obtained.

TABLE 2 Enterokinase digestion reaction System

Selecting Sephacryl S-100 for molecular sieve (gel filtration) chromatography; meanwhile, physiological saline (0.9% sodium chloride solution) is selected as an elution buffer; loading the gel column material into a chromatography column (inner diameter: height =1:20 to 1; and (3) closing the outflow port after buffer solution balance, 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. OD ₂₈₀ And detecting the peak of the eluted protein by ultraviolet, and collecting the eluted protein with the peak value. The purification results were determined by 15-cent SDS-PAGE and are shown in FIG. 3. As a result, it was found that the mixture of fusion proteins rfp-g4s-mrPCL2 and rfp-g4s-mrPCL5 cleaved with enzymes was dialyzed, concentrated and subjected to molecular sieve chromatography,obtaining the purified target proteins mrPCL2 and mrPCL5 with the molecular weight of about 11.9kDa.

Example 6 detection of coagulant Activity of mutant Polygonatum sibiricum lectin

The blood coagulation activity of the recombinant mutant polygonatum sibiricum lectins mrPCL2 and mrPCL5 obtained in example 5 was measured using fresh rabbit red blood cells. Healthy rabbit red blood cells are taken and made into 2% (v/v) red blood cell suspension by using normal saline. Weighing freeze-dried products of recombinant mutant type polygonatum agglutinin mrPCL2 and mrPCL5, preparing the freeze-dried products into 1mg/ml by using normal saline, and carrying out multiple dilution on 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, 25 mul is taken out from the first hole and mixed into a second hole, 25 mul is taken out from the second hole and added into a third hole, the dilution is continued by the same way, after all samples are diluted, 25 mul of 2 percent (V/V) rabbit blood erythrocyte suspension is added into each hole, and the mixture is vibrated on a micro mixer for 5min. All mixtures were incubated at room temperature for 2h and the agglutination results were observed microscopically for protein samples, with the lowest sample concentration that produced a clotting reaction representing the agglutination activity, and the results 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 maintain activity, and maintain usable activity intensity although the activity is slightly reduced compared with that of the wild type.

TABLE 3 clotting activity of wild type, recombinant and mutant Polygonatum sibiricum lectin

Note: * "+" indicates agglutination and "-" indicates no agglutination. "+" Agluting, "-" Non-agluting.

Example 7 sugar inhibition assay of mutant Polygonatum sibiricum lectin

After the recombinant mutant Polygonatum sibiricum lectins mrPCL2 and mrPCL5 (1 mg/ml) obtained in example 5 were diluted by a double ratio in 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 of the prepared sugar solutions 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 assay revealed that the mutant and wild-type polygonatum agglutinin showed similar sugar binding specificity, and both specifically bound to mannose (manna) and mannan (mannan), although the results of the sugar inhibition experiments were partially different. 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, to which 100. Mu.l of cells were added per well at a density of 5X 10 ³ Individual 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 ₂ After culturing in a 5% cell culture chamber for 24h and 48h, 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 used as blanks. A negative control was added to wells containing the same amount of cells and 10. Mu.l of CCK-8 solution (no drug) to the experimental group. 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 viable cell rate of normal cells and the inhibition rate of tumor cells were calculated according to the following formulas:

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 are less toxic to normal liver cells LO 2. mrPCL2, mrPCL5 are less toxic to normal cells than wild-type lectins.

As for tumor cells, as seen from FIG. 6, under the condition of 24h and 48h treatment time, mrPCL2 and mrPCL5 have substantially the same tumor cell inhibiting effect as wild-type Polygonatum sibiricum lectin. 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 HepG2 for 24h were stained with DAPI, and apoptosis was observed as shown in FIG. 8, and the results were found to be consistent with those of direct observation.

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 about 3.82 mg.

The titer of the prepared antiserum and Antibody is detected by ELISA, rabbit antibodies of mrPCL2 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. Mu.g/ml) was obtained by a matrix method, the optimal secondary antibody dilution concentration was 1. 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 mrPCL2 antibody titer was found to be about 256K, the mrPCL5 antibody titer 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 mrPCL2 purified antibody

Initial dilution: 1; titer, i.e. the highest dilution of the sample OD/blank OD ≥ 2.1

TABLE 5 detection results of mrPCL5 purified antibody

Initial dilution: 1; 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

TABLE 7 detection results of PCL purified antibody

Initial dilution: 1; 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 polygonatum agglutinin proteins

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Ile Tyr Gly Pro Val Val Trp Ala Thr Gly Ser Gly Pro Ala

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Ile Tyr Asp Gln Ser Asn Arg Val Ile Trp Gln Thr Lys Thr Asn Gly

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Ile Tyr Gly Pro Val Val Trp Ala Thr Gly Ser Gly Pro Ala

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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

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Ile Tyr Asp Gln Ser Asn Arg Val Ile Trp Gln Thr Lys Thr Asn Gly

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Lys Glu Asp His Tyr Val Leu Val Leu Gln Gln Asp Arg Asn Val Val

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Ile Tyr Gly Arg Val Val Trp Ala Thr Gly Ser Gly Pro Ala

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<211> 330

<212> DNA

<213> Polygonatum cyrtonema)

<400> 5

gtcaattctc tgtcttcccc caacagcctc ttcaccggcc attccctcga ggtggggccc 60

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gtttgggcgt ccaacaccgg cgggctcggc agtggctgcc gcttgacgtt gcacaacaac 180

gggaacctcg tcatctacga tcagagcaac cgtgtgattt ggcagaccaa gacgaacggg 240

aaggaggacc attacgtgct ggtgctgcag caagaccgca atgtggtcat ctacggccgt 300

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Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu

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Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys

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Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser

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Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly

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Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe

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Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro

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Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met

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Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys

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Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys

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Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys

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Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr

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Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr

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Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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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

1. A modified protein of sealwort lectin with low immunogenicity is characterized in that the modified protein is mrPCL2, and the amino acid sequence of the modified protein is shown as SEQ ID NO. 2.

2. A modified nucleotide molecule of polygonatum agglutinin is characterized in that the nucleotide molecule is shown as SEQ ID NO 3.

3. A modified protein of polygonatum sibiricum lectin with low immunogenicity is characterized in that the modified protein is mrPCL5, and the amino acid sequence of the modified protein is shown as SEQ ID NO. 4.

4. A modified nucleotide molecule of polygonatum agglutinin is characterized in that the nucleotide molecule is shown as SEQ ID NO. 5.

5. A method for preparing a polygonatum sibiricum lectin modified protein as claimed in claim 1 or 3, comprising the following steps:

(1) Performing single mutation on 19 th and 100 th amino acids of an amino acid sequence of PCL, wherein the replaced sequence is shown as SEQ ID NO:2 and SEQ ID NO; designing and synthesizing RFP-g4s-mrPCL2 gene, 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 vector p ET-21a respectively;

(3) Constructing a recombinant expression strain containing RFP-g4s-mrPCL2 gene and RFP-g4s-mrPCL5 gene: transforming the recombinant plasmid into a clone bacterium DH5 alpha to extract the plasmid, and finally transforming the plasmid into an expression bacterium Rosetta-gami B (DE 3) plys;

(4) Preparation of modified proteins mrPCL2 and mrPCL5 from polygonatum sibiricum lectin: selecting expression bacteria Rosetta-gami B (DE 3) plys for monoclonal inoculation in 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 shaking culture at 37 ℃ and 200rpm for overnight, and carrying out shaking culture on the next day according to a ratio of 1: transferring into LB culture medium containing Kan at a ratio of 100, performing shaking culture at 37 deg.C and 200rpm to OD value of 0.5-0.6, collecting partial bacterial liquid as control before induction, adding IPTG to make final concentration of 0.5 mmol/mlL, performing oscillation induction culture at 37 ℃ and 200rpm for 12 h to induce the expression of target protein, centrifuging at 8000rpm for 10min, collecting thalli after induction expression, and combining a nickel column with a Buffer solution Binding Buffer:20mMPB,500mMNaCl,10mM imidazole, pH =7.4, thalli is suspended, 1mM PMSF is added, ultrasonic treatment is carried out, the ultrasonic power is 52W, ultrasonic treatment is carried out for 3s, the interval is 7s and 90 times, ultrasonic treatment is carried out for two cycles, the total time is 30min, the ultrasonic treated total protein sample is 8000rpm and 4 ℃, centrifugal treatment is carried out for 10min, supernatant protein samples are collected, insoluble substances are removed through a 0.22 mu m filter membrane, and the sample application is started to be applied to Ni which is balanced by Binding Buffer in advance ²⁺ -Sepharose Fast Flow affinity column, after loading, washing the column with Wash Buffer to Wash the non-column-hanging protein, finally eluting with 250mM imidazole concentration, collecting the eluate; then, the fusion protein is subjected to enzyme digestion and separation by using enterokinase and molecular sieve chromatography to obtain a mutant protein, and the mutant protein is subjected to ultrafiltration by using dd H2O and then freeze-drying.

6. The use of a polygonatum sibiricum lectin modified protein as claimed in claim 1 or 3 in the preparation of a medicament with anti-tumor cell proliferation activity.