CN109456974A - A kind of Endoglin aptamer and its application - Google Patents

Abstract

The present invention discloses a kind of Endoglin aptamer and its application, belongs to genetic engineering, immunology and oncology technical field.The aptamers are one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 SEQ ID.Aptamer of the present invention is obtained using full Cell depletion Cell-SELEX technology screening, molecular weight is smaller, it is readily synthesized and modifies, can high specific identification and high-affinity combination Endoglin, in conjunction with other cells not occurrence features, non-immunogenicity stablizes easily modification, convenient for synthesis and saves.The Endoglin detection kit and reagent paper prepared with the aptamers is high to Endoglin detection sensitivity, and is easy to optimize.It is applied in diagnosing tumor, tumor prognosis and the assessment of monitoring, diagnosing tumor.

Description

A kind of Endoglin aptamer and its application

Technical field

The invention belongs to genetic engineering, immunology and oncology technical field, in particular to a kind of Endoglin nucleic acid is suitable Ligand and its application.

Background technique

Tumor vascular endothelial cell is the basis of Tumor Angiongesis, has very important work to tumour growth and transfer With.Early diagnosis of tumor and treatment are carried out for tumor vascular endothelial cell, it has also become tumour correlative study development necessarily becomes Gesture.Endoglin is a kind of transmembrane glycoprotein on tumor vascular endothelial cell surface, in the vascular endothelial cell of active proliferation It is overexpressed, and does not express in normal vascular endothelia cell or weak expression.Therefore, Endoglin is acknowledged as Tumor Angiongesis The specific marker formed with new vessels.

Aptamer is that one kind can have and anti-with the DNA or RNA sequence in conjunction with target molecule high specific high-affinity The similar effect of body, commonly referred to as " chemical antibody ".In recent years, with the continuous development of aptamer screening technique, more It is found come more tumor associated nucleic acid aptamers sequences by people.However, the relevant aptamer of these tumours is usually It is only capable of identifying a kind of tumour cell or a kind of tumour, largely limits the application of aptamer.Currently, can specific target It not yet appears in the newspapers to the screening of tumor vascular aptamers.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of Endoglin aptamer and its applications.

Technical solution used in purpose to realize the present invention are as follows:

A kind of Endoglin aptamer, the aptamers are in nucleotide sequence shown in NO:1 ~ 5 SEQ ID One or several any sequence.

Preferably, the aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or The sequence that the homology of several sequences is 60% or more.

Preferably, the aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or The sequence that several sequences are hybridized.

Preferably, the aptamers have with any one in nucleotide sequence shown in NO:1 ~ 5 SEQ ID or The sequence that several sequences are transcribed.

Preferably, several positions in the aptamers sequence be phosphorylated, methylate, amination, sulfhydrylation or Isotopologue.

Preferably, it is combined with biotin in the aptamers sequence, digoxin, fluorescent material, nano luminescent material, gathers Ethylene glycol, peptide fragment, albumen, folic acid or enzyme label.

Preferably, the Endoglin aptamer have in nucleotide sequence shown in NO:1 ~ 5 SEQ ID One or several any sequence alterations at corresponding peptide nucleic acid.

The present invention also provides a kind of Endoglin aptamers in preparation Endoglin detection kit or detection reagent The application of paper.

The application uses Cell-SELEX screening technique in the selection of screening technique.Although target molecules Endoglin For a kind of cell membrane surface protein, and traditional protein screening technique is not used, but uses Cell-SELEX screening technique, and The 293T cell line (293T-hE) of artificial constructed stable expression target molecules Endoglin is as positive screening cell.This method has Effect avoids the difference of external synthetic protein Yu epicyte protein native conformation, while also solving target cell and obtaining and being stranded Difficult problem.In addition, the positive and reverse screening cell that the present invention selects only has the differential expression of the single molecule of Endoglin, this makes Aptamers obtained are screened with more accurate targeting, target spot confirmation required when subsequent screening is completed is simplified and investigates Etc. correlation steps, optimize experimental arrangement.The stage is investigated in the verifying of candidate aptamers, the aptamers that the present invention obtains can also be with Other cells (such as HUVEC cell) with Endoglin expression other than target cell combine, and this demonstrates the present invention to screen Method is practical.

Present invention substantive distinguishing features outstanding and significant progress are:

Aptamer of the present invention using full bacterium abatement Cell-SELEX technology screening and obtain, molecular weight is smaller, be readily synthesized and Modification, can high specific identification and high-affinity combination Endoglin, with other bacteriums not occurrence features ining conjunction with, nothing be immunized Originality stablizes easily modification, convenient for synthesis and saves, can be used for detecting Endoglin, and effectively prevents and control Endoglin infection is of great significance.The Endoglin detection kit and reagent paper pair prepared with the aptamers Endoglin detection sensitivity is high, and is easy to optimize.

Detailed description of the invention

Fig. 1 is to be enriched with library for 293T-hE cell and 293T cell combination ability flow cytometer detection as a result, A:293T-hE is thin Cellular surface fluorescence intensity increases with number of screening round and is gradually increased, and B:293T cell surface fluorescence intensity increases with number of screening round and becomes Change not significant.

Fig. 2 is candidate aptamers and 293T-hE cell combination fluorescence imaging result (400x).

Fig. 3 is each aptamers truncated sequence situation streaming result in conjunction with target cell.

Fig. 4 is aptamer ENG-4c living imaging result in Mice Body.

Specific embodiment

The present invention program is described in further detail below with reference to embodiment, following the description is merely to explain this hair It is bright, its content is not defined.

Embodiment

Aptamers screening process is that duplicate, progressive process is taken turns one more, any one link mistake occurs all by shadow Ring to the end as a result, even resulting in entire the failure of an experiment when serious.Therefore, before in accordance with basic Cell-SELEX screening step It puts, it is also noted that the control of details, to improve the screening efficiency of aptamers.Specifically to follow following some points for attention:

(1) guarantee cell state

Whether aptamers screening is successful to have very close relationship with cell state.Cell state may will affect it when poor The normal expression of surface protein, leads to the missing or change of expected aptamers target, to influence the progress of aptamers screening.This Two groups of positive and reverse screening cells that invention is selected are both attached cells of 293T-hE and 293T respectively.Before each screening, It need to guarantee two kinds of cell culture times within 24-48 h, to may cause cell adherent not yet this is because incubation time is too short Reach perfect condition, and the too long cell that may cause of incubation time is adherent excessively securely, the link for scraping off cell and collecting compared with For difficulty.In addition, cell also should be at growing logarithmic growth phase the most vigorous, and maintain vigour greater than 90%.Cell viability Judgement is mainly to carry out according to trypan blue negative staining method, when cell activity is preferable, will not still can under mirror by Trypan Blue Observe the good cell of translucency；When cell is dyed to blue as the result is shown under the microscope, prompt cell state poor, very To death.

(2) selection of temperature is screened

Temperature can usually generate certain influence to the combination of cell and DNA.Therefore, suitable temperature is selected to screen aptamers Success or not plays important decisive action.The screening selection that the present invention is carried out carries out under 4 °C of environment, this is because with The extension of time can generate endocytosis to DNA in (such as 37 °C) cell under hot environment, affect the rich of library to a certain extent Fu Xing.And reduction screening temperature appropriate can effectively avoid this phenomenon.

(3) it is actively introduced counter-selection

To obtain the aptamers having compared with high specific as early as possible, it should counter-selection choosing be added before the 3rd wheel as far as possible.In the present invention, Anti- screening step is added in front of each round just screens, can not will directly select the DNA of cell combination to collect simultaneously with counter-selection in this way It puts into positive screening cell, without carrying out other washing steps, avoids screening while simplifying screening step The partial loss in library.

(4) increase of screening pressure

With the increase of number of screening round, it to be gradually increased screening pressure, can just effectively improve screening efficiency and success rate.Screening pressure The increase of power is mainly reflected in wash time, washing times, incubation time etc..It specifically, is exactly with number of screening round Increase, extend counter-selection as one sees fit and select the incubation time of cell and DNA library so that counter-selection choosing carry out more completely so that literary The poor DNA removal of specificity is more thorough in library；It reduces the dosage of DNA library simultaneously and is incubated for it with positive sieve cell Time shortens, and washing intensity increases.The screening efficiency of aptamers not only can be improved in this way, moreover it is possible to obtain screening suitable Ligand has more preferably binding ability.It is as shown in table 1 to the specific detail of the regulation of screening pressure herein.

1 screening pressure of table regulates and controls detail list

Number of screening round	Library dosage (pmol)	Positive sieve culture dish specification (mm)	Positive sieve incubation time (min)	Counter-selection culture dish specification (mm)	Counter-selection incubation time (min)	Washing buffer volume (mL)
							1	5000	10	60	-	-	2
2	278	10	60	-	-	2
							3	289	10	55	-	-	2
4	345	10	55	6	30	2
							5	336	10	50	6	30	3
6	356	10	50	6	35	3
							7	340	6	45	6	35	3
8	320	6	45	6	40	3
							9	300	6	40	6	40	4
10	280	6	40	10	45	4
							11	260	6	35	10	45	4
12	240	6	35	10	50	4
							13	220	6	30	10	50	5
14	200	6	30	10	55	5
							15	200	6	30	10	55	5
16	200	6	30	10	60	5
							17	200	6	30	10	60	5
18	200	6	30	10	60	5
							19	200	6	30	10	60	5

1. screening conditions selection and optimization

Screening process is constituted by repeating PCR process, and the selection of PCR condition is of crucial importance the selection result, PCR Condition selection fault usually makes amplified production have more nonspecific products, may cause screening when serious and loses completely It loses.Therefore, before being screened, the various aspects such as annealing temperature, thermal cycle number need to be optimized, to obtain optimal PCR Condition.

2. screening library enrichment degree to determine

To judge screening process of the invention, i.e., as number of screening round increases, the richness of the ssDNA in conjunction with target cell 293T-hE Collect situation, 12 wheels, 15 wheels, 17 wheels, 19 wheel screening products use simultaneously with the PCR amplification of FITC fluorescent marker Initial libraries with FITC label carry out Flow cytometry or fluorescence microscope image checking as negative control.Together When use counter-selection to select cell 293T as control cell.Experimental result as shown in Figure 1, with number of screening round increase, screening text Fluorescence curve of the library in conjunction with target cell 293T-hE gradually deviates to the right, indicates gradually increasing for fluorescence intensity.By in figure As can be seen that the degree that this fluorescence curve deviates to the right is gradually increased with the progress of screening, and reach in 17 wheel Peak.The degrees of offset of former wheels is compared therewith, and fluorescence curve of the 19 wheel screening products in conjunction with target cell is bent compared with 17 wheel fluorescence There was only faint degrees of offset for line, such phenomenon is exactly the mark of library enrichment degree judgement, this prompt screening library is rich Collection degree has reached saturation state substantially.And the fluidic cell after being incubated for each round screening library and control cell 293T In the detected result of art, the shift phenomenon of this fluorescence curve is not observed.And with number of screening round into Row, the detected fluorescence curve of counter-selection cell flow cytometry only have extremely faint right side degrees of offset.This is likely to only Certain non-specific adsorption for being only because DNA sequence dna and cell surface causes.The above results not only confirm that screening has reached Terminal, have also demonstrated the counter-selection choosing enrichment library that plays preferable effect really, therefore obtain being added in the present invention only with target Cell has stronger combination, and can hardly be in conjunction with control cell.

In order to carry out more intuitive observation, and the method for using cell climbing sheet to this fluorescence intensity change, use Fluorescence microscope investigates 19 wheel libraries with the case where positive and reverse screening cell incubation.As a result as shown in Fig. 2, fluorescence microscopy In mirror image, the visible obvious green fluorescence expression in the periphery target cell 293T-hE, and shown after light field picture is superimposed with fluorescence picture And cell membrane surface protein show, the distributing position of these green fluorescences meets design of the invention about around cell membrane, i.e., It combines.Conversely, can not observe green florescent signal on the surface of control cell 293T, it was demonstrated that 19 wheel libraries cannot with it is right Photo cell combines.Both the above laboratory facilities it can be proved that this screen it is obtained 19th wheel library can specificity With expression Endoglin positive cell 293T-hE combine, without with negative control cell 293T cell combination.

3 sequencing result secondary structure analysis

Since aptamers function in conjunction with target cell, the secondary structure of DNA sequence dna is depended greatly on, therefore, While investigating primary structure homology, the mould of secondary structure situation has been carried out to each family's DNA sequence dna using NUPACK Quasi-, simulated environment selects combination buffer condition, i.e. monovalent cation concentration is 150 mM, and divalent cation concentration is 4 mM, Temperature is 4 °C.The results show that the aptamers sequence of each family has the same or similar secondary structure mostly, therefore select It is glimmering to carry out FITC as candidate aptamers for the higher aptamers sequence of representative or frequency of occurrence in each family The candidate aptamers sequent synthesis of signal.Specific candidate's aptamers sequence is shown in Table 2.

The candidate aptamers sequence of table 2 and its equilibrium dissociation constant

Title	Sequence (5 ' -3 ')	Kd (nM)
			ENG-1	GAATTCAGTCGGACAGCGACGCAAGGATAGTAATTAGGTTTGGTGCGGTGGGGTAATTTCAGCGATGGACGAATATCGTCTCCC	46.79 ± 27.15
ENG-2	GAATTCAGTCGGACAGCGGAGAATGGCGTTAAGCTTTTCTTCCCTTGTGTTTGATTCTTAACCGATGGACGAATATCGTCTCCC	81.99 ± 26.93
			ENG-3	GAATTCAGTCGGACAGCGTATTACAAGGCTCTCAGCCGAGCGGCCCCGGCTCCTAGGGGAATAGATGGACGAATATCGTCTCCC	52.30 ± 46.78
ENG-4	GAATTCAGTCGGACAGCGGATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTCGATGGACGAATATCGTCTCCC	5.58 ± 3.11
			ENG-5	GAATTCAGTCGGACAGCGCAGTAATCCCTTGTGTTTGATGCTCATTTCTCTCTGAAATTGCTCGATGGACGAATATCGTCTCCC	38.52 ± 19.72

The design of 4 ENG-4 optimizations

The above confirms that the present invention screens the aptamers ENG-4 obtained in numerous aptamers candidate sequences with most strong Combination, therefore it can be expected that its in tumor associated target from now on to playing certain effect in Clinics and Practices.However, Identical as initial libraries, ENG-4 is the DNA sequence dna with 84 bp length, and in various functional studies, DNA sequence dna Length is longer, and stability is also poorer.Moreover, reasonably removal aptamers sequence in it is certain do not function, even because Space occupy-place and influence aptamers play combination base sequence, it is most important to the acquisition of more excellent aptamers.Therefore, originally Invention further carries out sequence optimisation to ENG-4, and this optimization is main by the way of sequence truncation, i.e., draws former sequence both ends Object carries out all or part of removal.Each aptamers sequence is as shown in table 3 after optimization truncates.

3 ENG-4 of table optimizes truncated sequence

Title	Sequence (5 ' -3 ')
		ENG-4	GAATTCAGTCGGACAGCGGATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTCGATGGACGAATATCGTCTCCC
ENG-4a	GATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTCGATGGACGAATATCGTCTCCC
		ENG-4b	GAATTCAGTCGGACAGCGGATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTC
ENG-4c	GATCAGTTTTCCATGCCAGTTGGTATTCCGCGACAGTTTGATCTC

5 ENG-4 optimization binding abilities are investigated

Flow cytometry is carried out to ENG-4 optimization, investigates the combination situation of itself and target cell 293T-hE.As a result such as Shown in Fig. 3, compared with former sequence, ENG-4a and ENG-4b have a partial reduction for the binding ability of target cell, and ENG-4c It is not substantially change with the binding ability of target cell.The results show that the aptamers sequence that the present invention obtains is playing combination When effect, forward and reverse primer sequence may be not rely on, thus after eliminating forward and reverse primer at both ends, have compared with The ENG-4c of short length also can play good combination with target cell 293T-hE.And compared with former sequence, truncated sequence ENG-4c can improve to a certain extent stability and avoid the ability being degraded since length shortens, thus may be subsequent More outstanding Targeting Effect is played in invention.

12 living imaging results

To investigate the targeting that the obtained aptamer ENG-4c of this screening is directed to tumor by local among complex environment in vivo Effect, the present invention investigate it using mouse living imaging method.In A549 Pulmonary carcinoma nude mice Transplanted tumor model living imaging In experiment, is injected through tail vein to tumor bearing nude mice have the random sequence of CY5 fluorescent marker and with CY5 label respectively ENG-4c aptamers, control group are injected PBS, are shot after 1h.As a result as shown in figure 4, the random sequence marked in CY5 In group, fluorescence can not be enriched in nude mouse tumor part, but be enriched among liver and be metabolized, this shows random sequence Do not have targeting to tumor by local；In the ENG-4c aptamers group of CY5 label, fluorescence is enriched in tumor by local mostly, Only small part is metabolized among liver, this shows in complicated biological vivo environment, what the present invention screened ENG-4c has good targeting for tumour；In PBS control group, any luciferase expression is not observed.The above reality Test the result shows that, the aptamers ENG-4c screened target in vivo in there is good effect, there is potential application Value.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.It is done within the spirit and principles of the present invention any to repair Change, equivalent replacement, improvement etc., should be included within scope of the invention.

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Claims (9)

1. a kind of Endoglin aptamer, which is characterized in that the aptamers are core shown in NO:1 ~ 5 SEQ ID One or several any sequence in nucleotide sequence.

2. Endoglin aptamer according to claim 1, which is characterized in that the aptamers have and SEQ The sequence that the homology of one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 ID is 60% or more.

3. Endoglin aptamer according to claim 1, which is characterized in that the aptamers have and SEQ The sequence that one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 ID is hybridized.

4. Endoglin aptamer according to claim 1, which is characterized in that the aptamers have and SEQ The sequence that one or several any sequence in nucleotide sequence shown in NO:1 ~ 5 ID is transcribed.

5. Endoglin aptamer according to claim 1, which is characterized in that the aptamers have and SEQ One or several any sequence in nucleotide sequence shown in NO:1 ~ 5 ID optimizes truncated sequence.

6. Endoglin aptamer according to claim 1, which is characterized in that if in the aptamers sequence A dry position be phosphorylated, methylate, amination, sulfhydrylation or isotopologue.

7. Endoglin aptamer according to claim 1, which is characterized in that combined in the aptamers sequence There are biotin, digoxin, fluorescent material, nano luminescent material, polyethylene glycol, peptide fragment, albumen, folic acid or enzyme label.

8. Endoglin aptamer according to claim 1, which is characterized in that the Endoglin nucleic acid adaptation Body have with one or several any sequence alterations in nucleotide sequence shown in NO:1 ~ 5 SEQ ID at corresponding peptide core Acid.

9. Endoglin aptamer described in claim 1-7 any one is in preparation Endoglin detection kit or detection The application of reagent paper.