阅读说明：本技术 用于检测肿瘤标志物pd-l1的高灵敏度量子点探针、制备方法及应用 (High-sensitivity quantum dot probe for detecting tumor marker PD-L1, preparation method and application ) 是由 施杰 吴志猛 李霞 刘金龙 于 2021-09-15 设计创作，主要内容包括：本发明公开了一种检测细胞表面肿瘤标志物PD-L1的量子点荧光探针、制备方法及应用,该探针荧光强度高,提高了相关检测的灵敏度。包括步骤：1)识别PD-L1的纳米抗体重组蛋白的设计、识别PD-L1的纳米抗体重组蛋白的原核表达和纯化；2)含有生物素基团多肽的制备；3)识别PD-L1的纳米抗体重组蛋白与生物素化多肽的体外酶法融合；生物素化的识别PD-L1的纳米抗体重组蛋白的纯化；4)生物素化的识别PD-L1的纳米抗体重组蛋白与链酶亲和素标记量子点的偶联探针制备。本发明的探针荧光强度高,结合流式细胞术、免疫荧光、酶联免疫吸附技术能有效检测细胞表面肿瘤标志物PD-L1的表达,方法简便,检测灵敏度高。(The invention discloses a quantum dot fluorescent probe for detecting a cell surface tumor marker PD-L1, a preparation method and application thereof. The method comprises the following steps: 1) designing a nano antibody recombinant protein for identifying PD-L1, and performing prokaryotic expression and purification on the nano antibody recombinant protein for identifying PD-L1; 2) preparing a polypeptide containing a biotin group; 3) the in vitro enzyme method of the nano antibody recombinant protein for recognizing PD-L1 and the biotinylated polypeptide is fused; purification of biotinylated nanobody recombinant protein recognizing PD-L1; 4) preparing a coupling probe of biotinylated nano antibody recombinant protein for recognizing PD-L1 and streptavidin marked quantum dots. The probe of the invention has high fluorescence intensity, can effectively detect the expression of a cell surface tumor marker PD-L1 by combining with flow cytometry, immunofluorescence and enzyme-linked immunosorbent assay technology, and has simple method and high detection sensitivity.)

1. A high-sensitivity quantum dot probe for detecting a tumor marker PD-L1, which is characterized by being formed by coupling the following two parts:

a. biotinylated nano-antibody recombinant protein RNB-MS-Bio for recognizing PD-L1,

b. streptavidin-labeled quantum dots;

wherein the biotinylated nano-antibody recombinant protein RNB-MS-Bio for recognizing PD-L1 is obtained by coupling the following components:

c. a nano antibody recombinant protein RNB-MSH for recognizing PD-L1,

d. biotinylation polypeptide GK-Bio;

wherein the nano-antibody recombinant protein RNB-MSH for identifying PD-L1 is obtained by identifying PD-L1 nano-antibody through carboxyl terminal modification, and the carboxyl terminal modification is as follows: modifying Myc tags, transpeptidase A recognition sites and His tag sequences at the carboxyl terminals of the nano antibodies for recognizing PD-L1;

the biotinylated polypeptide GK-Bio is a polypeptide sequence containing biotin groups and repeated glycine.

2. The high-sensitivity quantum dot probe for detecting the tumor marker PD-L1 as claimed in claim 1, wherein the amino acid sequence of the nano-antibody recombinant protein RNB-MSH for identifying PD-L1 is shown as SEQ ID No. 2.

3. The high-sensitivity quantum dot probe for detecting the tumor marker PD-L1, according to claim 1, wherein the biotinylated polypeptide is a polypeptide containing 1-3 glycin sequences in the amino acid sequence or having similar purpose based on the sequence change of the short peptide.

4. The high-sensitivity quantum dot probe for detecting the tumor marker PD-L1 as claimed in claim 3, wherein the amino acid sequence of the biotinylated polypeptide is shown as SEQ ID No. 4.

5. The method for preparing the high-sensitivity quantum dot probe for the tumor marker PD-L1 as claimed in any one of claims 1 to 4, characterized by comprising the following steps:

1) prokaryotic expression and purification of a nano antibody recombinant protein RNB-MSH for recognizing PD-L1;

2) preparing biotinylated polypeptide GK-Bio;

3) coupling the nanometer antibody recombinant protein RNB-MSH for identifying PD-L1 with biotinylated polypeptide to obtain biotinylated nanometer antibody recombinant protein RNB-MS-Bio for identifying PD-L1;

4) and coupling the biotinylated nano antibody recombinant protein RNB-MS-Bio for recognizing PD-L1 with streptavidin labeled quantum dots to prepare the probe.

6. The method for preparing the high-sensitivity quantum dot probe for detecting the tumor marker PD-L1 as claimed in claim 5, wherein in the step 2), the preparation of the biotinylated polypeptide GK-Bio adopts a polypeptide solid phase synthesis method, which comprises the following steps: 5-100mM amino resin is used as a solid phase synthesis carrier, a solid phase synthesis instrument is used for synthesis according to the sequence of SEQ No.3, wherein the side chain of lysine contains a biotin group, other amino acids are standard amino acids, the amino ends of all the amino acids contain Fmoc protecting groups, after the synthesis is finished, the protecting groups are removed under the condition of strong acid, and the product is obtained by purification through semi-preparative HPLC.

7. The preparation method of the high-sensitivity quantum dot probe for detecting the tumor marker PD-L1 as claimed in claim 5, wherein in the step 3), the coupling adopts an in vitro enzymatic fusion modification method, comprising the following steps: the enzyme reaction system comprises 50mM Tris, 150mM NaCl and 5mM CaCl₂Mixing the enzyme reaction system with 10-150 mu M of nano antibody recombinant protein RNB-MSH for identifying PD-L1, 50-500 mu M of biotinylated polypeptide GK-Bio and 1-10 mu M of transpeptidase A enzyme, and carrying out shake reaction at 4-37 ℃ for 1-12h to obtain a reaction solution; and incubating the reaction solution and nickel ion magnetic beads for 10-90min to obtain a supernatant solution, namely the biotinylated nano antibody recombinant protein RNB-MS-Bio for recognizing PD-L1.

8. The method for preparing a high-sensitivity quantum dot probe for detecting a tumor marker PD-L1 according to claim 5, characterized in that in step 4), the method for preparing the probe comprises: in a PBS buffer solution with the pH value of 6.0-8.0, forming a coupling probe reaction system of 0.1-10 mu M of biotinylated recombinant protein RNB-MS-Bio for identifying PD-L1 and 1-100 mu M of streptavidin marked quantum dots, keeping out of the sun at the temperature of 4-37 ℃, and carrying out oscillation reaction at the speed of 10-200rpm for 0.5-2 h.

9. The use of the high-sensitivity quantum dot probe for detecting the tumor marker PD-L1 according to any one of claims 1 to 4 or the high-sensitivity quantum dot probe for detecting the tumor marker PD-L1 prepared by the method according to any one of claims 5 to 8 is characterized in that the high-sensitivity quantum dot probe is used for detecting the tumor marker PD-L1 on the cell surface.

10. The application of the high-sensitivity quantum dot probe for detecting the tumor marker PD-L1, which is characterized in that the detection of the cell surface tumor marker PD-L1 comprises the flow cytometry detection of cell surface PD-L1, the enzyme-linked immunosorbent detection of cell surface PD-L1 and the immunofluorescence detection of cell surface PD-L1.

Technical Field

The invention belongs to the technical field of biomedicine/antigen detection, and particularly relates to a probe for detecting a cell surface tumor marker PD-L1 based on a nano antibody recombinant protein and a fluorescent quantum dot, a preparation method and application.

Background

Tumor treatment strategies based on the immune checkpoint inhibition principle of programmed death receptor 1(PD-1) and programmed death receptor ligand 1(PD-L1) have great application prospects in clinical treatment of various tumors, and therefore, accurate detection of the marker is the key to the development of relevant treatment. PD-1 belongs to the B7 immunoglobulin family and is an inhibitory transmembrane receptor on T cells. PD-L1 is the main ligand, widely expressed in immune cells and various malignant tumor cells, including malignant melanoma, lung cancer, liver cancer, renal cell carcinoma, ovarian cancer, colorectal cancer and the like. In the body normal immune negative regulation process, PD-1/PD-L1 plays an important role in regulating immune homeostasis. However, the high expression of PD-L1 on the surface of tumor cells can block T cell immune response and generate immunosuppressive tumor microenvironment, which is an important mechanism for tumor immune escape. The PD-L1 overexpressed on the surface of the tumor cell is not only an ideal effective tumor treatment target, but also an important marker molecule for accurate tumor diagnosis, and the development of an accurate detection method of PD-L1 has very important significance for developing related tumor treatment.

The existing PD-L1 clinical detection method is mainly based on monoclonal antibody Immunohistochemistry (IHC) technology, the method is not high in specificity and sensitivity, common in false negative, and poor in tissue penetrability of the used monoclonal antibody, particularly difficult to accurately detect solid tumors and thick sample tissues, and seriously restricts the curative effect and further popularization and application of the PD-L1/PD-1-based tumor accurate diagnosis and treatment. In addition, there is also a PD-L1 probe labeled with an isotope of a monoclonal antibody, but because of its large molecular weight and long half-life, it cannot be rapidly cleared from blood, and requires a long waiting time to acquire a radioactive image with a low background, increasing the imaging significance of the lesion site, and consuming a long time. The isotope-based probe also has some unavoidable defects, such as the harm of radioactive radiation, low spatial resolution and the like, and the technology relates to isotope or PET-CT and other large-scale equipment, has high requirements on equipment environment and the like, and is difficult to popularize clinically at present.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects and technical bottlenecks in the existing tumor marker PD-L1 detection technology and improve the sensitivity and accuracy of the PD-L1 detection technology, the invention provides a high-sensitivity probe for PD-L1 detection based on a nano-antibody recombinant protein and a fluorescent quantum dot, a preparation method and application thereof. The nano antibody has the advantages of small volume, good stability, strong affinity, easy modification, strong tissue penetration capability, low immunogenicity and the like, and is an ideal tumor marker and target recognition molecule. The quantum dot is a novel nano fluorescent dye, has the characteristics of high quantum yield, good stability, small particle size and the like, generates near infrared light with strong biological tissue penetration capability and small interference by organism autofluorescence, and is a hotspot fluorescent molecule for biomedical molecule detection research.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention relates to a novel high-efficiency detection technology of a tumor marker PD-L1, which comprises the steps of firstly exogenously expressing and identifying a nano antibody recombinant protein RNB-MSH of PD-L1, then preparing a polypeptide sequence GK-Bio containing biotin through solid phase synthesis, fusing the recombinant protein and the biotin polypeptide by using an in vitro enzyme method mediated by Sortase A enzyme to obtain a nano antibody recombinant protein RNB-MS-Bio of the biotinylated and identified PD-L1, finally coupling the recombinant protein RNB-MS-Bio and a streptoenzyme avidin labeled quantum dot to form a probe, and combining flow cytometry, immunofluorescence and enzyme-linked immunosorbent assay to detect the tumor marker PD-L1 on the cell surface.

The exogenous expression is carried out in prokaryotic escherichia coli E.coli BL21(DE 3).

The invention aims to provide a probe for detecting a cell surface tumor marker PD-L1 based on nanobody recombinant protein and quantum dot fluorescence, which consists of the following two parts:

a. biotinylated nano-antibody recombinant protein RNB-MS-Bio for recognizing PD-L1,

b. labeling quantum dots with streptavidin;

the invention also aims to provide a method for preparing the biotinylated nano-antibody recombinant protein RNB-MS-Bio for recognizing PD-L1, wherein the recombinant protein is obtained by coupling two parts:

c. a nano antibody recombinant protein RNB-MSH for recognizing PD-L1,

d. a polypeptide sequence containing biotin, namely biotinylated polypeptide GK-Bio;

wherein, the nano-antibody recombinant protein RNB-MSH for identifying PD-L1 is obtained by modifying the carboxyl terminal of a nano-antibody capable of identifying PD-L1, and the modification is as follows: MYC label, transpeptidase A (sortaseA) recognition site and His label sequence are added at the carboxyl terminal of the nano antibody capable of recognizing PD-L1.

The biotinylation polypeptide is a polypeptide sequence containing a biotin group and a glycine repeat, and the biotin group can be present at any position of the polypeptide in any mode.

In the embodiment of the invention, the amino terminal of the biotinylated polypeptide contains 1-3 glycine repetitive sequences, and the carboxyl terminal of the biotinylated polypeptide contains biotin side chain modified lysine. The biotinylated polypeptide may also be a polypeptide having a similar purpose based on the change in the sequence of the biotinylated short peptide, i.e., a polypeptide having a similar function based on the basic characteristics of the biotinylated short peptide.

In the embodiment of the invention, one of the biotinylated polypeptides is selected as shown in SEQ ID No. 4.

In the embodiment of the invention, the amino acid sequence of the recombinant protein RNB-MSH for recognizing the PD-L1 nano antibody is shown as SEQ ID No.2, and the nucleotide sequence of the recombinant protein RNB-MSH for encoding the nano antibody capable of recognizing the PD-L1 is shown as SEQ ID No. 3.

One objective of the present invention is to provide a preparation method of a high-sensitivity quantum dot probe for detecting a tumor marker PD-L1, comprising the following steps:

1) prokaryotic expression and purification of a nano antibody recombinant protein RNB-MSH for recognizing PD-L1;

2) preparing biotinylated polypeptide GK-Bio;

3) coupling the nanometer antibody recombinant protein RNB-MSH for identifying PD-L1 with biotinylated polypeptide to obtain biotinylated nanometer antibody recombinant protein RNB-MS-Bio for identifying PD-L1;

4) and coupling the biotinylated nano antibody recombinant protein RNB-MS-Bio for recognizing PD-L1 with streptavidin labeled quantum dots to prepare the probe.

In the embodiment of the invention, in the step 1), the expression of the nanobody recombinant protein RNB-MSH capable of recognizing PD-L1 comprises the following steps: cloning the nucleotide sequence of the coding nano antibody recombinant protein RNB-MSH capable of recognizing PD-L1 to a pET22b expression vector, transforming to escherichia coli E.coli BL21(DE3), wherein the concentration of an inducer is 0.2-1.0mM/L, the induction temperature is 16-37 ℃, and finally purifying by nickel ion affinity column chromatography.

In the embodiment of the invention, in step 2), the polypeptide sequence GK-Bio of biotin is obtained by solid phase synthesis, and comprises the following steps: 5-100mM amino resin is used as a solid phase synthesis carrier, a solid phase synthesizer is used for synthesis according to the sequence of SEQ No.3, wherein the side chain of lysine contains a biotin group, other amino acids are standard amino acids, the amino ends of all the amino acids contain Fmoc protecting groups, after the synthesis is finished, the protecting groups are removed under the condition of strong acid, and the product is obtained by semi-preparative HPLC purification.

In the embodiment of the invention, in the step 3), the in vitro enzyme coupling preparation method of the biotinylated recombinant protein RNB-MS-Bio for recognizing PD-L1 comprises the following steps: the enzyme reaction buffer system is 50mM Tris, 150mM NaCl and 5mM CaCl₂Mixing the enzyme reaction system with 10-150 mu M of the recombinant protein RNB-MSH for recognizing the PD-L1 nano antibody, 50-500 mu M of biotinylated polypeptide sequence GK-Bio and 1-10 mu M of transpeptidase A enzyme (Sortase A enzyme) and reacting for 1-12h at 4-37 ℃. Then, incubating the reaction solution and nickel ion magnetic beads for 10-90min to obtain supernatant solution, namelyThe biotinylated recombinant protein RNB-MS-Bio that recognizes PD-L1.

In the embodiment of the invention, in the step 4), the preparation method of the probe Strep-QDs based on the biotinylation recombinant protein RNB-MS-Bio for identifying PD-L1 and the fluorescent quantum dots comprises the following steps: the buffer solution for reaction is PBS buffer solution with pH6.0-8.0, two substances are added into the buffer solution to form a coupling probe reaction system of 0.1-10 mu M biotinylated recombinant protein RNB-MS-Bio for identifying PD-L1 and 1-100 mu M streptavidin marked quantum dots, and the coupling probe reaction system is protected from light at 4-37 ℃ and is subjected to oscillation reaction at the rotating speed of 10-200RPM for 30min-2h to prepare the probe.

The invention also aims to provide application of the probe based on the nano-antibody and the fluorescent quantum dot for detecting the tumor marker PD-L1, which is used for detecting the tumor marker PD-L1 on the cell surface.

In the embodiment of the invention, the detection of the cell surface tumor marker PD-L1 comprises flow cytometry detection of cell surface PD-L1, enzyme-linked immunosorbent assay of cell surface PD-L1 and immunofluorescence detection of cell surface PD-L1.

In the embodiment of the invention, the probe is used for detecting cell surface PD-L1 by flow cytometry, and comprises the following steps: mixing 10-100 mu L of nano antibody recombinant protein RNB-MS-Bio containing 10-100nM biotinylated recognition PD-L1 with cells to be detected, incubating at 4-37 ℃ for 30-90min, washing with PBS, adding 10-50 mu L of Strep-QDs solution containing 20-100nM, incubating at 4-37 ℃ for 30-90min, washing with PBS, resuspending the cells, and detecting with a flow cytometer, wherein the emission spectrum is 605nM +/-5 nM; .

In the embodiment of the invention, the probe is applied to enzyme-linked immunosorbent assay of cell surface PD-L1, and comprises the following steps: cells were fixed in a multi-well plate with 4% paraformaldehyde at room temperature for 10-30min and washed three times with PBST. And (2) sealing the cell at the temperature of a back sealing liquid for 1-2 hours, washing the cell by PBST three times, correspondingly adding 100 mu L of biotinylated recombinant protein RNB-MS-Bio for recognizing PD-L1 into each hole with the concentration of 10-50nM, incubating for 0.5-2 hours at the temperature of 4-37 ℃, washing the plate by the PBST three times after the completion, then adding 50 mu L of the streptavidin-labeled quantum dot containing 1-20nM into each hole, incubating for 0.5-2 hours at the temperature of 4-37 ℃, washing the hole by the PBST three times, and then detecting the absorption value of each hole by an enzyme labeling instrument at the excitation wavelength of 405nM and the emission wavelength of 605 nM.

In the embodiment of the invention, the probe is applied to immunofluorescence detection of cell surface PD-L1, and comprises the following steps: placing tumor tissue slices at 65 ℃ for 45-60min for dewaxing, soaking the tumor tissue slices in xylene and 100% -50% ethanol for 5-10min, washing the tissue slices with PBS for three times, then placing the tissue slices in 0.01M sodium citrate buffer solution with pH6.0, boiling for 10min, preserving heat for 10min, washing the tissue slices with PBS for three times, sealing the tissue slices with sealing liquid in a wet box at 37 ℃ for 2h after finishing, washing the tissue slices with PBS for three times, dropwise adding 20-50 mu L of biotinylated recombinant protein RNB-MS-Bio capable of identifying PD-L1 with the concentration of 25-100nM to the corresponding tissue slices, incubating the tissue slices at 4-37 ℃ for 1-12h, washing the tissue slices with PBS for three times after finishing, then dropwise adding 20-50 mu L of quantum dots containing 1-20nM streptavidin markers to the slices, incubating the tissue slices at 37 ℃ for 30min in a dark place, washing the tissue slices with PBS for three times, then, a drop of an anti-fluorescence quencher is dropped on the tissue section, and the tissue section is observed by a laser confocal microscope under different exciting lights and photographed.

Has the advantages that: compared with the prior art, the probe based on the nano-antibody recombinant protein and the fluorescent quantum dot for detecting the cell surface tumor marker PD-L1, the preparation method and the application have the following advantages: the invention successfully constructs the cell surface tumor marker PD-L1 detection method with high detection sensitivity, and the method has the advantages of good stability, simple and convenient operation, high detection signal intensity, high sensitivity and the like. The preparation method of the biotinylated recombinant protein RNB-MS-Bio for identifying PD-L1, disclosed by the invention, has the advantages of simplicity in operation, low cost, high conversion efficiency, greenness, environmental friendliness and no emission of toxic and harmful substances. The probe provided by the invention provides reliable and effective technical support for accurate diagnosis and treatment of tumors based on the PD-L1 tumor marker.

Drawings

FIG. 1 is a schematic diagram of the technical scheme of the present invention.

FIG. 2 is the SDS-PAGE electrophoresis picture of the expression and purification of the nano antibody recombinant protein RNB-MSH.

Wherein, Lane M is a molecular weight standard protein sample, Lane H is a whole bacteria solution, Lane S is a supernatant of a fermentation broth, Lane 1 is a sample eluted at 50mM imidazole concentration, Lane 2-4 are samples eluted at 100mM imidazole concentration, Lane 5-8 are samples eluted at 150mM imidazole concentration, Lane 9-10 are samples eluted at 250mM imidazole concentration, and Lane 11 is a sample eluted at 500mM imidazole concentration.

FIG. 3 is a mass spectrum containing biotinylated polypeptide GK-Bio.

FIG. 4 shows the HPLC chromatogram after purification of GK-Bio containing biotinylated polypeptide.

FIG. 5 is a key SDS-PAGE electrophoresis chart in the process of preparing biotinylated nano-antibody recombinant protein RNB-MS-Bio recognizing PD-L1 by enzyme method.

Wherein, Lane M is a molecular weight standard protein sample, Lane 1 is a control of purified nano antibody recombinant protein RNB-MSH, Lane 2 is a control of Sortase A enzyme protein, Lane 3 is a sample at the time of enzyme reaction 0, Lane 4 is a sample after enzyme reaction 3 hours, Lane 5 is biotinylated recognition PD-L1 nano antibody recombinant protein RNB-MS-Bio after nickel magnetic bead purification, and Lane 6 is a sample washed by nickel magnetic beads.

FIG. 6 is a Western Blot image for verifying biotinylated nanobody recombinant protein RNB-MS-Bio; lane M is a standard protein sample, and lane 1 is RNB-MS-Bio after incubation with HRP-Strep.

FIG. 7 shows key data of enzyme-linked immunosorbent assay application and specificity of the probe based on the nanometer antibody recombinant protein and quantum dot fluorescence for detecting the cell surface tumor marker PD-L1.

Wherein, CHO is Chinese hamster ovary cells which do not express PD-L1, and CHO-PD-L1 is Chinese hamster ovary cells which highly express PD-L1. PBS was also used as a blank control. RNB-MS-Bio + Strep-QDs are biotinylated nano antibody recombinant proteins RNB-MS-Bio and a fluorescent quantum dot probe treatment experimental group, and PBS + Strep-QDs are fluorescent quantum dot single treatment controls.

FIG. 8 is key data comparing the detection effect of the probe provided by the present invention and a conventional FITC probe based fluorescent dye.

Wherein CHO is a Chinese hamster ovary cell not expressing PD-L1, CHO-PD-L1 is a Chinese hamster ovary cell highly expressing PD-L1, and MDA-MB-231 is a tumor cell expressing PD-L1. PBS was also used as a blank control. RNB-MS-Bio + Strep-FITC is a traditional fluorescent dye FITC group, RNB-MS-Bio + Strep-QDs is a probe group of the invention, PBS + Strep-FITC and PBS + Strep-QDs are corresponding negative control groups (a single treatment control group of traditional fluorescent dye and a single treatment control group of fluorescent quantum dots).

FIG. 9 shows key data of flow cytometry application based on nanobody recombinant protein and quantum dot fluorescent probe for detecting cell surface tumor marker PD-L1.

Wherein CHO is a Chinese hamster ovary cell not expressing PD-L1, CHO-PD-L1 is a Chinese hamster ovary cell highly expressing PD-L1, and MDA-MB-231 is a tumor cell expressing PD-L1. The Control is a blank Control group with PBS, and the RNB-MS-Bio + Strep-QDs are probes of the invention.

FIG. 10 shows key data of immunofluorescence applications based on nanobody recombinant proteins and quantum dot fluorescent probes for detecting cell surface tumor marker PD-L1.

Wherein, 25-100nM RNB-MS-Bio is the experimental group of biotinylated nano antibody recombinant protein with different concentrations, and PBS is the control group. Strep-QDs is a quantum dot fluorescence map, DAPI is a nuclear fluorescence map, and Merge is a co-localization map of the two maps.

Detailed Description

The invention discloses a probe for detecting a cell surface tumor marker PD-L1, a preparation method and application, and particularly relates to a probe consisting of a biotinylation-based nano antibody recombinant protein RNB-MS-Bio capable of identifying PD-L1 and streptavidin-labeled fluorescence quantum, a preparation method and application, as shown in figure 1, the probe of the invention and the application comprise the following steps:

1) identifying the expression of a nano antibody recombinant protein RNB-MSH of PD-L1;

2) preparing a polypeptide containing a biotin group, namely a biotinylated polypeptide GK-Bio;

3) the in vitro enzymatic fusion between the nanometer antibody recombinant protein RNB-MSH capable of identifying PD-L1 and the polypeptide containing biotin groups, namely biotinylated polypeptide GK-Bio, to obtain biotinylated nanometer antibody recombinant protein RNB-MS-Bio capable of identifying PD-L1;

4) preparing a coupling probe of a biotinylated nano antibody recombinant protein RNB-MS-Bio for recognizing PD-L1 and a streptavidin labeled quantum dot.

5) The tumor marker PD-L1 on the cell surface is detected by using the probe through flow cytometry, enzyme-linked immunosorbent assay, immunofluorescence and the like.

Wherein, in the step 1), the preparation of the nanometer antibody recombinant protein RNB-MSH for identifying PD-L1 comprises the following steps:

1.1) designing a gene sequence for coding the recombinant protein, and adding a MYC label, a transpeptidase A (sortaseA) recognition site and a His label at the carboxyl terminal of the gene sequence.

1.2) using Escherichia coli BL21(DE3) to express the recombinant protein;

1.3) purifying by adopting a nickel ion affinity chromatography column to obtain the recombinant protein;

in the step 3), biotinylated nano-antibody recombinant protein RNB-MS-Bio for recognizing PD-L1 is prepared by the following steps:

2.1) coupling a nano antibody recombinant protein RNB-MSH capable of recognizing PD-L1 with a polypeptide GK-Bio containing a biotin group under the catalysis of transpeptidase A;

2.2) removing unreacted materials from the nickel ion affinity chromatography magnetic beads, and purifying to obtain biotinylated nano antibody recombinant protein RNB-MS-Bio capable of recognizing PD-L1.

The probe provided by the invention can effectively detect the expression of a cell surface tumor marker PD-L1 by combining enzyme-linked immunosorbent assay, flow cytometry and immunofluorescence, and can be applied to clinical diagnosis, treatment and post-cure monitoring of tumors related to the tumor marker PD-L1. Compared with the probe prepared by the traditional fluorescent dye, the detection method of the tumor marker PD-L1 has the advantages of high fluorescence intensity, high sensitivity, good specificity and lower preparation cost.

The invention is further described with reference to the following figures and examples. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 design of Nanobody recombinant protein RNB-MSH capable of recognizing PD-L1

In this example, part of the amino acid sequence of the protein capable of recognizing PD-L1 in the sequence of the nanobody recombinant protein RNB-MSH is derived from patent CN 106397592A (the original amino acid sequence is SEQ ID No.28 in this patent), and the amino acid sequence is shown in SEQ ID No. 1. In other embodiments, the amino acid sequence of the protein satisfying the requirement of recognition of PD-L1 can be used, and is within the scope of the present invention.

And adding a MYC label, a transpeptidase A recognition sequence and a nickel ion affinity chromatography purification histidine label to the carboxyl terminal of the sequence shown in SEQ ID No.1 to obtain the nano antibody recombinant protein RNB-MSH (SEQ ID No.2) which has an additional new function and can recognize PD-L1. The specific method comprises the following steps:

firstly, adding MYC label (EQKLISEEDLNGAA) and recognition site LPETGG (L: leucine; P: proline; E: glutamic acid; T: threonine; G: glycine) cut by transpeptidase A at the carboxyl terminal of a protein sequence of a nano antibody; then histidine purification label HHHHHHHHHHHH (H: histidine) is added to obtain the amino acid sequence (SEQ ID No.2) of the nano antibody recombinant protein RNB-MSH capable of identifying PD-L1. And reversely deducing the recombinant protein sequence according to the nucleotide codon of the escherichia coli to obtain the nucleotide sequence of the nano antibody recombinant protein RNB-MSH for identifying PD-L1, which is shown as SEQ ID No. 3. The nucleotide sequence (SEQ ID No.3) was synthesized and subcloned into the expression vector pET22b (+) plasmid and the plasmid was transformed into E.coli BL21(DE 3). The strain is a fermentation strain E.coli BL21(DE3) -RNB-MSH for producing a nano antibody recombinant protein RNB-MSH capable of recognizing PD-L1.

Example 2 expression and purification of Nanobody recombinant protein RNB-MSH capable of recognizing PD-L1

E.coli BL21(DE3) -RNB-MSH glycerol strain was inoculated into 5mL of LB fermentation medium (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L) containing ampicillin resistance, and cultured overnight at 200rpm in a shaker 37 ℃. Inoculating TB fermentation medium (peptone 12g/L, yeast powder 24g/L, glycerol 4g/L, KH) at an inoculation amount of 2% the next day₂PO₄23.1g/L，K₂HPO₄125.4g/L) when the density of the fermentation thallus reaches OD₆₀₀About 0.6-0.8 mM inducer IPTG was added to the mixture at a final concentration of 0.8mM and the fermentation was carried out at 37 ℃ on a shaker at 200rpm for 24 hours.

After the fermentation, the cells were collected by centrifugation at 9000rpm for 5min and the supernatant was discarded. After the mycelia were resuspended in a wall-breaking buffer (10mM Tris, 500mM NaCl, pH 8.0), the mycelia were disrupted by an ultrasonication apparatus under the following conditions: and (5) performing ice bath, running for 2s, stopping running for 3s, and running for 20-60 min. After the disruption was completed, centrifugation was carried out at 9000rpm for 10min, which was repeated three times to completely remove cell debris, thereby obtaining a clear cell-disrupted supernatant. The wall-broken supernatant was purified by a nickel ion affinity column, and the column was washed with a washing buffer containing 50mM imidazole to remove foreign proteins. The recombinant protein RNB-MSH is washed down in a gradient format with a wash buffer containing 50mM-500mM imidazole. The purified protein is desalted, freeze-dried and placed at-20 ℃ for later use.

FIG. 2 is an SDS-PAGE electrophoresis characterization chart of the purified recombinant protein, and the gray level analysis of Image J software shows that the purity of the purified RNB-MSH is more than 95%.

Example 3 preparation of polypeptide GK-Bio containing a Biotin group

The amino acid sequence of the biotinylated polypeptide GK-Bio selected in the examples of the present application is shown in SEQ ID No. 4. And preparing GK-Bio by adopting a solid phase synthesis technology based on an Fmoc protection strategy. All Fmoc-protected standard amino acids, containing a biotin groupLysine was dissolved in DMF at a concentration of 0.2M, and 0.22g of Rink Resin was taken as a solid phase Resin, and CEM Liberty Blue was used^TMAnd synthesizing by an automatic polypeptide synthesizer. Fmoc deprotection reagent was 20% piperidine solution and condensation reagent was 0.25M TBTU. Transferring the resin containing the polypeptide into a round-bottom flask after the synthesis is finished, and adding 5-10mL of a cutting reagent, wherein the cutting reagent is TFA/H₂O/TIPS (95: 2.5: 2.5, v/v/v), magnetically stirred at room temperature for 2-4 hours. Then filtering to remove resin, collecting supernatant, precipitating with ethyl acetate, centrifuging, and drying to obtain crude product. Purifying the crude peptide sample by semi-preparative High Performance Liquid Chromatography (HPLC) and reversed phase C18 with mobile phase A of 100% acetonitrile and mobile phase B of 100% ultrapure water, collecting target components to obtain purified polypeptide, lyophilizing, and weighing.

Weighing 1mg of the pure polypeptide, adding 500 mu L of mixed solution of acetonitrile and water for dissolving, filtering, detecting by RP-HPLC, and calculating the purity of the GK-Bio polypeptide by a peak area normalization method, wherein the purity is more than 95%.

FIG. 3 is a mass spectrum analysis chart of purified polypeptide GK-Bio containing biotin group, and the molecular weight is 999.1Da according to the expected value.

FIG. 4 is a HPLC analysis chart of purified polypeptide GK-Bio containing biotin group, and data analysis shows that the purity reaches more than 95%.

Example 4 preparation of biotinylated recognition PD-L1 Nanobody recombinant protein RNB-MS-Bio

Dissolving 20 μ M of recombinant protein RNB-MSH recognizing PD-L1 nano antibody, 100 μ M of recombinant protein containing biotinylated polypeptide GK-Bio and 5 μ M of Sortase A enzyme in 1mL of recombinant protein containing 50mM Tris, 150mM NaCl and 5mM CaCl₂The enzyme reaction system is reacted for 1 to 12 hours at the temperature of between 4 and 37 ℃, and samples are taken at regular time for analysis. And then, incubating the final reaction solution and nickel ion magnetic beads for 30min to obtain a supernatant solution, namely the biotinylated recombinant protein RNB-MS-Bio for recognizing PD-L1. The resulting recombinant protein and the crude reaction solutions at different times were analyzed by SDS-PAGE gel electrophoresis.

FIG. 5 is a data graph of SDS-PAGE analysis showing that the molecular weight of RNB-MS-Bio is significantly reduced compared to RNB-MSH, which corresponds to the expected molecular weight variation. The purity of the purified RNB-MS-Bio is more than 95%.

FIG. 6 is a graph showing the results of western blot analysis of RNB-MS-Bio. The result of horseradish peroxidase analysis marked by streptavidin shows that the recombinant protein is successfully subjected to biochemical modification.

Example 5 biotinylated recognition PD-L1 nano antibody recombinant protein RNB-MS-Bio and fluorescent quantum dot probe for detection of PD-L1 on cell surface by enzyme-linked immunosorbent assay

Cells to be detected are inoculated in a 96-well plate for 24 hours of culture, after supernatant is removed, the cells are fixed for 20min at 30 ℃ by 4 percent paraformaldehyde, and the PBST washes the cells for three times. Then using ELISA blocking liquid for blocking 2 hours under the condition of 30 ℃, washing cells three times by PBST, correspondingly adding 100 mu L of RNB-MS-Bio with the concentration of 25nM into each hole, incubating for 1 hour under the condition of 37 ℃, adding 100 mu LPBS into a control group, and washing plates three times by PBST after the incubation is finished. Then 50. mu.L of streptavidin-labeled Quantum Dots (QDs) at a concentration of 20nM were added to each well, incubated at 37 ℃ for 1 hour, and the plates were washed three times with PBST, and 50. mu.L of PBS was added to the control. And finally, carrying out fluorescence detection by using a microplate reader, wherein the excitation wavelength is 405nm, and the emission wavelength is 605 m.

FIG. 7 is key data for the specificity of the probe for cell surface PD-L1 detection. The background fluorescence intensity of the probe is very low when the Cell does not express PD-L1 (namely CHO Cell), and the fluorescence intensity of the probe is obviously higher when the Cell surface expresses PD-L1 (namely CHO/PD-L1 Cell) than when the Cell does not express PD-L1. The fluorescence intensity of the control group of streptavidin-labeled quantum dots only (Strep-QDs) was very low. The probe provided by the invention has obviously improved sensitivity.

FIG. 8 is key data comparing the probe with conventional fluorescence detection methods. By taking tumor cells MDA-MB-231 expressing PD-L1 as a reference group, when no expression exists in PD-L1 (CHO Cell), the nonspecific adsorption of the probe (RNB-MS-Bio + Strep-QDs) provided by the invention is lower than that of the probe of the traditional fluorescent dye (RNB-MS-Bio + Strep-FITC), and the probe has no obvious difference with a blank control group (PBS + Strep-FITC, PBS + Strep-QDs);

when the PD-L1 is highly expressed (CHO-PD-L1 Ce1L), the signal intensity of the probe (RNB-MS-Bio + Strep-QDs) provided by the invention is far stronger than that of the probe of the traditional fluorescent dye (RNB-MS-Bio + Strep-FITC) and a blank control group (PBS + Strep-FITC, PBS + Strep-QDs). The probe provided by the invention has obviously improved sensitivity.

Example 6 biotinylated recognition PD-L1 Nanobody recombinant protein RNB-MS-Bio and fluorescent Quantum dot Probe for detection of cell surface PD-L1 by flow cytometry

Transferring the cells to be detected into a flow tube, centrifuging to remove supernatant, washing the cells for 2 times by PBS, adding 100 mu L of PBS solution containing 50nM biotinylated recognition PD-L1 nano antibody recombinant protein RNB-MS-Bio into each tube, mixing uniformly, incubating on ice for 30min, and adding equivalent flow liquid into a control group. After incubation, adding 500 mu L of flow liquid, centrifuging at 1000g and 4 ℃ for 5min, removing supernatant, adding 50 mu L of streptavidin-labeled quantum dot QDs with the concentration of 20nM for resuspending cells, after ice incubation for 30min, adding 500 mu L of flow liquid, centrifuging at 4 ℃ for 5min, removing supernatant, washing the cells for 2 times by PBS, and centrifuging for 5 min; 200 μ of LPBS was added to each tube to resuspend the cells and examined by flow cytometry.

FIG. 9 shows key data for the probe used for flow cytometry for detection of PD-L1. In CHO samples of cells not expressing PD-L1, the result of detection using the probe RNB-MS-Bio + Strep-QDs is almost not different from that of Control.

In the CHO-PD-L1 and MDA-MB-231 samples with high expression of PD-L1, the signal displacement detected by the probe RNB-MS-Bio + Strep-QDs is obviously higher than that detected by a Control group Control, and particularly in the CHO-PD-L1 with high expression of PD-L1, the signal displacement detected is up to two orders of magnitude, which indicates that the probe has high sensitivity and specificity.

Example 7 biotinylated recognition PD-L1 Nanobody recombinant protein RNB-MS-Bio and fluorescent Quantum dot Probe for immunofluorescence detection of cell surface PD-L1

Placing the tumor tissue slices on a slice frame, then placing the slices in a 65 ℃ oven for dewaxing for 45min, soaking the slices twice in xylene for 10min each time, then soaking the slices in 100-50% ethanol for 5min, then placing the slices in 0.01M sodium citrate buffer solution with pH6.0, boiling the slices for 10min, preserving the heat for 10min, washing the slices with PBS for three times, after the completion, dripping 10% BSA confining liquid into a wet box at 37 ℃ for sealing the slices for 2h, washing the slices with PBS for three times, dripping 50 muL of biotinylated PD-L1-recognizing recombinant protein RNB-MS-Bio at the tumor tissue of the corresponding slices, incubating the slices for 12h at 4 ℃, then washing the slices with PBS for three times, dripping 20-50 muL of 10nM streptavidin-labeled quantum dots on the slices, incubating the slices at 37 ℃ for 30min in a dark place, tissue sections were washed three times with PBS, a drop of anti-fluorescence quencher was added dropwise to the sectioned tumor tissue, and the tissue sections were observed under different excitation lights with a confocal laser microscope and photographed.

FIG. 10 shows key data of the probe for immunofluorescence detection of PD-L1. In the experimental group of the probe, the luminescent positions of the quantum dots of the probe with different concentrations are overlapped with the luminescent positions of the cell nucleus stained by DAPI, while the fluorescence of the quantum dots cannot be detected in the PBS control group, which indicates that the probe can efficiently detect the expression of PD-L1 on the surface of the tumor cell.

The above embodiments illustrate that the probe provided by the invention based on the biotinylation recognition PD-L1 nano antibody recombinant protein RNB-MS-Bio and the fluorescent quantum dots can be specifically combined with a tumor marker PD-L1 receptor on the cell surface by combining enzyme-linked immunosorbent assay, flow cytometry and immunofluorescence, and the fluorescence intensity of the probe is significantly higher than that of the conventional fluorescent probe.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Sequence listing

<110> university of south of the Yangtze river

<120> high-sensitivity quantum dot probe for detecting tumor marker PD-L1, preparation method and application

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<170> SIPOSequenceListing 1.0

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<213> part of the Nanobody recombinant protein RNB-MSH sequence that can recognize PD-L1 ()

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

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Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

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Ala Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

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Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

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Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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<213> Nanobody recombinant protein RNB-MSH ()

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

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Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

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Ala Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

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Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

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Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Glu

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Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala Leu Pro Glu

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Thr Gly Gly His His His His His His

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<213> Nanobody recombinant protein RNB-MSH ()

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catatgcagg ttcagctgca ggaaagcggc ggcggtctgg tgcagccggg tggtagtctg 60

cgcctgagtt gtgccgccag tggcaaaatg agcagtcgcc gttgcatggc ctggtttcgc 120

caggcaccgg gtaaagaacg cgaacgcgtg gcaaaactgc tgaccaccag tggtagcacc 180

tatctggcag atagtgttaa aggtcgcttt accattagcc agaataatgc aaaaagtacc 240

gtttatctgc agatgaatag cctgaaaccg gaagataccg ccatgtatta ttgtgcagca 300

gatagctttg aagatccgac ctgtaccctg gttaccagta gcggtgcatt tcagtattgg 360

ggtcagggca cccaggttac cgtgagcgaa cagaaactga ttagtgaaga agatctgaat 420

ggtgcagcac tgccggaaac cggtggccat catcatcatc accattaact cgag 474

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Gly Gly Gly Ala Gly Ala Gly Ala Gly Ala Lys

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