CN117603336B

CN117603336B - Method for extracting tumor neoantigen from engineering bacteria expressing HLA protein

Info

Publication number: CN117603336B
Application number: CN202410091265.0A
Authority: CN
Inventors: 牛远杰; 于健鹏; 高文隆; 陈家童; 马媛; 张名浩; 杨洋; 刘洋
Original assignee: Panru Biotechnology Tianjin Co ltd
Current assignee: Panru Biotechnology Tianjin Co ltd
Priority date: 2024-01-23
Filing date: 2024-01-23
Publication date: 2024-04-05
Anticipated expiration: 2044-01-23
Also published as: CN117603336A

Abstract

The invention relates to the technical field of biology, in particular to a method for extracting tumor neoantigen from engineering bacteria expressing HLA proteins. The method for extracting tumor neoantigen from engineering bacteria expressing HLA protein comprises the following steps: the expressed HLA protein is displayed on the surface of engineering bacteria and is used for extracting antigens in tumor cells. The method avoids the defects that the prior method excessively depends on a mathematical model and is limited by lower HLA expression of tumor cells, can capture a large amount of tumor cell neoantigens, improves the screening efficiency of the tumor cell neoantigens, and has the advantage of high yield.

Description

Method for extracting tumor neoantigen from engineering bacteria expressing HLA protein

Technical Field

The invention relates to the technical field of biology, in particular to a method for extracting tumor neoantigen from engineering bacteria expressing HLA proteins.

Background

Mutations or aberrant cleavage of tumor cell genes can generate new autoantigen epitopes, referred to as neo-epitopes or neoantigens. The tumor neoantigen has high specificity and immunogenicity, and is an ideal target point of tumor vaccine. However, at present, part of tumor neoantigen recognition depends on bioinformatics algorithms, and the stability and the accuracy are lacking; the other part is based on protein co-precipitation technology, depends on the expression level of Human Leukocyte Antigen (HLA) in tumor cells, but the HLA expression in the tumor cells is inhibited, so that the method has high detection cost, and the quantity of captured tumor neoantigens is limited, so that the method is difficult to be applied in large-scale clinic.

Disclosure of Invention

The present invention is directed to solving at least one of the technical problems existing in the related art. Therefore, the invention provides a method for extracting tumor antigens by using engineering bacteria for expressing HLA proteins, which utilizes the HLA proteins expressed by the engineering bacteria to bind with the new antigens in tumor cells, avoids the defect that the existing method is limited by lower HLA expression of the tumor cells, can capture a large amount of tumor cell new antigens, improves the screening efficiency of the tumor cell new antigens, and has the advantage of high yield.

In one aspect of the present invention, there is provided a method for extracting tumor neoantigen from an engineering bacterium expressing HLA proteins, the method comprising:

the expressed HLA protein is displayed on the surface of engineering bacteria and is used for extracting antigens in tumor cells.

Further, the engineering bacteria include: probiotics and plasmids, transferring the plasmids into the probiotics;

the plasmid comprises a fluorescent protein coding gene, a promoter, an Lpp-OmpA gene sequence and an HLA protein expression sequence which are connected in sequence.

Further, the HLA protein expression sequence comprises an HLA-a 02:01 protein expression sequence.

Further, the HLA protein expression sequence is SEQ ID NO.1;

the Lpp-OmpA gene sequence is SEQ ID NO.2.

Further, the fluorescent protein coding genes comprise T7-GFP green fluorescent protein coding genes;

the promoter comprises Tac;

the probiotics comprise escherichia coli Nissle1917.

Further, the engineering bacteria are placed in tumor cell lysate to extract tumor neoantigen.

Further, the tumor cells include prostate cancer cells.

Further, the novel antigen comprises TP53 ^R175H 。

The above technical solutions in the embodiments of the present invention have at least one of the following technical effects:

and (3) combining HLA proteins expressed by engineering bacteria with new antigens in tumor cells, separating the engineering bacteria, identifying the polypeptide captured on the surface of the engineering bacteria, and simultaneously comparing the sequence information of the corresponding DNA sequencing gene mutation, and screening the new antigens generated by the gene mutation of the tumor cells. The method utilizes HLA functional peptide fragments displayed by engineering bacteria to bind antigens in tumor cells, avoids the defects that the prior method excessively depends on mathematical models and is limited by lower HLA expression of the tumor cells, can capture a large amount of tumor cell antigens, improves the screening efficiency of tumor cell neoantigens, and has the advantage of high yield. The invention overcomes the defects of changeable bioinformatics algorithm and lack of stability, and the constructed engineering bacteria can be amplified in large quantity with low cost and have the advantages of large quantity of application cost.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of PCR assay in example 1 of the present invention.

FIG. 2 shows the Western blot identification result in example 1 of the present invention.

FIG. 3 shows the results of the detection of HLA-A 02:01 protein on the surface of engineering bacteria by whole cell indirect ELISA in example 2 of the present invention.

FIG. 4 shows the results of flow cytometry identification of localization of HLA-A 02:01 on engineered bacterial surfaces in example 2 of the present invention.

FIG. 5 shows the results of identifying HLA-A 02:01 localization on the surface of engineering bacteria by laser confocal microscopy in example 2 of the present invention.

FIG. 6 shows the capture of P53 by engineering bacteria in example 2 of the present invention ^R175H Schematic of the process of neoantigenic peptides.

FIG. 7 shows the capture of P53 by engineering bacteria in example 2 of the present invention ^R175H Results of neoantigenic peptides.

FIG. 8 shows the peak intensities of the daughter ions identified by Parallel Reaction Monitoring (PRM) mass spectrometry after the target peptide fragment (P53R 175H neoantigenic peptide) was captured in the sample in example 3 of the present invention.

FIG. 9 is a chromatogram identified by Parallel Reaction Monitoring (PRM) mass spectrometry technique after capturing the target peptide fragment (P53R 175H neoantigenic peptide) in the sample in example 3 of the present invention.

FIG. 10 is a graph showing the sum of the areas of all the ion peaks detected by PRM technique of the target peptide according to example 3 of the present invention, which is the area of the ion peak of the target peptide, to show the corresponding content.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The following examples are illustrative of the invention but are not intended to limit the scope of the invention.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the embodiments of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

In one aspect of the present invention, there is provided a method for extracting tumor neoantigen from an engineering bacterium expressing HLA proteins, the method comprising: the expressed HLA protein is displayed on the surface of engineering bacteria and is used for extracting antigens in tumor cells.

In some embodiments of the invention, the engineering bacteria include: probiotics and plasmids, transferring the plasmids into the probiotics; the plasmid comprises a fluorescent protein coding gene, a promoter, an Lpp-OmpA gene sequence and an HLA protein expression sequence which are connected in sequence.

The Lpp-OmpA gene sequence and the HLA protein expression sequence expressed the fusion protein on the surface of the engineering bacterium.

In some embodiments of the invention, the method of detecting a tumor neoantigen by the engineering bacteria of the invention is as follows: and (3) combining new antigens in tumor cells by using fusion proteins on the surfaces of engineering bacteria, separating the engineering bacteria, identifying polypeptides captured on the surfaces of the engineering bacteria, and simultaneously comparing corresponding DNA sequencing gene mutation sequence information to screen new antigens generated by the gene mutation of the tumor cells.

The inventors of the present invention found that E.coli beta-barrel outer membrane proteins, which comprise the OmpA sequence of the present invention, can be assembled in a barrel-like structure in beta-sheet form across the outer membrane. Lipoprotein Lpp is also used for surface display as another outer membrane protein, which is bound to the cell outer membrane at the N-terminus and to the cell layer peptidoglycan at the C-terminus, and the foreign protein linked to the outer membrane OmpA fragment can be stably secreted to the cell outer membrane for surface expression. Lpp-OmpA can be used as a chimera for bacterial surface display, consisting of a signal peptide and the first nine amino acid residues of Lpp, responsible for targeting the outer membrane, fused to five of the eight transmembrane segments of OmpA porin (residues 46-159), in which case Human Leukocyte Antigen (HLA) proteins are fused at the C-terminus of the Lpp-OmpA chimera, and HLA proteins can be displayed on bacterial surfaces.

In some embodiments of the invention, the HLA protein expression sequence comprises an HLA-a 02:01 protein expression sequence. Therefore, HLA-A 02:01 is a high-frequency HLA of a population, fusion proteins are constructed by utilizing the HLA-A 02:01, and the fusion proteins are displayed on the surface of engineering bacteria, so that the defect that the HLA expression of tumor cancer cells is lower is overcome, the screening efficiency of tumor neoantigens is higher, and the yield is higher.

In some embodiments of the invention, the HLA protein expression sequence is SEQ ID No.1; the Lpp-OmpA gene sequence is SEQ ID NO.2.

In some embodiments of the invention, the fluorescent protein encoding gene comprises a T7-GFP green fluorescent protein encoding gene; the promoter comprises Tac; the probiotics comprise escherichia coli Nissle1917.

In some embodiments of the invention, the plasmid is T7-GFP+Tac-Lpp-OmpA-HLA, the engineering bacterial strain is Nissle1917, and the T7-GFP+Tac-Lpp-OmpA-HLA plasmid is transferred into the engineering bacterial strain; wherein, the T7-GFP green fluorescent protein is used for indicating that the plasmid transfer is successful; tac-Lpp-OmpA-HLA is a fusion protein expression sequence, and the expressed fusion protein can be displayed on the surface of engineering bacteria and is used for combining antigen polypeptides in tumor cells.

In some specific embodiments of the invention, the invention utilizes engineering bacteria to carry out biological safety improvement by synthetic biological technology, loads HLA fusion protein and displays on the surface of engineering bacteria, and extracts and screens the prostate cancer neoantigen in a mode independent of the endogenous HLA level and binding capacity of the prostate cancer cells so as to realize efficient capture of the prostate cancer neoantigen aiming at the scientific problems of enrichment and difficult recognition of the prostate cancer neoantigen caused by low abundance of Human Leukocyte Antigen (HLA) binding antigen, unstable biological informatics prediction neoantigen result, lack of accuracy and the like caused by the inhibition of the antigen presenting process of the prostate cancer cells.

The present invention will be further illustrated with reference to specific examples and experimental examples. The following examples and experimental examples are only for explaining the present invention, and are not to be construed as limiting the present invention.

Experimental example

Experimental example 1

Preparation of engineering bacteria (ECN)

The plasmid (T7-GFP+Tac-Lpp-OmaA-HLA-A 02:01, entrusted An Sheng to the company for sequence-based synthesis of the plasmid) was transferred into E.coli Nissle1917, comprising the following steps:

1. preparation of competent cells of E.coli Nissle1917 (performed under sterile conditions)

(1) In a super clean bench, using a sterile inoculating loop to select glycerol frozen bacteria Nissle1917 to streak on LB agar plates containing 50 mug/mL of DapA nutrient and 100 mug/mL of ampicillin, and simultaneously streaking on LB agar plates containing 100 mug/mL of ampicillin without DapA nutrient by a control group, and culturing at 37 ℃ overnight;

(2) The next day, single colonies in good condition were picked from LB agar plates, inoculated into 5mL of DapA nutrient LB liquid medium containing 50. Mu.g/mL, shake-cultured at 37℃for about 12 hours until logarithmic growth phase, and the bacterial suspension was prepared by mixing 1:100 is transferred into 50mL of LB liquid medium, shaking and expanding culture is carried out at 37 ℃, after the culture solution starts to be turbid, OD600 absorbance value is measured every 20-30 minutes, and the culture is stopped until OD600nm is about 0.5;

(3) Transferring 1mL of culture solution into 2mL of centrifuge tubes, adding three groups, cooling on ice for 20-30min, and centrifuging at 4000rpm (revolutions per minute) at 4deg.C for 10min (all operations are performed on ice at a speed as fast as possible from this step);

(4) The supernatant was decanted and cooled with 1mL ice-cold 0.1mol/L CaCl ₂ The (calcium chloride) solution gently suspended the cells, ice bath;

(5) Centrifuging at 4000rpm at 0-4deg.C for 10min;

(6) The supernatant was discarded and 500. Mu.L ice-cold 0.1mol/L CaCl was added ₂ The solution, carefully suspend the cells, centrifuge at 4000rpm for 10min at 0-4 ℃;

(7) The supernatant was discarded and 100. Mu.L ice-cold 0.1mol/L CaCl was added ₂ The solution is carefully suspended cells, and after a moment of standing on ice, competent cell suspension is prepared;

(8) The prepared competent cell suspension can be directly used for transformation experiments, or glycerol which accounts for about 15% of the total volume and is subjected to autoclaving can be added, and the mixture is packaged in a 1.5mL freezing tube and placed at the temperature of-80 ℃ for half a year to one year.

2. Plasmid transformed E.coli Nissle1917

(1) Taking out 200 mu L of prepared competent cell suspension from a refrigerator at-80 ℃, thawing the competent cell suspension at room temperature, and immediately placing the competent cell suspension on ice after thawing;

(2) Adding prepared T7-GFP+Tac-Lpp-OmaA-HLA-A 02:01 plasmid DNA solution (the content is not more than 50 mu g, the volume is not more than 10 mu L), shaking gently, and standing on ice for 30min;

(3) Heat-shocking in water bath at 42 deg.c for 60s, and cooling in ice for 3-5 min;

(4) 1mL of LB liquid medium containing 50 mug/mL of DapA nutrient and containing no ampicillin is added into the tube, and after uniform mixing, the bacteria are subjected to shaking culture at 37 ℃ for one hour, so that the bacteria are recovered to a growth state, and the ampicillin coded by the plasmid is expressed;

(5) Shaking the bacterial liquid uniformly by using a sterile coating rod, then taking 100 mu L of the bacterial liquid, coating the bacterial liquid on an LB screening agar plate containing 100 mu g/mL of ampicillin and 50 mu g/mL of DapA nutrient, standing the bacterial liquid for one hour upwards on the front surface, inverting a culture dish after the bacterial liquid is completely absorbed by a culture medium, and culturing the bacterial liquid for 16 hours at 37 ℃;

(6) The following day, single colonies in good condition were picked from LB agar plates containing 100. Mu.g/mL of ampicillin and 50. Mu.g/mL of DapA nutrient, inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL of DapA nutrient, and shake-cultured at 37℃for about 12 hours until logarithmic phase, and the bacterial suspension was prepared at a ratio of 1:100 is transferred into 50mL LB liquid medium, shaking and expanding culture is carried out at 37 ℃, after the turbidity of the culture solution begins, OD600 absorbance value is measured every 20-30 minutes, and the culture is stopped until OD600nm is about 0.5, thus obtaining the prepared escherichia coli Nissle1917 for expressing fusion protein, which is called engineering bacteria.

3. Engineering bacteria plasmid extraction

Using a plasmid small extraction kit, adopting an alkaline lysis method to lyse bacteria, and then specifically combining DNA in a solution under a high-salt state through centrifugal adsorption, wherein the following operation steps are applicable to 1-5mL of overnight cultured escherichia coli:

(1) Column balancing: adding 500 mu L of balance liquid BL into the adsorption column CP3, centrifuging at 12000rpm for 1min at normal temperature, pouring out waste liquid in the collecting pipe, and putting the adsorption column into the collecting pipe again;

(2) Adding 4.5mL of the overnight cultured bacterial liquid into a centrifuge tube, centrifuging for 1min at 12000rpm by using a conventional table-type centrifuge, and sucking the supernatant as much as possible (bacterial liquid can be deposited and collected into one centrifuge tube through multiple centrifugation when more bacterial liquid exists);

(3) Adding 50 mu L of solution P1 into a centrifuge tube with bacterial liquid, adding RnaseA, and thoroughly suspending bacterial precipitation by using a pipette or a shaker;

(4) Adding 350 mu L of solution P2 into the centrifuge tube, gently turning over up and down for 6-8 times to fully crack the thalli;

(5) Adding 350 mu L of solution P3 into a centrifuge tube, immediately and gently turning up and down for 6-8 times, fully and uniformly mixing, centrifuging at 12000rpm for 10min, and forming a precipitate at the bottom of the centrifuge tube;

(6) Transferring the supernatant collected in the previous step into an adsorption column CP3 by using a liquid transfer device, taking care that sediment is not sucked out as much as possible, centrifuging at 12000rpm for 30-60s, pouring out waste liquid in a collecting pipe, and placing the adsorption column P3 into the collecting pipe;

(7) Adding 600 μl of the rinse solution PW into the adsorption column CP3, centrifuging at 12000rpm for 30-60s, pouring out the waste liquid in the collecting tube, and placing the adsorption column CP3 into the collecting tube;

(8) Adding 600 μl of the rinse solution PW into the adsorption column CP3, centrifuging at 12000rpm for 30-60s, and pouring out the waste liquid in the collection tube;

(9) Placing the adsorption column CP3 into a collecting pipe, and centrifuging at 12000rpm for 2min to remove residual rinsing liquid in the adsorption column;

(10) The adsorption column CP3 was placed in a clean centrifuge tube, 80. Mu.L of elution buffer EB was added to the middle portion of the adsorption membrane, and the solution was placed at room temperature for 2min and centrifuged at 12000rpm for 1min, and the plasmid solution was collected in the centrifuge tube.

4. And (3) PCR identification of engineering bacteria plasmids:

(1) Primer synthesis was performed by hand from the company Shanghai, inc. of Biotechnology, according to published gene sequences of OmpA and HLA-A 02:01 in GeneBank;

(2) In an ice bath, the ingredients were added to a sterile 0.5mL centrifuge tube in the following order: 5. Mu.L of 10 XPCRbuffer, 4. Mu.L of dNTP mix, 2. Mu.L of primer F, 2. Mu.L of primer R, 1. Mu.L of LTaq enzyme, 1. Mu.L of LDNA template (50 ng-1000 ng/. Mu.L), 35. Mu.L of sterilized water;

(3) The reaction program is regulated, the mixed solution is slightly centrifuged, and is immediately placed on a PCR instrument to perform amplification, and the mixture is usually pre-denatured at 93 ℃ for 3-5min and enters a cyclic amplification stage: cycling for 30-35 times at 93 deg.C 40-58 deg.C 30-70 deg.C 60s, and maintaining at 72 deg.C for 7min;

(4) Finishing the reaction, and placing the PCR product at 4 ℃ for electrophoresis detection or preserving at-20 ℃ for a long time;

(5) Weighing 0.4g agarose powder, putting into a triangular flask, adding 50mL TAE electrophoresis buffer (1 x), putting into a microwave oven for boiling for about 1min, observing the agarose powder in the flask, and stopping the microwave oven after complete melting;

(6) Putting on gloves, taking out the triangular flask from the microwave oven, and placing the triangular flask on a tabletop for cooling until the triangular flask does not scald hands (50-60 ℃);

(7) After the comb is inserted into the correct position of the gel casting mould, slowly pouring the gel solution until the gel solution is leveled with the short edge of the mould. Standing on the tabletop for 10-20min, and completely solidifying the glue;

(8) Filling 1x TAE electrophoresis liquid in the horizontal electrophoresis tank, regulating the voltage to 170v, and noticing that the position connection of the anode and the cathode is correct;

(9) After the gel is completely solidified, about 20-30min, carefully pulling out the comb, pinching the high edges at two sides of the mold with fingers, taking out the mold and the gel, placing the mold and the gel on a platform in the middle of the electrophoresis tank, and immersing the gel in the electrophoresis liquid;

(10) Adding 10 mu L of DNA and DNA molecular standard;

(11) Turning on a power switch, and allowing the sample to move forward to form a blue mold belt, and performing electrophoresis for about 30min; when the blue bromophenol blue moves to 1cm from the edge of the gel, the power supply is turned off, the mold and the gel are taken out, and the gel is placed into a gel imaging system for photographing.

Experimental example 2

Induction expression and Western blot analysis of engineering bacteria

(1) Engineering bacteria ECN and escherichia coli Nissle1917 were inoculated in 1% of LB liquid medium (containing 50. Mu.g/mL of DapA nutrient and 100. Mu.g/mL of ampicillin) and cultured at 37℃overnight. The following day, 1% was inoculated into 10 mL of LB medium for expression. Culturing 10 mL bacteria until OD600 is 0.8, adding 47.6 mu L IPTG, inducing at 37 ℃ for 24h, collecting 1mL bacterial liquid, centrifuging at 12000 rpm/min for 1min, and cleaning with sterile PBS for 3 times;

(2) Extraction of bacterial proteins using a bacterial protein extraction kit: adding 2 mu L of protease inhibitor mixture, 2 mu L of phosphatase inhibitor mixture and 5 mu L of protein stabilizing solution into every 500 mu L of lysate according to the required sample size, fully mixing uniformly, and placing on ice for standby; centrifuging the bacterial liquid at 4 ℃ and 10000g for 5min, discarding the supernatant, sucking the residual liquid as much as possible, and collecting bacterial cells; washing thallus with PBS once, adding 250-500 μl of extractive solution per 50-100mg wet weight thallus sample, blowing, mixing, and oscillating at 2-8deg.C for 45-60min; centrifuging the bacterial liquid for 5min at 4 ℃ under 12000g, and transferring the supernatant into a clean centrifuge tube to obtain a bacterial total protein sample; the total protein sample is quantitatively packaged in a refrigerator at the temperature of minus 80 ℃ or directly used for downstream experiments;

(3) The polyacrylamide separating gel with the concentration of 10% and the concentrated gel with the concentration of 5% are prepared by adopting conventional SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the specific preparation method is referred to the third edition of molecular cloning experiment guidelines. Samples were added to the loading wells at 10 μl/well. The sample is electrophoresed by selecting 7V/cm voltage in the upper concentrated gel layer, and electrophoresis is performed by adopting 15V/cm voltage in the lower separating gel layer. And stopping electrophoresis when bromophenol blue electrophoresis reaches about 0.5 and cm at the bottom of the separation gel. The method comprises the steps of carrying out a first treatment on the surface of the

(4) The wet transfer method is adopted for transferring the film, and the specific steps are as follows: cutting 2 Bio-Rad Mini Trans-Blot Filter Paper and 1 nitrocellulose membrane (NC membrane), the sizes of which are matched with the gel, and soaking in a membrane transfer buffer solution for 30-60 min; taking out the separation gel after electrophoresis, rinsing for 1 time by using TBST, sequentially placing 1 Filter Paper, PVDF film, separation gel and 1 Filter Paper on an anode plate of a transfer device, switching on a power supply, and carrying out electric transfer in an ice bath of 120. 120V for 2.5 h; after the transfer is finished, the transfer device is disassembled, the PVDF film is taken out, then is rinsed for 1 time by ddH2O and 3 times by TBST, and is placed in a sealing liquid which is 5% skim milk and is sealed for 1 h at 37 ℃; rinsing TBST 3 times, adding primary antibody diluted 500 times by TBST buffer solution, and carrying out shaking table incubation at room temperature for 1 h; rinsing with TBST 3 times, adding HRP-labeled goat anti-mouse IgG diluted 10000 times with TBST buffer, and incubating in a shaking table at room temperature for 1 h; rinsing with TBST for 3 times, sequentially placing PVDF film on fresh-keeping film, spreading, rapidly and uniformly adding the prepared luminescent agent on the film, reacting at room temperature in dark place for 3min, observing the result, and photographing.

Experimental example 3

With P53 ^R175H Expressed prostate cancer cell model

The most frequently altered gene in human tumors is TP53, which encodes the p53 protein. TP53 mutations are associated with poor prognosis for many sporadic cancers, and germline TP53 mutations are the etiology of a rare familial cancer susceptibility Li Fraumeni syndrome. The tumor suppressor p53 is encoded by the TP53 gene (or Trp53 in mice) and is critical for normal cell growth and tumor prevention. Typically the p53 protein is maintained at a low level in normal tissues by its negative regulator mouse two minutes 2/X (MDM 2/X). Many endogenous and exogenous stressors activate p53, triggering it to further regulate a series of cellular responses necessary to maintain homeostasis. Activation of p53 in a variety of stress responses is critical for normal cell survival and protection from tumorigenesis. However, TP53 is frequently mutated in most human cancers, resulting in loss of functions required for tumor Suppression (LOFs) and even acquisition of functions required for tumor Growth (GOFs). The most common p53 mutation is a DNA Binding Domain (DBD) missense mutation, which affects only one amino acid in the p53 protein, but has a significant effect on the function of the protein. Tumors containing p53 mutations tend to progress faster, respond poorly to anticancer therapies, and are poorly predicted. The mutated TP53 allele-derived proteins are proteolytically degraded, processed, and presented by the major histocompatibility complex MHC, producing novel antigens that are recognized by the T cell receptor TCR. Substitution of arginine at position 175 in TP53 with histidine (R175H) is the most common mutation observed in TP53 and is also the most common mutation in all tumor suppressor genes. HMTEVVRHC (mutant amino acid underlined) short peptides derived from p53 ^R175H Can bind to one of the most common Human Leukocyte Antigen (HLA) allele (a x 02:01) subtypes, present in more than 40% of the us population. The P53 gene is the most common mutant gene in human cancers, P53 mutation occurs in more than 50% of cancers, targeting P53 for cancer treatment is an attractive strategy. Among the prostate cancer cell lines, the PC3 cell line and the LNCaP cell line are the most commonly used cell lines, with LNCaP cellsHLA typing of the line is consistent with fusion protein HLA-A 02:01 expressed by constructed engineering bacteria, and the HLA-A 02 is: type 01, thus selecting the neoantigen p53 ^R175H As a breakthrough point, a new antigen cell model of the prostate cancer is constructed.

Example 1

Randomly selecting 4 monoclonal engineering bacteria, respectively marking as engineering bacteria monoclonal 1 (ECN 1), engineering bacteria monoclonal 2 (ECN 2), engineering bacteria monoclonal 3 (ECN 3) and engineering bacteria monoclonal 4 (ECN 4), and transferring the T7-GFP+Tac-Lpp-OmaA-HLA-A 02:01 plasmid into escherichia coli Nissle1917 through Polymerase Chain Reaction (PCR) and immunoblotting (Western blot) experiments, wherein the results are shown in figures 1 and 2.

Example 2

Identification of HLA-A 02:01 protein localization on engineering bacteria surface

1. Detection of HLA-A 02:01 protein expression on engineering bacteria surface by whole thallus indirect ELISA

(1) Firstly, preparing 2.5% glutaraldehyde by using 0.1mol/L sodium bicarbonate solution, adding 150 mu L of aldehyde 96-hole ELISA plates into each hole, reacting for 2 hours at a temperature of 37 ℃, taking out, cleaning each hole by using 200 mu L of deionized water, and preparing the solution for 5min/4 times;

(2) Formaldehyde was diluted 10-fold with PBS, then IPTG was slowly added to induce 24 hours followed by resuspension of Nissle1917 bacterial liquid to be inactivated (negative control group) and 4 engineering bacteria monoclonal amplified cultures in PBS to a final concentration of 1x 10 ⁸ In CFU/mL bacterial liquid, shaking is carried out while adding so as to avoid overhigh local formaldehyde concentration, and the formaldehyde concentration is 0.3% after inactivation for 24 hours at 37 ℃;

(3) 100 mu L of bacterial liquid is added into each hole, a blank control group is PBS solution, the blank control group is coated overnight at 37 ℃,200 mu L/hole PBST is used for washing ELISA plates the next day for 5min;

(4) Adding 100 mu L of 5% BSA blocking solution into each hole, blocking for 1.5 hours at 37 ℃, washing the plate 3 times by using 200 mu L/hole of PBST, and beating the plate as dry as possible after the plate is washed for the last time;

(5) mu.L of primary antibody HLA-A 02:01 (1000 fold dilution of 0.5% PBST) was added per well, incubated at 37℃for 1 hour, and plates were washed 3 times 5min each with 200. Mu.L/well of PBST;

(6) 100 mu LHRP-labeled goat anti-mouse IgG secondary antibody (0.5% PBST diluted 5000-fold) was added to each well, incubated at 37℃for 1 hour, and plates were washed 3 times with 200. Mu.L/well of PBST for 5min each;

(7) After the mixture is patted dry, 100 mu L of substrate color development liquid TMB is added into each hole, and color development is carried out for 15-30min in a dark place; after completion of the color development, 50. Mu.L of 2M H2SO4 solution was added to each well to terminate the reaction, and the OD450 value was measured by reading the plate, and the result is shown in FIG. 3.

2. Identification of localization of HLA-A 02:01 on engineered bacteria surfaces by flow cytometry

In order to further determine the localization of the foreign protein HLA-A 02:01 on the surface of the engineering bacteria, the detection of flow cytometry is carried out, and the specific steps are as follows:

(1) Firstly, taking 1mL of Nissle1917 bacterial liquid induced by IPTG for 24 hours and 1mL of bacterial liquid cultured by 4 ECN monoclonal amplification, filtering the bacterial liquid by a 200-mesh screen for 1 time, centrifuging the bacterial liquid at the rotating speed of 12000rpmn and the temperature of 4 ℃ for 3min, and collecting bacterial bodies;

(2) After discarding the supernatant, PBS was added to purge three times to adjust the bacterial concentration to 5X 10 ⁵ Centrifuging again at 12000rpm and 4 ℃ for 3min at the speed of each mL, and collecting thalli;

(3) Adding PBS containing 5% BSA at 4 ℃ to block bacteria solution overnight;

(4) The next day, centrifugal for 1min at 12000rpm, purge three times with PBS, add in the first anti-APC anti-human HLA-A 02:01 (1:1000 dilution), light-proof at room temperature for 1 hour;

(5) centrifuging the bacterial liquid at 5000rpm/min, and flushing with PBS for three times;

(6) finally, the cells were resuspended with 1ml fbs and subjected to flow detection, with excitation light wavelengths of 633nm and emission light wavelengths of 660nm, resulting in red fluorescence, the results of which are shown in fig. 4.

3. Identification of HLA-A 02:01 positioning on engineering bacteria surface by laser confocal microscope observation

Firstly, taking 1mL of Nissle1917 bacterial liquid induced by IPTG for 24 hours and 1mL of bacterial liquid cultured by monoclonal amplification of 4 engineering bacteria, filtering the bacterial liquid by a 200-mesh screen for 1 time, centrifuging the bacterial liquid at the speed of 12000rpm and the temperature of 4 ℃ for 3min, and collecting thalli; and detecting the thalli by adopting an indirect immunofluorescence technology. The method comprises the following specific steps:

(1) Soaking the glass slide with 75% ethanol overnight, taking out, and naturally drying;

(2) Dripping 20 mu L of sample to be detected on a glass slide, placing the glass slide in an incubator at 37 ℃ at room temperature, and naturally drying;

(3) Fixing the glass slide in 4% tissue fixing liquid for 30min;

(4) PBST washes and then blocked overnight in BSA blocking solution;

(5) PBST washes the slide, and absorbs the water by using absorbent paper, drop monoclonal antibody of anti-HLA-A 02:01 on the sample to be detected, place in wet box, act 5h at 4 ℃;

(6) PBST washes the slide, absorbs the slide with absorbent paper, drops FITC-labeled fluorescent secondary antibody, and places the slide in a wet box at room temperature and in a dark place 2 h; the slides were washed with PBST, once with deionized water, blocked with 10. Mu.L of glycerol/PBS [ 75mL of glycerol, 0.01M PBS (pH=9.0-9.5) 25mL ] buffer, and subjected to microscopic examination, the results of which are shown in FIG. 5.

4. Indirect ELISA method for verifying engineering bacteria capture P53 ^R175H New antigenic peptides

The operation flow is shown in fig. 6, and the specific operation steps are as follows:

(1) Antigen coating: will know antigen P53 ^R175H Diluting the neoantigenic peptide to a concentration of 2-10 mu g/mL by using a phosphate coating solution, adding 100 mu l into a 96-well ELISA plate, coating overnight at 4 ℃, discarding the suspension the next day, and washing once by using 200 mu l/well of PBST for 5min;

(2) Closing: blocking with 5% BSA blocking solution, adding 100 μl of each to the wells, standing at room temperature for 1.5 hr, discarding the blocking solution, and washing with PBST once, 200 μl of each well for 5min;

(3) Sample addition incubation: nissle1917 bacteria solution (negative control group) to be inactivated after 24 hours of induction by adding IPTG and re-suspension of 4 engineering bacteria monoclonal amplified culture in PBS to a final concentration of 1×10 ⁸ Adding 100 μl of the culture medium into CFU/mL bacterial liquid, culturing in a 37 ℃ incubator for 4-8 hours, discarding bacterial liquid, adding PBST, washing for three times, 200 μl/hole, and 5min;

(4) Reading a plate: according to the procedure of the bacterial activity detection kit, bacterial activity detection reagents were added to each well, mixed well and incubated at room temperature for 15 minutes, protected from light, ELISA plates were placed on a microplate reader and OD values were measured at OD450, as shown in FIG. 7.

Example 3

1. Protein mass spectrum technology for identifying P53 in recombinant engineering bacteria captured prostate cancer cell model ^R175H New antigens

(1) Sample preparation: will be 6X 10 ⁷ Overexpression of P53 ^R175H After digestion and centrifugation, re-suspending with 2ml PBS, performing ultrasonic disruption, taking cell supernatant after centrifugation for 30min at 14000rpm under the condition of 4 ℃ under the ultrasonic condition (100W, 3s, intermittent 7s for 10 min), and dividing one part into two parts for subsequent mass spectrum identification and the other part for subsequent experiments for later use; taking 20mL of engineering bacteria liquid subjected to monoclonal amplification culture after IPTG induction for 24 hours, centrifuging and re-suspending to 2mL volume, uniformly mixing with the cell supernatant extracted in the step 1, and co-culturing for 8 hours; centrifuging the bacterial liquid after co-culture at 5000rpm for 5min, discarding the supernatant, and collecting bacterial cells; washing thalli for 1-2 times by using an equal volume of PBS buffer solution, and then carrying out ultrasonic crushing under the ultrasonic condition: the power is 300W, the working time is 2s, the gap is 4s, the temperature is controlled below 30 ℃, the ultrasonic crushing is started under the ice bath condition, and the total ultrasonic time is 15min; taking out the sample after the ultrasonic treatment is finished, balancing the solution, and centrifuging at 14000rpm for 30min at 4 ℃; after centrifugation, transferring the supernatant into a new centrifuge tube to be used as working solution for breaking engineering bacteria for mass spectrometry.

(2) PRM targeting quantitative proteomics (Targeted Proteomics) identification of recombinant engineering bacteria to capture P53 in prostate cancer cell model ^R175H The new antigen:

sample preparation: transferring a proper amount of sample into a 10KD ultrafiltration centrifuge tube, centrifuging 12000g, adding 0.25% acetic acid solution after centrifuging, centrifuging 12000g for 20min, and repeating for 2 times. The filtrate was collected, dried, and desalted by adding an appropriate amount of 0.1% TFA solution and C18 Cartridge. The peptide was dried and reconstituted with 0.1% FA and the peptide concentration was determined for LC-MS analysis.

LC-PRM/MS analysis: according to the target Shotgun mass spectrum analysis result, the target peptide has reliable identification information and good chromatographic separation behavior (the chromatographic elution peak is sharply symmetrical), and can be used for PRM quantitative analysis. The peptide fragment information suitable for PRM analysis is imported into the software Xcalibur for PRM method setting. Taking 2ug of peptide fragment of each sample for LC-PRM/MS analysis; after loading, the nano-rising flow rate Easy nLC is used

1200. The chromatographic system (Thermo Scientific) performs chromatographic separation. Buffer solution: solution A was 0.1% formic acid in water, and solution B was 0.1% formic acid, acetonitrile and water mixed solution (wherein acetonitrile was 95%). The column was equilibrated with 95% solution a. Samples were taken into a Trap Column (100 μm. Times.20 mm, 5 μm, C18, dr. Maisch GmbH) and subjected to gradient separation by chromatography Column (75 μm. Times.150 mm, 3 μm, C18, dr. Maisch GmbH) at a flow rate of 300 nl/min. The liquid phase separation gradient is as follows: 0. minutes-5 minutes, linear gradient of liquid B from 2% to 5%; 5. minutes-45 minutes, linear gradient of liquid B from 5% to 23%; 45. minutes-50 minutes, linear gradient of liquid B from 23% to 40%; 50. minutes-52 minutes, liquid B linear gradient from 40% to 100%; 52. and the time is between minutes and 60 minutes, and the solution B is maintained at 100 percent. The peptide fragments were separated and then analyzed by targeted PRM mass spectrometry using a QExactive HF-X mass spectrometer (Thermo Scientific). Analysis duration was 60min, detection mode: positive ion, parent ion scan range: 300-1200m/z, a primary mass spectrum resolution of 60000@m/z 200, AGC target:3e6, primary mass spectrum maximumit: 50 ms. Peptide fragment secondary mass spectrometry was collected as follows: and after each Full scan (Full MS scan), sequentially selecting the preform/z of the target peptide according to the Inclusion list for secondary mass spectrometry (MS 2), wherein the resolution of the MS2 is 30000@m/z 200, the AGC target is 1e6, and the secondary mass spectrometry is Maximum IT:100 MS, MS2 Activity Type: HCD, isolation window:1.6 Th, normalized collision energy:28. the resulting mass spectrum RAW file was analyzed for PRM data using the software Skyline 4.1 pair.

Statistical analysis of mass spectrometry data: skyline is a targeted proteomic analysis software developed by MacCoss team at the university of washington, and has gradually become one of the standard solutions in the field of targeted proteomics in recent years. Performing LC-PRM/MS analysis on candidate peptide fragments of the target protein, wherein the LC-PRM/MS analysis comprises a chromatographic peak contrast chart (Skyline analysis chart) of each peptide fragment in different samples; 3-5 sub-ions with higher abundance and as continuous as possible in the secondary mass spectrogram of the candidate peptide fragment are selected for quantitative analysis, the peak area result of each target peptide fragment sub-ion after Skyline analysis is exported, and the result is shown in a target peptide fragment PRM quantitative Skyline data analysis table form-Transition Results, wherein the target peptide fragment PRM quantitative Skyline data analysis table form comprises a target protein name, a peptide fragment sequence, a parent ion charge number, selected sub-ions, a sub-ion charge number and the original peak area of each sub-ion for quantification. And adding all the ion peak areas of each peptide fragment to obtain the ion peak area of the peptide fragment. And adding all the ion peak areas of each peptide fragment to obtain the ion peak area of the peptide fragment.

Quantitative results of target peptide fragment: from the experimental results, candidate peptide fragments with strong quantitative information in all samples are finally selected, and the target peptide fragment Skyline analysis patterns (shown in figures 8-10) are shown. The target peptide PRM quantitative Skyline data analysis table comprises peptide peak areas. The target peptide Skyline quantitative results are shown in the following table 1:

TABLE 1

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting tumor neoantigen from engineering bacteria expressing HLA proteins, which is characterized by comprising the following steps:

displaying the Lpp-OmpA gene sequence and the HLA protein expression sequence on the surface of engineering bacteria for extracting antigens in tumor cells;

the engineering bacteria comprise: probiotics and plasmids, transferring the plasmids into the probiotics;

the plasmid comprises a fluorescent protein coding gene, a promoter, an Lpp-OmpA gene sequence and an HLA protein expression sequence which are connected in sequence;

the HLA protein expression sequence is SEQ ID NO.1;

the Lpp-OmpA gene sequence is SEQ ID NO.2;

the tumor cells comprise prostate cancer cells;

the neoantigen includes TP53 ^R175H 。

2. The method of claim 1, wherein the HLA protein expression sequence comprises an HLA-a 02:01 protein expression sequence.

3. The method of claim 2, wherein the fluorescent protein encoding gene comprises a T7-GFP green fluorescent protein encoding gene;

the promoter comprises Tac;

the probiotics comprise escherichia coli Nissle1917.

4. The method of claim 1, wherein the engineered bacteria are placed in tumor cell lysate to extract tumor neoantigens.