阅读说明：本技术 一种sumo化修饰捕获探针的合成方法及应用 (Synthetic method and application of SUMO modified capture probe ) 是由 伍会健 王甜甜 李淑晶 于 2020-12-01 设计创作，主要内容包括：本发明属于生物技术领域,涉及一种SUMO化修饰捕获探针、其合成方法及应用。所述的SUMO化修饰捕获探针分子式为SUMO-C_6H_8NO_3,所述的SUMO为人源SUMO-1,其核苷酸序列如SEQ ID NO：1所示,其氨基酸序列如SEQ ID NO：2所示。本发明采用基因克隆得到编码人源SUMO-1蛋白的多肽,通过化学合成方法得到SUMO-C_6H_8NO_3,SUMO-C_6H_8NO_3结构稳定,方便化学合成及检测应用。SUMO-C_6H_8NO_3荧光探针具有高度特异性灵敏性较高。(The invention belongs to the technical field of biology, and relates to a SUMO modified capture probe, a synthetic method and application thereof. The molecular formula of the SUMO modified capture probe is SUMO-C 6 H 8 NO 3 The SUMO is human-derived SUMO-1, and the nucleotide sequence of the SUMO is shown in SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively. The invention adopts gene cloning to obtain polypeptide for coding human-derived SUMO-1 protein, and obtains SUMO-C by a chemical synthesis method 6 H 8 NO 3 ，SUMO‑C 6 H 8 NO 3 Stable structure and convenient chemical synthesisAnd detection application. SUMO-C 6 H 8 NO 3 The fluorescent probe has high specificity and high sensitivity.)

1. The SUMO modified capture probe is characterized in that the molecular formula of the SUMO modified capture probe is SUMO-C₆H₈NO₃The structural formula is shown as the following formula (I):

2. the SUMO modified capture probe of claim 1, wherein SUMO is human SUMO-1, and its nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

3. The method for preparing the SUMO modified capture probe of claim 1 or 2, wherein the method comprises the following steps:

step 1, obtaining a full-length cDNA sequence of human-derived SUMO through gene cloning;

step 2, recombining and expressing the SUMO protein polypeptide in escherichia coli by the plasmid containing the SUMO full-length cDNA sequence obtained in the step 1), and performing affinity purification to obtain a pure SUMO protein polypeptide;

step 3, obtaining the SUMO-C from the pure SUMO protein polypeptide obtained in the step 2) by a full chemical synthesis method₆H₈NO₃A fluorescent probe.

4. The method according to claim 3, wherein the step 3) further comprises:

3.1) putting the pure SUMO protein polypeptide, ethyl chloroformate and N-methylmorpholine in tetrahydrofuran, cooling to-20 ℃, and stirring for reaction;

3.2) after the reaction in the step 3.1), heating to-5 ℃, incubating, dripping sodium azide aqueous solution at the temperature, and reacting at room temperature;

3.3) extracting the product obtained in the step 3.2) with ethyl acetate, adding anhydrous magnesium sulfate for drying, and concentrating under reduced pressure to recover the obtained white solid;

3.4) dissolving the white solid obtained in the step 3.3) in toluene and heating to 100 ℃;

3.5) when bubbles are released from the reaction liquid in the step 3.4), adding allyloxycarbonyl, and stirring for reaction at 100 ℃;

3.6) after the reaction in the step 3.5), cooling to below 65 ℃ to separate out product crystals, performing suction filtration to obtain a crude product, and performing recrystallization by using methanol as a solvent to obtain crystals;

3.7) dissolving the crystals obtained in the step 3.6) in dry trichloromethane;

3.8) after the mixture is fully dissolved in the step 3.7), adding mCPBA under ice bath, immediately replacing nitrogen, and stirring and reacting in ice bath;

3.9) TLC monitoring until the reaction is finished, adding water to quench the reaction, extracting with ethyl acetate, and washing with saturated salt water;

3.10) drying the organic phase obtained in the step 3.9) by using anhydrous sodium sulfate, decompressing, spin-drying the solvent, and separating and purifying by using silica gel column chromatography to obtain white powder; after dissolution, after trypsin digestion, LC-MS identification.

5. Use of the SUMO modified capture probe of claim 1 or 2 or the SUMO modified capture probe prepared by the method of claim 3 or 4 for detecting SUMO modification.

6. The use of claim 5, wherein said detecting the SUMO modification comprises cellular localization, quantitative analysis and/or dynamic modification change analysis of SUMO modification.

7. Use of the SUMO modified capture probe of claim 1 or 2 or the SUMO modified capture probe obtained by the method of claim 3 or 4 for the preparation of a reagent for the diagnosis and/or prognosis of cancer.

Technical Field

The invention belongs to the technical field of biology, and relates to SUMO (surface acoustic wave) modificationA synthetic method and application of a decoration capture probe, in particular to a SUMO (SUMO- (E) 4-methyl-2-methyl crotonate) (SUMO-C) decoration capture probe₆H₈NO₃) A synthetic method thereof and application thereof in tumor detection.

Background

Breast cancer is a malignant tumor occurring in breast glands and ducts, is cancer with the highest incidence in women, is one of the most common causes of morbidity and mortality in human diseases in the world today, and seriously threatens human life and health. Therefore, the prevention and treatment of breast cancer is a worldwide health care problem and is a disease which is mainly prevented and treated by the world health organization. According to the ACS data, the incidence of breast cancer is about 113.3 in 10 thousands of people in the United states, the incidence of breast cancer is rising year by year in China, and the first place of female tumors in many big cities is high, thus bringing huge property loss and mental pain to the society and families.

At present, the pathogenesis of breast cancer is not completely clear, and no effective prevention measures exist. It is known that most cancers, including breast cancer, occur and develop in a long and chronic pathological change process, and the current methods for diagnosing breast cancer can only diagnose the cancer with tumor mass growing to a certain volume, which is too late in many cases. Currently, the treatment of breast cancer is mainly drug therapy, radiotherapy and surgical resection, but no effective diagnosis and treatment method exists yet. Although the tumor focus is removed in the operation, the corresponding tissue and organ are partially or completely removed, and the normal function of the tissue and organ is lost; while the radiotherapy kills tumor cells by radioactive rays, the radiotherapy also has an injury effect on normal tissue cells of an organism, so that the tumor treatment is limited to a great extent; the drug therapy causes great physical and psychological trauma to patients due to weak curative effect and strong toxic and side effects. As with other cancers, effective prevention and treatment of breast cancer is also in need of elucidating and elucidating the pathogenesis of breast cancer, and gene therapy is of great importance.

Small ubiquitin-related modifier (SUMO) modification is an important dynamic reversible protein posttranslational modification, which has been discovered for more than 20 years. Over 3000 SUMO-modified proteins have been found to be identified so far, and SUMO-modified proteins have important regulatory effects on the function of target proteins, such as cell sublocalization, protein stability, signal transduction, enzyme activity, gene transcription regulation, cell cycle regulation, cell differentiation, and the like. Currently, in mammals, 4 different SUMO protein subtypes are identified, namely SUMO-1, SUMO-2, SUMO-3 and SUMO-4, and in the presence of E1 activating enzyme, E2 binding enzyme and E3 linking enzyme, the double Gly at the C terminal of the SUMO molecule is covalently bonded with the lysine side chain epsilon-NH 2 on a target protein through isopeptide bonds to regulate the structure and the function of the substrate protein. For example, sumoylation modification of p53 occurs at lysine residue 386, and members of the pias (protein inhibitor of activated stats) family can enhance the stability of p53, and sumoylation has been shown to enhance the transcriptional activity of p53, leading to apoptosis. The lysine residues at positions 254, 266 and 289 of PTEN (phosphatydinositol-3, 4, 5-triphosphate-3-phosphatase) protein can be modified by SUMO-1 and SUMO-2 to down-regulate PI3K/AKT pathway, thereby inhibiting cell proliferation and tumor growth. A plurality of proteins interacting with p53 can be SUMO, and the core factors of a signal channel such as NF-kappa B, PTEN and the like play a role in the development of tumors at least partially depend on the activity of the SUMO proteins. SUMO is therefore one of the most potential targets for future cancer detection and therapy. By researching the related SUMO fluorescent probe, the dynamic change of SUMO formation level in the process of generating and developing tumors is known, and a new idea can be developed for early diagnosis and treatment of tumors.

SUMO modification of proteins is a dynamic and reversible process, and therefore dessumo enzymes are considered as drug therapy targets. The SUMO-removing enzyme (DSP) is composed of a group of SUMO-specific protease family SENP (Sentrin/SUMO-specific protease), SENPs can regulate the SUMO modification level and activity of target protein, and the expression or activity of the SENPs is regulated by some regulating factors. Based on the characteristics of DSP, SENPs have an enzymatic activity region of about 200 amino acids, belong to C48 cysteine protease, and can cut off the isopeptide bond between SUMO and a target protein.

Because SUMO modification is a dynamic process, positioning and dynamic modification detection of SUMO modification in cells are very difficult, and a detection means for accurately positioning and dynamically monitoring SUMO modification is urgently needed at present. Particularly in some tumor cells, the degree of SUMO modification is closely related to the malignancy and prognosis of the tumor, so that the localization, quantitative analysis and tracking of dynamic modification changes of SUMO modified cells can be helpful for diagnosis and prognosis evaluation of malignancy.

Disclosure of Invention

In order to solve the problems in SUMO detection, the invention aims to provide a SUMO modified capture probe which can rapidly detect the subcellular localization, the modification degree and the dynamic modification change process of a SUMO modified protein in a cell.

The technical scheme of the invention is as follows:

in a first aspect, the invention provides a SUMO modified capture probe, the molecular formula of which is SUMO-C₆H₈NO₃The structural formula is shown as the following formula (I):

further, the SUMO is human-derived SUMO-1, and the nucleotide sequence of the SUMO is shown in SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

In a second aspect, the present invention provides a method for preparing the SUMO modified capture probe, which includes the following steps:

step 1, obtaining a full-length cDNA sequence of human-derived SUMO through gene cloning;

step 2, recombining and expressing the SUMO protein polypeptide in escherichia coli by the plasmid containing the SUMO full-length cDNA sequence obtained in the step 1), and performing affinity purification to obtain a pure SUMO protein polypeptide;

step 3, obtaining the SUMO-C from the pure SUMO protein polypeptide obtained in the step 2) by a full chemical synthesis method₆H₈NO₃A fluorescent probe.

Specifically, step 3 of the preparation method of the SUMO modified capture probe further comprises:

3.1) putting the pure SUMO protein polypeptide, ethyl chloroformate and N-methylmorpholine in tetrahydrofuran, cooling to-20 ℃, and stirring for reaction;

3.2) after the reaction in the step 3.1), heating to-5 ℃, incubating, dripping sodium azide aqueous solution at the temperature, and reacting at room temperature;

3.3) extracting the product obtained in the step 3.2) with ethyl acetate, adding anhydrous magnesium sulfate for drying, and concentrating under reduced pressure to recover the obtained white solid;

3.4) dissolving the white solid obtained in the step 3.3) in toluene and heating to 100 ℃;

3.5) when bubbles are released from the reaction liquid in the step 3.4), adding allyloxycarbonyl, and stirring for reaction at 100 ℃;

3.6) after the reaction in the step 3.5), cooling to below 65 ℃ to separate out product crystals, performing suction filtration to obtain a crude product, and performing recrystallization by using methanol as a solvent to obtain crystals;

3.7) dissolving the crystals obtained in the step 3.6) in dry trichloromethane;

3.8) after the mixture is fully dissolved in the step 3.7), adding mCPBA under ice bath, immediately replacing nitrogen, and stirring and reacting in ice bath;

3.9) TLC monitoring until the reaction is finished, adding water to quench the reaction, extracting with ethyl acetate, and washing with saturated salt water;

3.10) drying the organic phase obtained in the step 3.9) by using anhydrous sodium sulfate, decompressing, spin-drying the solvent, and separating and purifying by using silica gel column chromatography to obtain white powder; after dissolution, after trypsin digestion, LC-MS identification.

In a third aspect, the invention provides the SUMO modified capture probe and the use of the SUMO modified capture probe prepared by the method for detecting SUMO modification.

Further, the detecting SUMO modification comprises cell localization, quantitative analysis and/or dynamic modification change analysis of SUMO modification.

In a fourth aspect, the present invention provides the SUMO modified capture probe and the use of the SUMO modified capture probe prepared by the above method in preparing a reagent for cancer diagnosis and/or prognosis.

The invention has the advantages that:

the invention adopts gene cloning to obtain polypeptide for coding human-derived SUMO-1 protein, and obtains SUMO-C by a chemical synthesis method₆H₈NO₃，SUMO-C₆H₈NO₃Stable structure, convenient chemical synthesis and detection application. SUMO-C₆H₈NO₃The fluorescent probe has high specificity and high sensitivity.

Drawings

FIG. 1 is a three-dimensional structure diagram of a human-derived SUMO-1 molecule.

FIG. 2 is a mass spectrum of SUMO protein identification

FIG. 3 is SUMO-1-C₆H₈NO₃Schematic diagram of chemical total synthesis method for preparing fluorescent probe molecule.

FIG. 4 is SUMO-1-C₆H₈NO₃And (3) identifying a mass spectrogram by using a fluorescent probe.

FIG. 5 is SUMO-1-C₆H₈NO₃The imaging effect of the fluorescent probe molecules in MCF7 is shown schematically.

FIG. 6 is SUMO-1-C₆H₈NO₃The imaging effect of the fluorescent probe molecule in a nude mouse is shown schematically.

Detailed Description

The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Cloning and expression of human-derived SUMO-1 gene

Extracting total RNA of human breast cancer MCF-7 cells, purifying mRNA, carrying out reverse transcription on the mRNA, constructing a cDNA library, designing a primer, and screening a human SUMO1 gene by using a PCR method. The 5 '-end primer was 5'-ATGTCTGACCAGGAGGCAAAACCT-3'(SEQ ID NO: 3), and the 3' -end primer was 5'-CTAAACTGTTGAATGACCCCCCGT-3' (SEQ ID NO: 4). The obtained positive monoclonal was subjected to gene nucleotide sequence determination. The gene sequencing result shows that the coding human-derived SUMO-1 sequence consists of 306 nucleotides, and the sequence from the 5 'end to the 3' end is (SEQ ID NO: 1):

the human SUMO-1 protein molecule is coded, and the amino acid sequence is (SEQ ID NO: 2; NP-001005781.1):

Met-Ser-Asp-Gln-Glu-Ala-Lys-Pro-Ser-Thr-Glu-Asp-Leu-Gly-Asp-Lys-Lys-Glu-Gly-Glu-Tyr-Ile-Lys-Leu-Lys-Val-Ile-Gly-Gln-Asp-Ser-Ser-Glu-Ile-His-Phe-Lys-Val-Lys-Met-Thr-Thr-His-Leu-Lys-Lys-Leu-Lys-Glu-Ser-Yyr-Cys-Gln-Arg-Gln-Gly-Val-Pro-Met-Asn-Ser-Leu-Arg-Phe-Leu-Phe-Glu-Gly-Gln-Arg-Ile-Ala-Asp-Asn-His-Thr-Pro-Lys-Glu-Leu-Gly-Met-Glu-Glu-Glu-Asp-Val-Ile-Glu-Val-Tyr-Gln-Glu-Gln-Thr-Gly-Gly-His-Ser-Thr-Val(MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV)

specifically, the cloning of the gene of the SUMO-1 molecule includes:

1) extracting total RNA of human MCF7 cells:

first, MCF7 cells cultured in a 35mm dish were added to 1m1 RNAasso Plus (Trizol, TAKARA, Japan), and lysed on a decolorizing shaker at room temperature for 5 min.

② transferring the cell lysate into a 1.5ml centrifuge tube, adding 200 mul of chloroform into the tube, violently shaking for 15s, and standing for 5min at room temperature.

Thirdly, placing the centrifuge tube in the second step into a low-temperature refrigerated centrifuge, and centrifuging for 15min at 12000rpm under the condition of 4 ℃.

Transferring the supernatant into a centrifuge tube of 1.5ml RNase-Free, adding 500 mu l of isopropanol, gently turning the centrifuge tube to mix the liquid evenly, and standing for 10min at room temperature.

Fifthly, putting the centrifuge tube in the fourth step into a low-temperature refrigerated centrifuge, and centrifuging for 10min at 12000rpm under the condition of 4 ℃.

Sixthly, removing the supernatant, adding 1ml of 75% ethanol into the centrifugal tube, and gently blowing and beating the RNA for precipitation.

Seventhly, placing the centrifugal tube in the sixth step into a low-temperature refrigerated centrifuge, and centrifuging for 3min at the temperature of 4 ℃ and the rpm of 5000.

The supernatant is carefully removed by a pipette, left at room temperature for several minutes, and the RNA is dried.

Ninthly, 30 ul of RNase-Free water was added to the centrifuge tube in the vessel to dissolve RNA.

2) Constructing a human cDNA library:

first strand cDNA Synthesis (reverse transcription of mRNA):

adding 1.0. mu.l of human MCF7 cell total RNA, 1.0. mu.l of Oligo (dT) primer and 5.0. mu.l of RNase-Free ddH to the RNase-Free PCR tube₂O, leading the total volume to reach 7 mu l, mixing uniformly, centrifuging for a short time (2000rpm for 30s), and preserving the temperature for 10 minutes at 72 ℃; after incubation, the tubes were incubated at 4 ℃ for 2 minutes.

② adding the following reagents, 2.0. mu.l of 5 XM-MLV Buffer, 0.5. mu.l of 10mM dNTP Mix, 0.5. mu.l of 40U/. mu.l RNase Inhibitor and 0.25. mu.l of RTase M-MLV into the centrifuge tube, mixing the reagents in the centrifuge tube and centrifuging for a short time (2000rpm, 30s), keeping the temperature at 42 ℃ for 60min, and then keeping the temperature at 70 ℃ for 15 s. After heat preservation treatment, the centrifugal tube is placed on ice to stop synthesis for standby.

Amplification of SUMO1 gene using Polymerase Chain Reaction (PCR):

first, 1. mu.l of cDNA template, 0.25. mu.l of Taq enzyme, and 10. mu.l of Taq enzymeMu.l of 5 XPCR buffer, 1. mu.l of 10mM dNTP, 1.0. mu.l of 5 'PCR primer, 1.0. mu.l of 3' PCR primer and 35.75. mu.l of ddH₂O was mixed in a PCR tube, and the mixture was centrifuged to collect the mixture at the bottom of the tube.

Amplifying in a PCR instrument according to the following procedures: 95 ℃ for 5 min; 35 cycles: 94 deg.C, 30sec, 56 deg.C, 30sec, 72 deg.C, 1 min. After the circulation is finished, incubation is carried out for 10min at 72 ℃, and the mixture is stored in a refrigerator at minus 80 ℃ after PCR is finished.

3) Cloning and screening the human SUMO-1 gene:

and after the PCR reaction is finished, carrying out electrophoresis on the PCR reaction product by using agarose gel with the concentration of 1%, carrying out constant pressure of 120V for 30min, placing the product in a gel imager for observation and photographing, cutting gel containing the SUMO-1 target fragment, and recovering by using an agarose gel DNA recovery kit. The recovered target fragment was ligated to pGEX-4T-3 vector and transformed into DH 5. alpha. competent cells. Plating and double screening of ampicillin and blue-white spot, picking single colony and detecting the size of the insert by PCR with M13 primer. The positive colonies were picked, shaken to extract plasmids, sent to Biotechnology engineering (Shanghai) GmbH for nucleotide sequencing, and translated into amino acid sequences.

4) SUMO protein expression and in vitro purification

pGEX-4T-3-SUMO1 is transformed into escherichia coli BL21, positive clones are screened out, shake flask culture is carried out, the strips grow to the logarithmic phase, after IPTG induction for 4 hours, continuous culture is carried out for 16 hours, ultrasonic disruption is carried out, and proteins are purified through an affinity purification column and MS identification is carried out (as shown in figure 2).

Example 2: SUMO-C₆H₈NO₃Synthesis of Probe molecules

SUMO-C₆H₈NO₃The fluorescent probe molecule is prepared by a chemical total synthesis method (as shown in FIG. 3), and the target SUMO protein is divided into 3 fragments, wherein Ala6 is selected as a connecting point and is substituted by cysteine to promote the connection reaction.

Putting 0.5mmol of the obtained SUMO protein, 1.1mmol of ethyl chloroformate and 1.1mmol of N-methylmorpholine in 10mL of tetrahydrofuran, cooling to-20 ℃, and stirring for 20 min;

② after the reaction, heating to-5 ℃, incubating for 5min, dripping sodium azide aqueous solution (2.1 mmol sodium azide is dissolved in 2mL water) at the temperature, reacting for 15min at room temperature;

③ the product was extracted with ethyl acetate (15 mL. times.3), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to recover a white solid.

In SUMO-N-terminal fragment synthesis, 2-aminobutyric acid (Dab) with side chain amino acid protected by allyloxycarbonyl (Alloc) is used for replacing lysine at a modification site, and a label molecule (such as Biotin) is condensed at the N-terminal.

Dissolving the white solid in 10mL of methylbenzene, and heating to 100 ℃;

adding allyloxycarbonyl (Alloc) when bubbles are released from the reaction solution, and stirring and reacting for 20min at 100 ℃;

sixthly, after the reaction is finished, cooling to below 65 ℃ to separate out product crystals, performing suction filtration to obtain crude products, and recrystallizing by using methanol as a solvent to obtain crystals.

After the amino acid sequence condensation is completed, the Alloc protecting group is orthogonally removed, and then the SUMO connecting arm is condensed. Because the existence of sulfydryl or activated unsaturated double bond in the connecting arm cannot be compatible with free radical desulfurization reaction, the synthetic strategy of connecting three fragments in sequence is selected. Firstly, carrying out N-to-C sequential connection and desulfurization to obtain a linker modified SUMO molecule, then removing Acm protecting group, connecting with SUMO hydrazide, and finally activating the linker to obtain SUMO-C₆H₈NO₃And (3) a probe.

Seventhly, dissolving the crystal (500mg) in dried trichloromethane (5 mL);

after the materials are fully dissolved, adding mCPBA (98.5mg, 0.57mmol) under ice bath, immediately replacing nitrogen, and stirring in ice bath for reaction;

ninthly, monitoring by TLC until the reaction is finished, adding water to quench the reaction, extracting with ethyl acetate (20mL), and washing with saturated salt water (10 mL);

r drying the organic phase with anhydrous sodium sulfate, decompressing, drying the solvent, separating and purifying by silica gel column chromatography (dichloromethane: ethyl acetate ═ 5:1) to obtain white powder; 100mg of the DNA was dissolved in 2.5ml of buffer and identified by LC-MS after tryptic cleavage (see FIG. 4).

Example 3: SUMO-C₆H₈NO₃Application of probe molecule in MCF7 cells and mice

MCF7 cells were seeded into 35mm cell culture plates and the fusion experiments were performed until the degree of cell fusion was about 40%. Mixing SUMO-C₆H₈NO₃The probe molecules were incubated with MCF7 cells and detected by fluorescence microscopy after 36h, the results are shown in FIG. 5.

The probe detection method comprises the following steps:

because the SUMO protein can specifically recognize lysine residue of substrate protein, the modified protein can react with SUMO-C through the action of enzyme related to SUMO process₆H₈NO₃The probe molecules are directly combined with the substrate, and can perform autofluorescence, wherein the excitation wavelength is 475nm, and the emission wavelength is 507nm, so that the intracellular SUMO modification can be positioned, quantified and dynamically changed and monitored by detecting the autofluorescence emission intensity and position of the probe molecules.

Will contain SUMO-C₆H₈NO₃MCF7 cells as probe molecules were injected into the left and right ventral sides of nude mice by subcutaneous injection, and the light intensity was measured at different times, and the results are shown in FIG. 6, in which the light intensity was still high 7 days after injection.

The above description of exemplary embodiments has been presented only to illustrate the technical solution of the invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those skilled in the art. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to thereby enable others skilled in the art to understand, implement and utilize the invention in various exemplary embodiments and with various alternatives and modifications. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Sequence listing

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