CN111116718A - Protein connecting peptide and application thereof in modification of TP recombinant antigen - Google Patents

Protein connecting peptide and application thereof in modification of TP recombinant antigen Download PDF

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CN111116718A
CN111116718A CN202010097789.2A CN202010097789A CN111116718A CN 111116718 A CN111116718 A CN 111116718A CN 202010097789 A CN202010097789 A CN 202010097789A CN 111116718 A CN111116718 A CN 111116718A
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protein
linker
recombinant
stability
specificity
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兰军
李萌萌
徐慧珍
张娜
钟士博
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Beijing Boshengfu Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention discloses a protein connecting peptide and application thereof in transforming TP recombinant antigen. The TP recombinant antigen uses linker proteins with different lengths to connect TPN15, TPN17 and TPN 47. The Linker is in two forms of (EAAAK) n and (GGGGS) n, and when the length of the Linker is n-5 and n-6, the protein stability and specificity are best. The invention discovers the influence of the length of the Linker on the stability and the specificity of the protein for the first time, and opens up a new idea for modifying the recombinant protein to improve the activity of the recombinant protein in the future.

Description

Protein connecting peptide and application thereof in modification of TP recombinant antigen
Technical Field
The invention belongs to the technical field of enzyme-linked immunosorbent assay, and particularly relates to a protein connecting peptide and application thereof in modification of TP recombinant antigen.
Background
Syphilis (i.e., treponema pallidum) is a sexually transmitted disease, mainly transmitted through sexual contact and blood, and is a serious sexually transmitted disease next to aids. The currently widely applied treponema pallidum serum detection methods are enzyme-linked immunosorbent assay (ELISA), lateral chromatography immunization and chemiluminescence methods. The TP detection kit at the present stage has certain false positive samples, and the false positive samples are clinically tested and are derived from technical false positive and biological false positive. The technical false positive refers to the false positive of the specimen under the conditions of preservation, transportation or laboratory operation, and statistics shows that about 25% of the false positive belongs to the category, and the false positive can be confirmed by repeated tests; the biological false positive is the false positive of the detection result caused by the age of the person to be detected and other diseases (such as other infectious diseases, connective tissue diseases, autoimmune diseases and the like) infected by the person to be detected, and the false positive is still obtained after repeated tests. How to improve the specificity of TP recombinant antigen, eliminate biological false positive and further improve the stability of TP recombinant antigen is a research focus in the field.
At present, TPN15-TPN17-TPN47 is adopted as a TP recombinant antigen for chimeric expression of polypeptides, and polypeptide fragments are directly connected. In order to improve the stability and specificity of the TP recombinant antigen, a method for modifying protein structures and core mutation sites of TPN15, TPN17 and TPN47 in the TP recombinant antigen is generally adopted, but the current TP recombinant antigen is continuously improved and optimized, and the problems of false positive detection and low specificity still cannot be solved.
The function of the Linker in the recombinant protein is to maintain the conformation of each active protein of the recombinant antigen, so that the recombinant protein has the effects of presenting an antigen epitope and promoting the combination of antigen and antibody, and the selection of the Linker polypeptide sequence and the selection of the length are generally randomly set according to experience without a certain rule. For example, 201510990710.8 discloses a TP recombinant antigen, its preparation method and application, wherein the TP recombinant antigen uses TPN15-TPN17-TPN47 to chimeric express polypeptide, the linker is flexible linking peptide with length of 110aa, and the patent does not discuss the relationship between the length of flexible linking peptide and the stability and specificity of recombinant active protein. Patent 201310669798.4 discloses the sequences of (EAAAK) m and (GGGGS) n, but the disclosed flexible linker peptide combinations are too short to be suitable for TP recombinant antigens of the invention, and the patent is random for the length selection of the linker and does not discuss the effect of the length change of the linker on the stability and specificity of the protein.
Disclosure of Invention
The invention aims to provide a protein connecting peptide and application thereof in transforming TP recombinant antigen.
A protein linker peptide comprising (EAAAK) n and/or (GGGGS) n, n-5-7.
Preferably, n-5 or n-6.
A chimera of a TP protein comprising the protein linker peptide.
Preferably, the chimera of TP protein is TPN15-TPN17-linker-TPN47, and the linker is the protein connecting peptide.
An ELISA kit for detecting syphilis, which comprises the chimera of the TP protein.
The invention has the beneficial effects that: the invention reforms TP recombination antigen and adopts Linker as (EAAAK) n and (GGGGS) n; when the length of Linker, namely n is 0, n is 2 and n is 4, the protein stability and specificity are poor; when n is 5 or 6, the protein stability and specificity are improved; when n is 8 and n is 10, the protein stability and specificity are poor; the introduction of the linker has great influence on the whole protein structure, and the influence on the protein properties is different along with the change of the length of the linker; when the length of the amino acid residue of the linker is 25-35, the protein stability and specificity of the obtained recombinant TP antigen are the best. The invention discovers the influence of the length of the Linker on the stability and the specificity of the protein for the first time, and opens up a new idea for modifying the recombinant protein to improve the activity of the recombinant protein in the future.
Drawings
FIG. 1 shows the experimental electrophoretogram of the stability of the recombinant proteins TP, TH4S and TH6S at 37 ℃ and-20 ℃.
FIG. 2 is the experimental electrophoresis chart of the stability of the recombinant proteins TH4M and TH6M at 37 ℃ and-20 ℃.
FIG. 3 shows the experimental electrophoretogram of the stability of the recombinant proteins TH4L, TH6L, TH4U and TH6U at 37 ℃ and-20 ℃.
FIG. 4 shows the experimental electrophoretograms of the stability of the recombinant proteins TH4M2, TH6M2, TH4L2 and TH6L2 at 37 ℃ and-20 ℃.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 Gene splicing and vector construction
The dominant epitopes were selected by consulting the sequence information of TPN15, TPN17, TPN47 at NCBI, as shown in table 1:
TABLE 1
TPN15 TPN17 TPN47
GeneBank NO. U73115.1 M74825 QBC41615.1
Dominant epitope sequences 27aa-142aa 37aa-125aa 36aa-434aa
The following 13 proteins described in table 2 were designed to be prepared:
TABLE 2
Figure BDA0002385774810000031
Figure BDA0002385774810000041
After determining the gene fragment, obtaining the chimeric gene by a gene synthesis mode, inserting the gene into pET-28a, selecting NcoI and XhoI enzyme cutting sites, transforming Escherichia coli DH5 α, identifying the correct positive plasmid by bacterial liquid PCR, extracting the correct plasmid DNA, sequencing, and selecting the recombinant plasmid with correct sequencing (namely the result is consistent with the designed sequence).
EXAMPLE 2 protein expression and purification
Transforming the recombinant plasmid into Escherichia coli BL-21(DE3), and coating the Escherichia coli BL-21 in LB solid culture medium with kanamycin resistance; picking single colony, transferring the single colony to a fresh LB culture medium containing kanamycin resistance, and culturing at 37 ℃ overnight; transferred to 600mL of LB medium containing kanamycin resistance, cultured to an OD of about 0.6, and induced at 16 ℃ for 22 hours at 100rpm with the addition of IPTG (final concentration of 0.1-0.4 mM). Taking 2-5mL of thallus, centrifuging, collecting the thallus, suspending in 1mL of PBS buffer solution, ultrasonically crushing, (ultrasonically stopping for 3', 1min), centrifuging at 12000rpm for 1min, and taking supernatant and precipitate; a40 uL sample was taken, 10uL of 5 Xloadingbuffer was added, boiled in boiling water for 5min, and subjected to SDS-PAGE. Coomassie brilliant blue staining.
Selecting Ni filler; adjusting the flow rate to 1-3ml/min, and washing the column with water for 5 column volumes; equilibrating and stabilizing the column with equilibration buffer at a constant flow rate using 20 column volumes of equilibration buffer; reducing the flow speed to the sampling flow speed, and sampling; the column was allowed to equilibrate after the sample was loaded. Adjusting the flow rate to a maximum flow rate of about 80% that can be tolerated by the column, washing the column with equilibration buffer for about 10 column volumes; when the reading of the ultraviolet detector drops to a near baseline, the collection of the passing peaks is stopped. Starting elution, collecting elution peaks, and marking sequence numbers; after the base line is stable, inserting the second eluent into the first eluent, starting elution, collecting elution peaks, and marking serial numbers; repeating the steps according to the type and the number of the eluent until the elution is finished; a40 uL sample was taken, 10uL of 5 Xloadingbuffer was added, boiled in boiling water for 5min, and subjected to SDS-PAGE. Coomassie brilliant blue staining. And selecting the protein with higher purity to carry out next experimental verification.
EXAMPLE 3 stability testing of proteins
Adding 0.05% sodium azide into 13 proteins in total, and placing the proteins in a 37-oven and a refrigerator at-20 ℃; after 6 days, 40uL of the sample was taken, 10uL of 5 Xloadingbuffer was added, and after boiling in boiling water for 5min, SDS-PAGE was performed. Coomassie brilliant blue staining.
Results as shown in fig. 1-4, when the length of Linker, i.e., n-0, n-2, n-4, the TP recombinant protein oven treated at 37 ℃ degraded severely and protein stability was poor; when n is 5 and n is 6, the TP recombinant protein treated by a refrigerator at the temperature of-20 ℃ and an oven at the temperature of 37 ℃ is not obviously degraded, and the protein stability is good; when n is 8 and n is 10, the TP recombinant protein treated by a refrigerator at-20 ℃ and an oven at 37 ℃ is seriously degraded, and the protein stability is poor.
Example 4 detection of TP recombinant proteins
Diluting protein to coating concentration (0.005-0.1ug/ml) with 50mM CB (pH8.5-10), adding mercaptoethanol (volume ratio 1:1 k-1: 3k) for pretreatment, and treating at room temperature (15-25 deg.C) for 30min-1 h. The processed protein coating solution is added into a high-adsorption 96-well plate according to the coating amount of 100 ul/well for coating, and is wrapped by preservative film to prevent the coating solution from evaporating and losing, and the coating solution is coated for 16 to 22 hours at room temperature (15 to 25 ℃) overnight. The next day, the fresh-keeping film is washed for 2-5 times by a plate washing machine (washing solution: 10mM PBST, pH6.8-8.0), then the fresh-keeping film is patted dry, 200 ul/hole of the fresh-keeping film is added with antigen sealing solution for sealing, and the fresh-keeping film is wrapped on the fresh-keeping film and placed in a constant-temperature oven at 37 ℃ for incubation for 2-4 hours. And after the incubation is finished, taking out the coated plate, standing at room temperature (15-25 ℃) for balancing for 30min-1h, then throwing off the sealing liquid in the coated plate, drying, and then placing in a fume hood or drying in drying equipment for later use.
The proteins TP15, TP17 and TP47 to be labeled are diluted to a concentration of 1.0-2.0 mg/ml with 50mM CB (pH8.5-10), and then put into dialysis bags respectively, and dialyzed (2-8 ℃) with 50mM CB (pH8.5-10) buffer solution, and the solution is changed once every 3-4 hours for 10-12 hours. Weighing arrangementHorse Radish Peroxidase (HRP)10mg/ml, preparation NaIO4Concentration 13mg/ml, then according to V (NaIO)4): v (hrp) 1:2 addition of NaIO4Oxidation of HRP (room temperature 15-25 ℃) is carried out for 25-30 min in the dark. Then 20 times diluted ethylene glycol was added to terminate the oxidation. Then, the protein and HRP are crosslinked in 50mM CB (pH8.5-10) buffer solution under a certain mass ratio (mTP15: mHRP 1: 1; mTP17: mHRP 1: 6; mTP47: mHRP 1:2) and in a dark place for 12h, and then 10mg/ml NaBH is added4Adding the solution with the volume 0.04 times of the mass of the HRP, terminating crosslinking, and reacting for 2-4h at 2-8 ℃. After the reaction, the enzyme-labeled proteins (TP15-HRP, TP17-HRP and TP47-HRP) were obtained by dialysis against 20mM Tris +150mM NaCl (0.85% w/v) pH7.5 to 8.2.
Preparing enzyme-labeled TP15-HRP, TP17-HRP and TP47-HRP into monomer mixed enzyme by using enzyme diluent, enabling the final dilution ratio of the enzyme-labeled TP15-HRP, the TP17-HRP and the enzyme-labeled TP47-HRP to be 1: 10000-1: 40000, 1: 30000-1: 80000 and 1: 10000-1: 40000 respectively, adding the prepared monomer mixed enzyme according to 100 ul/hole, and carrying out incubation reaction for 30min at 37 ℃.
Adding blood sample to be tested into a coated 96-well plate according to 100 ul/hole, taking a reaction hole with 10% of calf serum as a blank control, standing at 37 ℃ for reacting for 1h at constant temperature, taking out, washing for 5 times by a plate washing machine (washing solution: 10mM PBST, pH6.8-8.0), and drying by beating; adding 100ul enzyme-labeled protein per hole in a proper proportion, placing the mixture at 37 ℃ for constant-temperature reaction for 30min, taking the mixture out, washing the mixture for 5 times by using a plate washing machine, and drying the mixture by beating; adding TMB color developing agent into the mixture according to 100 ul/hole, reacting at 37 ℃ for 30min, taking out the mixture, adding 50 ul/hole dilute sulfuric acid solution, and measuring the absorption values at 450nm and 630nm to obtain the increment between the two. The increment is negative below 0.11, gray area is 0.11-0.15, and positive above 0.15.
Selecting 8 false positive samples from nearly 5000 serum samples, namely (-2, -4, -9, -10, -11, -13, -14 and-16), and detecting and confirming all the false positive samples to be negative samples by various methodologies such as a TPPA method and the like; selecting 3 parts of positive serum, namely (mixed positive, 07-3 and 116) as strong, medium and weak positive samples; 6mIU is a quality control serum sample; fixing marker protein and protein concentration, and coating 13 kinds of above antigens. The results of the above 13 antigens and ELISA detection methods were shown in Table 3.
As can be seen from table 3, when the length of Linker, i.e., n-0, n-2, n-4, the specificity of TP recombinant protein is poor; when n is 5 or 6, the specificity of the TP recombinant protein becomes good; when n is 8 and n is 10, the specificity of the TP recombinant protein is poor; the introduction of the linker really has great influence on the whole protein structure, and the influence on the protein properties is different along with the change of the length of the linker; the specificity of the recombinant TP antigen obtained is best when the linker is 25 or 30 amino acid residues in length.
TABLE 3
Mixed yang 07-3 116 -2 -4 -9 -10 -11 -13 -14 -16 6miu
TP 3.501 1.724 0.655 0.294 0.295 0.297 0.573 0.270 0.418 0.292 0.299 1.456
TH4S 3.501 1.638 0.711 0.287 0.256 0.311 0.612 0.243 0.452 0.283 0.234 1.543
TH6S 3.501 1.654 0.694 0.277 0.267 0.322 0.599 0.279 0.396 0.256 0.289 1.687
TH4M 3.501 2.115 0.702 0.231 0.201 0.137 0.339 0.239 0.285 0.480 0.114 1.506
TH6M 3.501 2.327 0.718 0.372 0.208 0.085 0.391 0.250 0.430 0.247 0.114 1.534
TH4L 3.501 1.626 0.697 0.037 0.042 0.043 0.042 0.039 0.032 0.043 0.040 1.786
TH6L 3.225 1.924 0.665 0.042 0.047 0.040 0.041 0.020 0.031 0.033 0.021 1.893
TH4U 3.501 1.770 0.657 0.033 0.040 0.038 0.024 0.036 0.034 0.041 0.034 2.029
TH6U 3.501 1.789 0.989 0.022 0.021 0.012 0.020 0.027 0.004 0.037 0.007 1.914
TH4M2 3.501 1.645 0.776 0.111 0.221 0.145 0.234 0.123 0.245 0.153 0.112 1.896
TH6M2 3.501 1.755 0.656 0.131 0.261 0.125 0.264 0.163 0.215 0.143 0.122 1.766
TH4L2 3.501 1.822 0.616 0.171 0.291 0.345 0.434 0.223 0.445 0.253 0.212 1.798
TH6L2 3.501 1.645 0.776 0.189 0.321 0.375 0.454 0.245 0.478 0.267 0.234 1.876
Description of the drawings: the values in the table are all OD450 values detected, and the test was negative when the value was less than 0.15 and positive when the value was 0.15 or more.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (5)

1. A protein linker peptide comprising (EAAAK) n and/or (GGGGS) n, wherein n is 5-7.
2. The protein linker peptide according to claim 1, wherein n-5 or n-6.
3. A chimera of a TP protein comprising the protein linker peptide of claim 1.
4. The chimera of TP protein according to claim 3, wherein the chimera of TP protein is TPN15-TPN17-linker-TPN47, and the linker is the protein linking peptide according to claim 1.
5. An ELISA kit for detecting syphilis, comprising a chimera of the TP protein of claim 3 or 4.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248331B1 (en) * 1995-12-25 2001-06-19 Fujirebio, Inc. Treponema pallidum fused antigen and assay for anti-treponema pallidum antibodies using the same fused antigen
CN102183646A (en) * 2010-12-16 2011-09-14 孙爱华 Preparation method of rTpN15-17-47-ELISA for detecting syphilis serum antibody
EP2617731A1 (en) * 2012-01-19 2013-07-24 Roche Diagnostics GmbH Soluble immunoreactive Treponema pallidum TpN47 antigens
US20140186861A1 (en) * 2011-07-11 2014-07-03 Uvic Industry Partnerships Inc. Soluble treponema pallidum protein tp0453, tp0453-tp0326 fusion protein, and use in syphilis diagnosis
CN104693270A (en) * 2013-12-10 2015-06-10 清华大学 Connecting peptide for fusion protein
CN105542014A (en) * 2015-12-24 2016-05-04 菲鹏生物股份有限公司 TP recombinant antigen and preparing method and application thereof
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Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248331B1 (en) * 1995-12-25 2001-06-19 Fujirebio, Inc. Treponema pallidum fused antigen and assay for anti-treponema pallidum antibodies using the same fused antigen
CN102183646A (en) * 2010-12-16 2011-09-14 孙爱华 Preparation method of rTpN15-17-47-ELISA for detecting syphilis serum antibody
US20140186861A1 (en) * 2011-07-11 2014-07-03 Uvic Industry Partnerships Inc. Soluble treponema pallidum protein tp0453, tp0453-tp0326 fusion protein, and use in syphilis diagnosis
EP2617731A1 (en) * 2012-01-19 2013-07-24 Roche Diagnostics GmbH Soluble immunoreactive Treponema pallidum TpN47 antigens
CN104693270A (en) * 2013-12-10 2015-06-10 清华大学 Connecting peptide for fusion protein
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CN108059683A (en) * 2017-12-21 2018-05-22 江汉大学 Syphilis restructuring multivalent fusion antigen, syphilis recombinant antigen plasmid and syphilis recombinant antigen bacterial strain and preparation method thereof and kit

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Title
BRENDEN C.SMITH等: "New proteins for a new perspective on syphilis diagnosis", 《JCM.》 *
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