CN114085873A

CN114085873A - Cancer cell state identification gene circuit group and preparation method thereof

Info

Publication number: CN114085873A
Application number: CN202111355874.5A
Authority: CN
Inventors: 余裕; 姜长安
Original assignee: Zhuhai Institute Of Advanced Technology Chinese Academy Of Sciences Co ltd
Current assignee: Zhuhai Institute Of Advanced Technology Chinese Academy Of Sciences Co ltd
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2022-02-25

Abstract

The invention provides a cancer cell state identification gene circuit group and a preparation method thereof, belonging to the technical field of genetic engineering. The cancer cell state identification gene circuit group comprises a first identification gene circuit and a second identification gene circuit which are used for identifying the cancer cells in the same cancer cell state. microRNA sequences are inserted into the 3' UTR segment, and a positive promoter, a negative promoter and microRNA are from cancer cells in a cancer cell state. The invention also discloses a preparation method of the cancer cell state identification gene circuit group. The invention can accurately mark the state of the cancer cells by obtaining the positive promoters, the negative promoters and the microRNAs which can represent different phenotypes of the cancer cells and combining the bar code regions which can assist in judgment, and can analyze the proportion information of the cancer cells in various states by means of high-throughput cell experiments and the like, thereby being beneficial to analyzing the heterogeneity among the cancer cells.

Description

Cancer cell state identification gene circuit group and preparation method thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a cancer cell state identification gene circuit group and a preparation method thereof.

Background

With the continuous evolution of medical means and the rapid development of genetic technology, medical workers find that compared with the traditional identification means, the artificially synthesized genetic circuit has the advantages of higher accuracy, stronger modulation and the like, and can accurately identify various types of cancer cells. The artificially synthesized gene circuits can play excellent roles in screening cancer cells and killing cancer cells in a targeted manner.

The subgroup heterogeneity of tumor cells is always a key difficulty in tumor diagnosis, prognosis and effective treatment, and is also a hotspot of basic research on tumor drug resistance. Although the current sequencing technology from single cells can distinguish potential drug-resistant subgroups of tumor parts.

However, due to the sparse gene expression and the complexity of the subsets of the current single cell sequencing technology, it is difficult to truly realize the accurate grouping of multiple cancer cell subsets based on signal paths, and it is difficult to accurately and completely obtain tumor subsets with different signals and phenotypic characteristics. The invention can accurately calibrate, extract and collect specific tumor subgroups according to specific genes, protein signal pathway characteristics and protein interaction characterization information, and provides a high-efficiency intelligent analysis tool for tumor heterogeneity research.

Disclosure of Invention

The present invention is directed to a cancer cell status recognition gene circuit set and a method for making the same, which at least partially solve the above-mentioned problems.

According to one aspect of the present invention, the present invention provides a cancer cell status identification gene circuit set, comprising a first identification gene circuit and a second identification gene circuit which are used in combination, wherein the first identification gene circuit and the second identification gene circuit are used in combination to identify a cancer cell;

the first identification gene circuit at least comprises a first 3'UTR segment, a first promoter, a barcode sequence, a second promoter and a second 3' UTR segment which are connected in sequence;

the second identification gene circuit at least comprises a third 3'UTR segment, a bait protein sequence, a first fluorescent protein C-terminal sequence, a PGK promoter, a barcode sequence, a PGK promoter, a capture protein sequence, a first fluorescent protein N-terminal sequence and a fourth 3' UTR segment which are connected in sequence;

microRNA sequences are inserted into the first 3'UTR segment, the second 3' UTR segment, the third 3'UTR segment and the fourth 3' UTR segment, and the first promoter, the second promoter and the microRNA sequences are all from the cancer cells.

Preferably, the first promoter is an active promoter in the cancer cell.

Preferably, the second promoter is an inactive promoter in the cancer cell.

Preferably, the barcode sequence is shown as SEQ ID NO.1, one of the capture protein sequence and the bait protein sequence is shown as SEQ ID NO.5, and the other is shown as SEQ ID NO. 6.

Preferably, the first gene signature circuit further comprises a second fluorescent protein sequence disposed between the first 3'UTR segment and the first promoter and a third fluorescent protein sequence disposed between the second promoter and the second 3' UTR segment.

Preferably, the first identifier circuit comprises a first 3'UTR segment, a second fluorescent protein sequence, a first promoter, a barcode sequence, a CMV promoter, a mScarlet sequence, a WPRE, a second promoter, a third fluorescent protein sequence, and a second 3' UTR segment connected in sequence.

Preferably, the second identification gene circuit comprises a third 3'UTR segment, a bait protein sequence, a GGGs protein sequence, a first fluorescent protein C-terminal sequence, a PGK promoter, a barcode sequence, a CMV promoter, an mScarlet sequence, a WPRE, a PGK promoter, a capture protein sequence, a GGGs protein sequence, a first fluorescent protein N-terminal sequence, and a fourth 3' UTR segment, which are connected in sequence.

Preferably, the first fluorescent protein sequence, the second fluorescent protein sequence, the third fluorescent protein sequence, and the mScarlet sequence are all different.

The embodiment of the present application further provides a method for preparing a circuit group of cancer cell state recognition genes, comprising the following steps:

step 1: obtaining a first promoter, a second promoter and a microRNA sequence of a cancer cell;

step 2: respectively compiling a first 3'UTR segment, a second 3' UTR segment, a third 3'UTR segment and a fourth 3' UTR segment which are inserted into the microRNA sequence, and compiling a barcode sequence;

and step 3: sequentially connecting the first 3'UTR segment, the first promoter, the barcode sequence, the second promoter and the second 3' UTR segment to obtain a first identification gene circuit;

and 4, step 4: and sequentially connecting a third 3'UTR section, a bait protein sequence, a first fluorescent protein C-terminal sequence, a PGK promoter, a barcode sequence, a PGK promoter, a capture protein sequence, a first fluorescent protein N-terminal sequence and a fourth 3' UTR section to obtain a second identification gene circuit, and inserting the first identification gene circuit and the second identification gene circuit in parallel in a virus vector sequence to obtain a cancer cell state identification gene circuit group.

Preferably, in step 1, the first promoter is an active promoter in the cancer cell and the second promoter is an inactive promoter in the cancer cell.

The invention provides a first identification gene circuit and a second identification gene circuit which are used for identifying cancer cells under the same cancer cell state. microRNA sequences are inserted into the 3' UTR segment, and a positive promoter, a negative promoter and microRNA are from cancer cells in a cancer cell state. The invention can accurately mark the state of the cancer cells by obtaining the positive promoters, the negative promoters and the microRNAs which can represent different phenotypes of the cancer cells and combining the bar code regions which can assist in judgment, and can analyze the proportion information of the cancer cells in various states by means of high-throughput cell experiments and the like, thereby being beneficial to analyzing the heterogeneity among the cancer cells.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram of a method for obtaining a circuit set of cancer cell state recognition genes according to the present invention;

FIG. 2 is a schematic diagram of a first signature circuit according to the present invention;

FIG. 3 is a schematic diagram of a second gene recognition circuit according to the present invention.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. For convenience of description, only portions related to the invention are shown in the drawings.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The embodiment of the application provides a cancer cell state identification gene circuit group and a method for preparing the cancer cell state identification gene circuit, and it needs to be noted that the cancer cell state identification gene circuit in the gene circuit group can be applied to identification of cancer cells in various states, such as various states of immune escape, skeleton recombination, invasion and migration and the like generated by a SMMC-7721 or MHCC997H cancer cell line under a drug-resistant condition, and can also be combined with a targeted drug to prepare the targeted drug capable of accurately killing certain subtype of cancer cells, and the specific application of the targeted drug is not limited by the application. Accordingly, the cancer cell status recognition gene circuit described in the embodiments of the present application, i.e., a gene segment composed of a promoter or an element including a promoter and having a recognition function, is called a gene circuit because it is responsible for recognizing a specific target gene segment and initiating a subsequent recognition display or treatment process.

The cancer cell line in the present embodiment mainly refers to a group of cancer cells with similar gene expression, such as SMMC-7721 or MHCC997H line, and the cancer cells in the present embodiment may be liver cancer, glioblastoma, biliary tract cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colorectal cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma or brain cancer, more preferably liver cancer, glioblastoma or biliary tract cancer, and most preferably liver cancer.

The cancer cell state in the present application mainly refers to various states of tumor cancer stem-stem maintenance differentiation, immune escape, skeleton recombination, invasion and migration, cell division cycle speed and the like, which are expressed by cancer cells under the condition of drug action or continuous culture, and the gene expression of the cancer cells is different from the cancer cell lines, especially, the enhancer in the gene sequence of the cancer cells under different cancer cell states is greatly different. Such cancer cells of different cancer cell states are often the cause of repeated recurrence during cancer therapy, and are also the main target of the present invention, because they have drug resistance and may have other malignant properties such as high metastasis.

In the examples of the present application, the viral vector used for testing and practicing the cancer cell status recognition gene circuit group provided by the present invention may be derived from a retrovirus, such as, but not limited to, Human Immunodeficiency Virus (HIV), Mouse Leukemia Virus (MLV), Avian sarcoma/leukemia virus (ASLV), Spleen Necrosis Virus (SNV), Rous Sarcoma Virus (RSV), Mouse mammary tumor virus (motor tumor virus, tv), etc.), Adenovirus (Adenovirus), adeno-associated virus (AAV), or Herpes Simplex Virus (HSV), etc. The techniques for constructing promoters based on retroviruses for testing lentiviruses are well known to those skilled in the art and will not be described in detail herein.

In the present embodiment, the vector of the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression regulatory elements such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer, and can be prepared in various ways according to the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector comprises a selectable marker for selecting host cells containing the vector, and when the expression vector is a replicable expression vector, a replicon may be included. The vector may be self-replicating or integrated into the host DNA.

In the present examples, the terms "linked" and "linking" are used to refer to the functional linkage of a nucleic acid expression control sequence that performs a conventional function to a nucleic acid sequence encoding a gene of interest. For example, when a promoter is inserted into a gene sequence of a retrovirus, the expression of the gene sequence of the retrovirus may be under the influence or control of the promoter. The "ligation" or "ligation" in the embodiments of the present application can be considered to be completed when the ligation property between the gene sequence of the promoter and the gene sequence of the retrovirus does not induce a frame shift mutation and the expression of the ribozyme is not inhibited by the expression regulatory sequence. This "joining" or "linking" process can be accomplished using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes well known in the art.

The fluorescent protein in the present embodiment may be selected from luciferase (luciferase), Green Fluorescent Protein (GFP), modified green fluorescent protein (mGFP), Enhanced Green Fluorescent Protein (EGFP), Red Fluorescent Protein (RFP), modified red fluorescent protein (mRFP), Enhanced Red Fluorescent Protein (ERFP), Blue Fluorescent Protein (BFP), Enhanced Blue Fluorescent Protein (EBFP), Yellow Fluorescent Protein (YFP), enhanced yellow fluorescent protein (ebp), cyan enhanced fluorescent protein (cfyfp), cyan fluorescent protein (ECFP), and the like, but is not limited thereto. By inserting a fluorescent protein into the gene of interest, the expression level of the cancer cell-specific ribozyme can be observed. It will be appreciated by those skilled in the art that this method can be used to determine whether a cancer cell is present in the presence of a particular type of cancer cell.

In the present invention, the enhancer may be obtained by a method described in a literature in which cancer cell lines of various cancer cell states have been already clarified, or by a method in which cancer cells of different cancer cell states are cultured by a combination of a drug-resistant culture or the like, and the enhancer contained in the cancer cells of different cancer cell states is analyzed and obtained.

The barcode regions (barcode regions) in the embodiments of the present application may be essentially any length and sequence suitable for uniquely identifying a gene type-associated feature. The barcode region can be used to represent any type of unique characteristic of a cancer cell, including the type of modification (e.g., DNA methylation, histone variants, etc.), the amount of replication (e.g., technical replication), or the concentration. The barcode region may also indicate the absence of any modification. In some embodiments, a barcode region has a length of about 6 to about 50 base pairs, such as about 7 to about 30 base pairs, for example about 8 to about 20 base pairs, such as about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs, or any range therein.

The present invention provides a cancer cell status identification gene circuit set, which comprises a first identification gene circuit and a second identification gene circuit, as shown in fig. 1 and 2, the first gene recognition circuit and the second gene recognition circuit are used to recognize cancer cells in the same cancer cell state, for example, for MHCC97H line cancer cells, the cancer cell states that have been ascertained at present include 8 different cancer cell states, namely, highly invasive cells, cytoskeleton recombination active cells, highly stem cells, highly differentiated cells, fast-dividing periodic cells, slow-dividing periodic cells, immune escape cells and anti-apoptotic cells, and the first and second recognition gene circuits in the embodiment of the present application can be, for example, MHCC97H line cancer cells in the highly stem cell state, and have a technical goal of accurately recognizing and reducing recognition probability as a main goal. Of course, the cancer cell state recognition gene circuit group in the embodiment of the present application may be only for cancer cells in a certain cancer cell state, or may be configured according to specific needs by using a plurality of cancer cell state recognition gene circuit groups provided in the embodiment of the present application in combination so as to be able to target all or part of possible cancer cell states of a cancer cell line. The present application is not limited to the number of cancer cell status recognition gene circuit groups provided in the embodiments of the present application.

In an embodiment of the present application, the cancer cell status recognition gene circuit includes at least a first promoter, a barcode region, and a second promoter connected in sequence and corresponding to a targeted cancer cell status, the first promoter including a positive promoter possessed by a cancer cell in the cancer cell status, and the second promoter including a negative promoter possessed by a cancer cell in the cancer cell status. The positive promoter and the negative promoter are also called as a strong enhancer and a weak enhancer or a positive enhancer and a negative enhancer, the former has positive enhancement effect on gene transcription, the latter has negative regulation effect on gene transcription, and the combination of the two can complete a complete transcription control process. Since both the positive and negative promoters are derived from a cancer cell in a certain cancer cell state, the cancer cell in the cancer cell state can be accurately identified, thereby achieving the technical effect of targeted identification.

The invention provides a cancer cell state identification gene circuit group, which comprises a first identification gene circuit and a second identification gene circuit which are used in a combined way, wherein the first identification gene circuit and the second identification gene circuit are used in a combined way to be matched with each other to identify cancer cells;

microRNA sequences are inserted into the first 3'UTR segment, the second 3' UTR segment, the third 3'UTR segment and the fourth 3' UTR segment, and the first promoter, the second promoter and the microRNA sequences are from cancer cells. Preferably, the first promoter is an active promoter in a cancer cell state.

The combination in the invention means that when the state of the cancer cell is identified, the carrier virus loaded with the first identification gene circuit and the carrier virus loaded with the second identification gene circuit are used successively or simultaneously to infect the target cancer cell in one identification, and the two identification gene circuits are used in combination, so that a more accurate identification effect is provided.

Preferably, the second promoter is an inactive promoter in a cancer cell state.

Preferably, the first gene identification circuit further comprises a second fluorescent protein sequence and a third fluorescent protein sequence, the second fluorescent protein sequence is arranged at the upstream of the first promoter, and the third fluorescent protein sequence is arranged at the downstream of the second promoter.

Preferably, the first signature circuit comprises a 3'UTR segment, a second fluorescent protein sequence, a first promoter, a barcode sequence, a second promoter, a fluorescent protein sequence and a 3' UTR segment connected in sequence.

Preferably, the first identifier circuit comprises a 3'UTR segment, a second fluorescent protein sequence, a first promoter, a barcode sequence, a CMV promoter, an mScarlet sequence, a WPRE, a second promoter, a fluorescent protein sequence, and a 3' UTR segment, connected in sequence.

Preferably, the second recognition gene circuit comprises a 3'UTR segment, a bait protein sequence, a GGGs protein sequence, a first fluorescent protein C-terminal sequence, a PGK promoter, a barcode sequence, a CMV promoter, a mSacrlet sequence, a WPRE, a PGK promoter, a capture protein sequence, a GGGs protein sequence, a first fluorescent protein N-terminal sequence and a 3' UTR segment which are connected in sequence.

Preferably, the first fluorescent protein sequence, the second fluorescent protein sequence, and the third fluorescent protein sequence are all different and different from the mScarlet sequence.

According to another aspect of the present invention, there is provided a method of preparing a cancer cell state recognition gene circuit, comprising the steps of:

step 2: compiling a first 3'UTR segment, a second 3' UTR segment, a third 3'UTR segment and a fourth 3' UTR segment, and inserting microRNA sequences into the first 3'UTR segment, the second 3' UTR segment, the third 3'UTR segment and the fourth 3' UTR segment;

and step 3: sequentially connecting the 3'UTR segment, the first promoter, the barcode sequence, the second promoter and the 3' UTR segment to obtain a first identification gene circuit;

and 4, step 4: and sequentially connecting the 3'UTR section, the bait protein sequence, the first fluorescent protein C-terminal sequence, the PGK promoter, the barcode sequence, the PGK promoter, the capture protein sequence, the first fluorescent protein N-terminal sequence and the 3' UTR section to obtain a second identification gene circuit, and respectively inserting the first identification gene circuit and the second identification gene circuit into the virus vector sequence to obtain the cancer cell state identification gene circuit group.

Preferably, in step 1, the first promoter is an active promoter in the cancer cell state and the second promoter is an inactive promoter in the cancer cell state.

The invention provides a cancer cell state identification gene circuit group, which comprises at least two cancer cell state identification gene circuits, wherein each cancer cell state identification gene circuit is respectively aimed at one cancer cell state. For example, for MHCC97H cancer cells, the cancer cell states that have been detected at present include 8 different cancer cell states, namely, highly invasive cells, cytoskeletal recombination active cells, highly dry cells, highly differentiated cells, fast-dividing periodic cells, slow-dividing periodic cells, immune escape cells, and anti-apoptotic cells, and of course, the cancer cell state recognition circuit in the embodiment of the present invention may be all possible cell states of a certain cancer cell line, or only some of them.

Taking malignant metastatic non-small cell lung cancer H1299 cells as an example, the first promoter sequence can be the following SEQ ID No.1, specifically:

SEQ ID NO.1：gacttgcaaagggccgactgaccctggtggccaagacagaagagatgtgggtttcctgccaaagatattgccacctccaggaaattgccagtgagctggaagttcccactattacaagccataaggccatgttgccatggacaccagaatatctgtagtcagagcacctatcagttgcaaaagccatgcctgcaaccgatggaaaatgtaagagggagttcttaaggttcttggtggcatcacccaaggcattctgggaaaacctagggcctggccccaaaacttccctactctgtggctagtcctgctgccaacaaaatcgtagcgacctggcttttcacagctttgcttttatttccaagtcaaggacaagccgctttgtgatattcctcctgggcattcttcttcaaatactgtgatattccttgctttccagggagaatgtgcttggcaaggtctggagaactaattcagaatcttaggggaaggggagagatggaaatacaaacctgcttactggaaaggtgcaaatatatgggttgagctggaggtaggaatacaggtaattaaggtttctagtttaagggaaaacagatctattgccatttaaataaggtaactgggatttggttaagttcacaaagatagcagaagatttatttacaggccctatcagttgcaaatactgtcagggcaagagggtaaaccagctacagcagtttaccagtgtgatggctgtgacacagctccactccacgggtggacacagcagagggcaactgggctggcctggaagtttgtgaatcaaaccgcttaactggcttatggtacatgtgattttcttttgtgagccttacaccaagccaaactattgtcaaagcatcatttctatagaaataaagccttatcttgacctgttctattaaaacctgccacacccgccctttcctacctagatttaatgagcccaagtttttaaaatggaagaaatgactctggggcaaagacccctaatgaactagtggcagagccaggaataacaaagaagtaactaatgagtccaaaatgggcagagtatgcaaaaaccttaagtggaaaccaaatagaccctggtatcatattaatagaagtttctggttggggtgatctaggttcaacagaaataagatgaggagtgtaaagtataaagccatttaagaacagagtagggtgggaaagcaaaaaaataaccagctctgaacaggtaacagctacatggtgactgagtctatgggcaaaagttcttgcatcacaggcttttgggactagcctatcacagggccctgtacaaataaacttggctgcaatcccagctctccctctgatgttgtgtgaccttaaggagtgtaaatggcaccttatcagggtcacttgggtatgagcattggatattcccatccctacctcagtaactga。

taking malignant metastatic non-small cell lung cancer H1299 cells as an example, the second promoter sequence can be the following SEQ ID No.2, specifically:

SEQ ID NO.2：tagacgaggctttgtttgactccgtgtagcgacaacaagagaaacaaaactacctatttgtaacggacgtgctgccattgccctccgcattgagcgcctacctattgaaatctttacgtcgggacaatgggagagcggctaaaattaccctcttgggtcctgggcgggcaagattcctgagcccctacccccgcccccatctcatcctcctctaacccgggccttgctgggctcccccttccccagtcccggccgccttctcccagtgtgcgctgcctgcacctgtgcctggagagcatcgaccccgcctcccaggccttgagcccctttgcggcgcagccccagccttgcgcggcctgggctttgcggccaccacaatggaaatctacggggaaaatgccaggcgtgggtggttctgctggagtcctgggaactctgcgtgggagggagtttgtgactgcggcccaaaagccacctccttattttccgtgggatgccaggaagttgaaatcaccctccctagacgaggctttgtttgactccgtgtagcgacaacaagagaaacaaaactacctatttgtaacggacgtgccacgaccgaaacccttcttacggggacagccccgggaatggggagtgggggcagacagtagaagcatcccctttgctacggttgaatgaagacagtctagtgggagatggctaagaggaagagctgcagtttcctgggccaaagagctgagttggacagggagatgcgtggggcttaccaaggcctgctggttctcagctctagagtctgccttatggtccgagcaggatttatttttaagaacagagcaagttacgtggaagcaaggaaggttttgaggacagaggtttgggtctcctaacttctagtcgggactgtgagaagggcgaggttggcacctgtaaggtaagagaggagagcggaagagcgcagtacgggagtggctgggggctggagttgggggggagtgctgtggatgagcgggagaacaatgacacaccaactcctgcactggctgtttccagaaatacgagttggacagcccttagctgcgagccacccactgtgccctgccccacccccgcaccttagctgcttcccgcgtcccatcctcatttaagtaccctgcaccaatgtatcaatattaagtttaaagaaaaaaaaacccacgtagtcttagtgctgtttacccacttccttcgaaaaggcgtgtggtgtgacctgttgctgcgagaggggatacaaaggtttctcagtggctggcaggctggctctgggagcctcctaggggatctcgcctgccccctcctcccccggcctcccccgcgcggccggcggcgcgggaggccccgccccctttcatgcaaaacccggcagcggagcctcctccggctcgagtggaggagccgccgcgcgctgattggtcgctagaaacccatttattccctgacagcccccgtcacatgg。

taking malignant metastatic non-small cell lung cancer H1299 cells as an example, the microRNA sequence can be SEQ ID NO.3 or SEQ ID NO.4 as follows, specifically:

SEQ ID NO.3：tccatcatacccatgcagtatta；

SEQ ID NO.4：actccatacaccactacctca。

the bait protein sequence and the prey protein sequence in the examples of the present application may preferably be SEQ ID No.5 and SEQ ID No.6 below, wherein the bait protein sequence is one of the sequences and the prey protein sequence is the other, in particular:

SEQ ID NO.5：atggatcccgggcagcagccgccgcccgggcagcaaccggccccgggcagcaggccaagggcagccgccttcgcagcccccgcaggggcagggctcccgggcgggcagcacccgggcaaccggcacccgcggccaccccggcgccgcaggcaccccccgcacccatactacgtccgcggggactcatactaacctggaggcgctcttcaacgccgtcatgaaccccaagacggccaacgtgccccagaccgtgcccatgaggctccggaagctgcccgactccttcttcaagccgccggagcccaaatcccactcccgacaggccagtactgatgcaggcactgcaggagccctgactccacagcatgttcgagctcatactactctccagcttctctgcagttgggagctgtttctcctgggacactgacccccactggagtagtctctggcccagcagctacacccacagctcagcatcttcgacagtcttcttttgagatacctgatgatgtacctctgagaattgccagcccagcaggttgggagatggcaaagacatcttctggtcagagatacttcttaaatcacatcgatcagacattgccacaacacagcacagcaaaaccataagaacaagaccacctcttggctagacccaaggcttgaccctcgttttgccaggtatgggtggcagcaactccaaccagcagcaacagatgcgactgcagcaactgcagatggagaaggagaggctgcggctgaaacagcaagaactgcttcggcaggtgaggccacaggcaatgcggaatatcaatcccagcacagcaaattctccaaaatgtcaggagttagccctgcgtagccagttatactaacactggagcaggatggtgggactcaaaatccagtgtcttctcccgggatgtctcaggaatctgccaatactaggttgagaacaatgacgaccaataatggagaaggagaggctgcggctgaaacagcaagaactgcttcggcaggtgaggccacaggcaatgcggaataagctacagtgtccctcgaaccccagatgacttcctgaacagtgtggatgagatggatacagaaagcaggtgatactatcaaccaaagcaccctgccctcacagcagaaccgtttcccagactaccttgaagccattcctgggacaaatgtggaccttggaacactggaaggagatggaatgaacatagaaggagaggagctgatgccaagtctgcaggaagctttgagttctgacatccttaatgacatggagtctgttttggctgccaccaagctagataaagaaagctttcttacatggttatag；

SEQ ID NO.6：atggaggtcccgggggccggcaccattacctccaacgagtggagctctcccacctcccctgaggggagcaccgcctctgggggcagtcaggcactggacaagcccatcgacaatgacgcagagggcgtgtggagcccggatattgagcagagtttccaggaggccctcgcaggcccgccctgtggcaggcgcaaaatcatcctgtcggacgagggcaagatgtatggtcgcacgaagatgtccaggtgctggctcgtcgcaaagctcgcgagatccaggccaagctaaaggaccaggcagctaaggacaaggccctgcagagcatggaggatgtcgtctgcacagatcatctccgccacggccttccacagtagcccacggcgccctcgcccggggccccggccgcccagccacggcctcagggttttggcaaggagctttgccaggccaagccggaattcccatgatgtgaagcctttctctcagcaaacctatgctgtccagcctccgctgcctctgccagggtttgagtctcctgcagggcccgccccatcgccctctgcgcccccggcaaagggtgcatggcagggccgcagcgtggccagctccaagctctggatgttggagttctctgccttcctggagcagcagcaggacccggacacgtacaacaagcacctgttcgtgcacattggccagtccagcccaagctacagcgacccctacctcgaagccgtggacatccgccaaatctatgacaaattcccgccttaaaaagggtggactcaaggatctcttcgaacggggaccctccaatgccttttttcttgtgaagttctgggcagacctcaacaccaacatcgaggatgaaggcagggcaagctctatggggtctccagccagtatgagagccccgagaacatgatcatcacctgctccacgaaggtctgctctttcggcaagcaggtggtggagaaagttgagacagagtatgctcgctatgagaatggacactacgaactcttacaagatgtcaccggtccccgctctgtgagtacatgatcaacttcatccacaagctcaagcacctccctgagaagtacatgatgaacagcgtgctggagaacttcaccatcctgcaggtggtcaccaacagagaagatgtcaggagaccttgctgtgcattgcctatgtctttgaggtgtgtcccagtgggctccacggggctcagcaccacatctacaggctggtgaaacatctacgaatga。

in the implementation of the application, the bait protein sequence and the capture protein sequence are arranged by dividing the fluorescent protein sequence into two sections without fluorescence according to a certain rule, and respectively fusing the two sections with the bait protein sequence and the capture protein sequence, and only under the condition that the bait protein and the capture protein interact after translation, the two sections of incomplete fluorescent reporter proteins can form complete reporter proteins and emit fluorescence. Detection in the flow cytometer is performed in real time after collection of viable cells, and signals are captured as long as fluorescence is generated in the cells, so that neither weak-affinity binding nor transient binding is missed.

In an embodiment of the present application, the cancer cell status recognition gene circuit includes at least a first promoter, a barcode region, and a second promoter connected in sequence and corresponding to a targeted cancer cell status, the first promoter including a positive promoter possessed by a cancer cell in the cancer cell status, and the second promoter including a negative promoter possessed by a cancer cell in the cancer cell status. The positive promoter and the negative promoter have positive enhancement effect on gene transcription, and negative regulation effect on gene transcription, and can complete a complete transcription control process by combining the positive promoter and the negative promoter. Since both the positive and negative promoters are derived from a cancer cell in a certain cancer cell state, the cancer cell in the cancer cell state can be accurately identified, thereby achieving the technical effect of targeted identification.

In the embodiment of the present application, a specific implementation manner of constructing the first promoter and the second promoter based on the positive promoter and the negative promoter may be based on common knowledge in the art that the enhancer and the core sequence of the promoter are relatively close, and the first promoter and the second promoter including the corresponding enhancer may be prepared by using a technical means commonly used by those skilled in the art, such as overlapping the enhancer sequence with the CpG island sequence and performing protein modification. The present application is not particularly limited as to the specific manner of synthesizing the first promoter and the second promoter, and the means thereof is well known to those skilled in the art.

The barcode sequence in the embodiment of the present application may be SEQ ID No.1, specifically:

SEQ ID NO.1：gggcgagcgcgcgcaaatggcggcgactagtgatatccgtctctcggcgctgtgctcgttccacacaccgg。

the cancer cell state recognition gene circuit group in the present application mainly refers to a group including at least two different cancer cell state recognition gene circuits, so that a group integrating cancer cell state recognition gene circuits for a plurality of different cancer cell states can distinguish cancer cells in different states in one test.

The invention can accurately calibrate the state of the cancer cells by obtaining the positive promoter and the negative promoter which can represent different phenotypes of the cancer cells to form the first promoter and the second promoter and combining the bar code regions which can assist in judgment, can analyze the proportion information of the cancer cells in various states by means of high-throughput cell experiments and the like, and is helpful for analyzing the heterogeneity among the cancer cells.

Preferably, the first promoter may be arranged to be single in protein binding region, while the second promoter may also be arranged to be single in protein binding region. The single protein binding region, namely the single binding site of the promoter, can ensure that the cancer cell state recognition gene circuit provided by the embodiment of the application can accurately position a target site and reduce the off-target rate. Here, the binding sites of the first promoter and the negative promoter, which are constructed in advance, may be queried and estimated based on the existing databases such as the JASPAR database and the NCBI database, and modified according to the query result so as to set the protein binding regions of the first promoter and the second promoter to be single.

Preferably, the cancer cell status recognition gene circuit further comprises a fluorescent protein sequence. After the infection is completed, cancer cells which need to be further distinguished and counted subsequently can be screened out according to the fluorescence effect of the infected cancer cells, and the workload required for overall identification is reduced.

In a preferred implementation manner, as shown in fig. 2, the fluorescent protein sequence, the first promoter, the barcode region and the second promoter are sequentially connected in the cancer cell status recognition gene circuit provided in the embodiment of the present application.

As shown in FIG. 2, the circuit of cancer cell status recognition gene further comprises a Lac promoter and a Lac terminator, and the fluorescent protein sequence, the Lac promoter, the first promoter, the barcode region, the second promoter and the Lac terminator are connected in sequence. More specifically, the cancer cell status recognition gene circuit provided in the embodiments of the present application includes a 3'UTR segment, a fluorescent protein sequence, a Lac promoter, a first promoter, a barcode region, a CMV promoter, a mscaret sequence, a WPRE, a second promoter, a Lac terminator, and a 3' UTR segment, which are sequentially connected.

According to another aspect of the present invention, as shown in FIG. 1, there is provided a method for preparing a cancer cell state recognition gene circuit, comprising the steps of:

step 1: obtaining a first promoter, a second promoter and a microRNA sequence of a cancer cell in a cancer cell state;

step 2, compiling a 3'UTR segment, and inserting a microRNA sequence into the 3' UTR segment;

and sequentially connecting the 3'UTR segment, the bait protein sequence, the first fluorescent protein C-terminal sequence, the PGK promoter, the barcode sequence, the PGK promoter, the capture protein sequence, the first fluorescent protein N-terminal sequence and the 3' UTR segment to obtain a second identification gene circuit.

In step 1, the method for obtaining the enhancer in the embodiment of the present invention may be a method that is described in a literature on a cancer cell line in which various cancer cell states have been ascertained, or a method that cancer cells in different cancer cell states are cultured by means of combination drug-resistant culture or the like, and the enhancer contained in the cancer cells in different cancer cell states is analyzed and obtained therefrom.

In step 2, the specific implementation manner of constructing the first promoter and the second promoter based on the positive promoter and the negative promoter may be based on the common knowledge in the art that the enhancer and the core sequence of the promoter are relatively close, and the first promoter and the second promoter including the corresponding promoters may be prepared by using the technical means commonly used by those skilled in the art, such as overlapping the enhancer sequence with the CpG island sequence and performing protein modification. The present application is not particularly limited as to the specific manner of synthesizing the first promoter and the second promoter, and the means thereof is well known to those skilled in the art.

The invention provides a cancer cell state identification gene circuit group and a preparation method thereof. microRNA sequences are inserted into the 3' UTR segment, and a positive promoter, a negative promoter and microRNA are from cancer cells in a cancer cell state. The invention can accurately mark the state of the cancer cells by obtaining the positive promoters, the negative promoters and the microRNAs which can represent different phenotypes of the cancer cells and combining the bar code regions which can assist in judgment, and can analyze the proportion information of the cancer cells in various states by means of high-throughput cell experiments and the like, thereby being beneficial to analyzing the heterogeneity among the cancer cells.

Hereinafter, the present invention will be described in detail with reference to examples. However, the embodiments according to the present specification may be modified into various other forms, and the scope of the present specification should not be construed as being limited to the embodiments described below. The examples of this specification are provided to more fully describe the specification to those skilled in the art.

Example (b):

materials:

cell:

MHCC97H human highly metastatic hepatoma cell line provided by Shanghai institute of cell biology, Chinese academy of sciences.

Drugs and reagents:

doxorubicin (Doxorubicin), specification: 10mg, batch number: KFS276, supplier: (ii) Baiolaibo.

Sorafenib (Sorafenib) specification: 10mg, batch number: bay 43-9006, supplier: gonghai Ruihui chemical technology Co., Ltd.

Los Wei-1640 (RPMI-1640) Medium and Fetal Bovine Serum (FBS), purchased from GIBCO, USA.

Doxycycline (Doxycycline hydrochloride), specification: 10mM/mL, batch number: ID0390-10mM × 1ml (in water), supplier: solibao.

Plasmids were synthesized from general organisms (Anhui), the classifier plasmid backbone used the SWB-Blas tidin lentiviral backbone, and the synthetic promoter vector backbone used SWP-Puromycin.

Lipofectamine 3000, Specification: 1mL, batch number: l3000015, supplier: ThermalFisher.

Lentivirus packaging kit, specification: 100mL, batch number: GM-040801-: is filled with organisms.

The instrument comprises the following steps:

CX41 inverted phase contrast microscope and BX51 fluorescence microscope, both available from Olympus, Japan; DTX880 ELISA, available from Beckman, USA; 751GD ultraviolet spectrophotometer, available from Hangzhou Hull instruments, Inc.; CytoFLEX flow cytometer available from beckmann coulter international trade (shanghai) ltd.

The implementation method comprises the following steps:

1. 8 plasmids of 8 phenotypic cell state identifiers were mixed in equal mass ratio as target mixed plasmids, and the cell state identifier mixed plasmids were subjected to lentivirus packaging by co-transfecting HEK293T cells with 4 plasmids in a 10cm cell culture dish using a third generation lentivirus packaging system at a transfection ratio of gag/pol: rev: vsv-g: target plasmid 5:2:3:8, wherein gag/pol is a genome integrin, rev is a reverse transcriptase, and vsv-g is a packaging protein.

2. After successful lentivirus packaging, the lentivirus supernatant was collected and filtered through a 0.45 μm filter to remove cells and debris.

Note: the filter suggests the use of cellulose acetate or Polyethersulfone (PES) filters (low protein binding) and no nitrocellulose filter can be used.

3. The lentivirus supernatant (4 parts) and the 5 Xlentivirus concentrate (1 part) were mixed at a volume ratio of 4:1, left at 4 ℃ for 2 hours or overnight, and initially mixed every 30min for 3 times.

Note: the longer the period of time at 4 ℃ (e.g., overnight), the higher the yield of virus may be; viral particles can be stored stably in a concentrate for several days, typically at 4 ℃.

4. Centrifuge at 4000g for 25min at 4 ℃.

5. The supernatant was carefully removed without vigorous shaking of the tube, and a white precipitate was generally visible (sometimes the precipitate was not visible).

6. The appropriate volume of DMEM (1/10 volume of the original supernatant) was added, carefully pipetted and the pellet resuspended.

7. The resuspended virus was dispensed in 50. mu.L tubes and stored in a freezer at 80 ℃.

The frozen stock solution of lentivirus can not be frozen and thawed repeatedly, otherwise the virus titer can be reduced.

MHCC97H cells were infected with packaged lentiviruses in 8 wells of a six-well plate with lentiviruses of different cell status identifiers, infected and antibiotic-screened (Puromycin resistance screen, 2. mu.g/mL) for about 10 days. Then, the targeting drug Sorafenib is used for adding drugs to MHCC97H cells stably expressing identifiers of different cell states for 10 days, 20 days and 30 days, the cells expressing EYFP fluorescent protein (excitation light wavelength 513nm and emission light wavelength 527nm) are analyzed and sorted by flow cytometry, genomes of the cells are extracted, barcode regions such as a barcode region sequence recorded by SEQ ID NO.1 are amplified by setting amplification primers, second-generation NGS150bp single-end sequencing is carried out on amplified fragments, the ratio of each phenotype is obtained by comparing the barcode region sequence with a set phenotype characterization barcode region sequence through bowtie2 and calculating the abundance of the barcode region, and the ratio data of the phenotype of each phenotype are obtained by the following steps:

based on the above data, and the principle of maximum coverage of each subset, candidate promoters for sample 7, sample 8, and sample 9 were selected as driving elements for subsequent targeting of therapeutic gene circuits of multiple resistant subsets in the case of a three-promoter configuration; candidate promoters for sample 6, sample 7, sample 8, and sample 9 were selected as driving elements for subsequent targeting of therapeutic gene circuits of multiple resistance subpopulations in the case of a four promoter configuration.

According to the invention, a plurality of different preselected promoters are obtained by carrying out drug-resistant culture on cancer cells, the preselected promoters are used for constructing the promoter test lentivirus, and different cell phenotype state estimators are combined, so that a dual stable transgenic cancer cell line is obtained by infecting the cancer cells respectively by using the cell phenotype state estimator and the promoter test lentivirus, and a preselected promoter can be used for highly identifying various subtype cancer cell lines based on the drug-resistant culture result of the dual stable transgenic cancer cell line, so that the preselected promoter can be used as a driving element in a gene circuit constructed subsequently.

The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the invention herein disclosed is not limited to the particular combination of features described above, but also encompasses other arrangements formed by any combination of the above features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.

Sequence listing

<110> Zhuhaizhongke advanced technology research institute Co., Ltd

<120> cancer cell state identification gene circuit group and preparation method thereof

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ataaggccat gttgccatgg acaccagaat atctgtagtc agagcaccta tcagttgcaa 180

aagccatgcc tgcaaccgat ggaaaatgta agagggagtt cttaaggttc ttggtggcat 240

cacccaaggc attctgggaa aacctagggc ctggccccaa aacttcccta ctctgtggct 300

agtcctgctg ccaacaaaat cgtagcgacc tggcttttca cagctttgct tttatttcca 360

agtcaaggac aagccgcttt gtgatattcc tcctgggcat tcttcttcaa atactgtgat 420

attccttgct ttccagggag aatgtgcttg gcaaggtctg gagaactaat tcagaatctt 480

aggggaaggg gagagatgga aatacaaacc tgcttactgg aaaggtgcaa atatatgggt 540

tgagctggag gtaggaatac aggtaattaa ggtttctagt ttaagggaaa acagatctat 600

tgccatttaa ataaggtaac tgggatttgg ttaagttcac aaagatagca gaagatttat 660

ttacaggccc tatcagttgc aaatactgtc agggcaagag ggtaaaccag ctacagcagt 720

ttaccagtgt gatggctgtg acacagctcc actccacggg tggacacagc agagggcaac 780

tgggctggcc tggaagtttg tgaatcaaac cgcttaactg gcttatggta catgtgattt 840

tcttttgtga gccttacacc aagccaaact attgtcaaag catcatttct atagaaataa 900

agccttatct tgacctgttc tattaaaacc tgccacaccc gccctttcct acctagattt 960

aatgagccca agtttttaaa atggaagaaa tgactctggg gcaaagaccc ctaatgaact 1020

agtggcagag ccaggaataa caaagaagta actaatgagt ccaaaatggg cagagtatgc 1080

aaaaacctta agtggaaacc aaatagaccc tggtatcata ttaatagaag tttctggttg 1140

gggtgatcta ggttcaacag aaataagatg aggagtgtaa agtataaagc catttaagaa 1200

cagagtaggg tgggaaagca aaaaaataac cagctctgaa caggtaacag ctacatggtg 1260

actgagtcta tgggcaaaag ttcttgcatc acaggctttt gggactagcc tatcacaggg 1320

ccctgtacaa ataaacttgg ctgcaatccc agctctccct ctgatgttgt gtgaccttaa 1380

ggagtgtaaa tggcacctta tcagggtcac ttgggtatga gcattggata ttcccatccc 1440

tacctcagta actga 1455

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gggacaatgg gagagcggct aaaattaccc tcttgggtcc tgggcgggca agattcctga 180

gcccctaccc ccgcccccat ctcatcctcc tctaacccgg gccttgctgg gctccccctt 240

ccccagtccc ggccgccttc tcccagtgtg cgctgcctgc acctgtgcct ggagagcatc 300

gaccccgcct cccaggcctt gagccccttt gcggcgcagc cccagccttg cgcggcctgg 360

gctttgcggc caccacaatg gaaatctacg gggaaaatgc caggcgtggg tggttctgct 420

ggagtcctgg gaactctgcg tgggagggag tttgtgactg cggcccaaaa gccacctcct 480

tattttccgt gggatgccag gaagttgaaa tcaccctccc tagacgaggc tttgtttgac 540

tccgtgtagc gacaacaaga gaaacaaaac tacctatttg taacggacgt gccacgaccg 600

aaacccttct tacggggaca gccccgggaa tggggagtgg gggcagacag tagaagcatc 660

ccctttgcta cggttgaatg aagacagtct agtgggagat ggctaagagg aagagctgca 720

gtttcctggg ccaaagagct gagttggaca gggagatgcg tggggcttac caaggcctgc 780

tggttctcag ctctagagtc tgccttatgg tccgagcagg atttattttt aagaacagag 840

caagttacgt ggaagcaagg aaggttttga ggacagaggt ttgggtctcc taacttctag 900

tcgggactgt gagaagggcg aggttggcac ctgtaaggta agagaggaga gcggaagagc 960

gcagtacggg agtggctggg ggctggagtt gggggggagt gctgtggatg agcgggagaa 1020

caatgacaca ccaactcctg cactggctgt ttccagaaat acgagttgga cagcccttag 1080

ctgcgagcca cccactgtgc cctgccccac ccccgcacct tagctgcttc ccgcgtccca 1140

tcctcattta agtaccctgc accaatgtat caatattaag tttaaagaaa aaaaaaccca 1200

cgtagtctta gtgctgttta cccacttcct tcgaaaaggc gtgtggtgtg acctgttgct 1260

gcgagagggg atacaaaggt ttctcagtgg ctggcaggct ggctctggga gcctcctagg 1320

ggatctcgcc tgccccctcc tcccccggcc tcccccgcgc ggccggcggc gcgggaggcc 1380

ccgccccctt tcatgcaaaa cccggcagcg gagcctcctc cggctcgagt ggaggagccg 1440

ccgcgcgctg attggtcgct agaaacccat ttattccctg acagcccccg tcacatgg 1498

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<213> Homo sapiens

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atggatcccg ggcagcagcc gccgcccggg cagcaaccgg ccccgggcag caggccaagg 60

gcagccgcct tcgcagcccc cgcaggggca gggctcccgg gcgggcagca cccgggcaac 120

cggcacccgc ggccaccccg gcgccgcagg caccccccgc acccatacta cgtccgcggg 180

gactcatact aacctggagg cgctcttcaa cgccgtcatg aaccccaaga cggccaacgt 240

gccccagacc gtgcccatga ggctccggaa gctgcccgac tccttcttca agccgccgga 300

gcccaaatcc cactcccgac aggccagtac tgatgcaggc actgcaggag ccctgactcc 360

acagcatgtt cgagctcata ctactctcca gcttctctgc agttgggagc tgtttctcct 420

gggacactga cccccactgg agtagtctct ggcccagcag ctacacccac agctcagcat 480

cttcgacagt cttcttttga gatacctgat gatgtacctc tgagaattgc cagcccagca 540

ggttgggaga tggcaaagac atcttctggt cagagatact tcttaaatca catcgatcag 600

acattgccac aacacagcac agcaaaacca taagaacaag accacctctt ggctagaccc 660

aaggcttgac cctcgttttg ccaggtatgg gtggcagcaa ctccaaccag cagcaacaga 720

tgcgactgca gcaactgcag atggagaagg agaggctgcg gctgaaacag caagaactgc 780

ttcggcaggt gaggccacag gcaatgcgga atatcaatcc cagcacagca aattctccaa 840

aatgtcagga gttagccctg cgtagccagt tatactaaca ctggagcagg atggtgggac 900

tcaaaatcca gtgtcttctc ccgggatgtc tcaggaatct gccaatacta ggttgagaac 960

aatgacgacc aataatggag aaggagaggc tgcggctgaa acagcaagaa ctgcttcggc 1020

aggtgaggcc acaggcaatg cggaataagc tacagtgtcc ctcgaacccc agatgacttc 1080

ctgaacagtg tggatgagat ggatacagaa agcaggtgat actatcaacc aaagcaccct 1140

gccctcacag cagaaccgtt tcccagacta ccttgaagcc attcctggga caaatgtgga 1200

ccttggaaca ctggaaggag atggaatgaa catagaagga gaggagctga tgccaagtct 1260

gcaggaagct ttgagttctg acatccttaa tgacatggag tctgttttgg ctgccaccaa 1320

gctagataaa gaaagctttc ttacatggtt atag 1354

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atggaggtcc cgggggccgg caccattacc tccaacgagt ggagctctcc cacctcccct 60

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gagggcgtgt ggagcccgga tattgagcag agtttccagg aggccctcgc aggcccgccc 180

tgtggcaggc gcaaaatcat cctgtcggac gagggcaaga tgtatggtcg cacgaagatg 240

tccaggtgct ggctcgtcgc aaagctcgcg agatccaggc caagctaaag gaccaggcag 300

ctaaggacaa ggccctgcag agcatggagg atgtcgtctg cacagatcat ctccgccacg 360

gccttccaca gtagcccacg gcgccctcgc ccggggcccc ggccgcccag ccacggcctc 420

agggttttgg caaggagctt tgccaggcca agccggaatt cccatgatgt gaagcctttc 480

tctcagcaaa cctatgctgt ccagcctccg ctgcctctgc cagggtttga gtctcctgca 540

gggcccgccc catcgccctc tgcgcccccg gcaaagggtg catggcaggg ccgcagcgtg 600

gccagctcca agctctggat gttggagttc tctgccttcc tggagcagca gcaggacccg 660

gacacgtaca acaagcacct gttcgtgcac attggccagt ccagcccaag ctacagcgac 720

ccctacctcg aagccgtgga catccgccaa atctatgaca aattcccgcc ttaaaaaggg 780

tggactcaag gatctcttcg aacggggacc ctccaatgcc ttttttcttg tgaagttctg 840

ggcagacctc aacaccaaca tcgaggatga aggcagggca agctctatgg ggtctccagc 900

cagtatgaga gccccgagaa catgatcatc acctgctcca cgaaggtctg ctctttcggc 960

aagcaggtgg tggagaaagt tgagacagag tatgctcgct atgagaatgg acactacgaa 1020

ctcttacaag atgtcaccgg tccccgctct gtgagtacat gatcaacttc atccacaagc 1080

tcaagcacct ccctgagaag tacatgatga acagcgtgct ggagaacttc accatcctgc 1140

aggtggtcac caacagagaa gatgtcagga gaccttgctg tgcattgcct atgtctttga 1200

ggtgtgtccc agtgggctcc acggggctca gcaccacatc tacaggctgg tgaaacatct 1260

acgaatga 1268

<210> 7

<211> 71

<212> DNA

<213> Escherichia coli

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gggcgagcgc gcgcaaatgg cggcgactag tgatatccgt ctctcggcgc tgtgctcgtt 60

ccacacaccg g 71

Claims

1. A cancer cell state identification gene circuit group is characterized by comprising a first identification gene circuit and a second identification gene circuit which are used in combination, wherein the first identification gene circuit and the second identification gene circuit are used in combination to identify cancer cells;

2. The circuit set of cancer cell status recognition genes of claim 1, wherein the first promoter is an active promoter in the cancer cell.

3. The circuit set of cancer cell status recognition genes according to claim 1, wherein said second promoter is an inactive promoter in said cancer cell.

4. The circuit panel of claim 1, wherein the barcode sequence is represented by SEQ ID No.1, one of the capture protein sequence and the bait protein sequence is represented by SEQ ID No.5, and the other is represented by SEQ ID No. 6.

5. The set of cancer cell status identifier gene circuits of claim 1 wherein the first identifier gene circuit further comprises a second fluorescent protein sequence disposed between the first 3'UTR segment and the first promoter and a third fluorescent protein sequence disposed between the second promoter and the second 3' UTR segment.

6. The set of cancer cell status recognition gene circuits of claim 5, wherein the first recognition gene circuit comprises a first 3'UTR segment, a second fluorescent protein sequence, a first promoter, a barcode sequence, a CMV promoter, a mScarlet sequence, a WPRE, a second promoter, a third fluorescent protein sequence, and a second 3' UTR segment connected in sequence.

7. The set of cancer cell status recognition gene circuits of claim 6, wherein the second recognition gene circuit comprises a third 3'UTR segment, a bait protein sequence, a GGGs protein sequence, a first fluorescent protein C-terminal sequence, a PGK promoter, a barcode sequence, a CMV promoter, a mScalet sequence, a WPRE, a PGK promoter, a capture protein sequence, a GGGs protein sequence, a first fluorescent protein N-terminal sequence, and a fourth 3' UTR segment connected in sequence.

8. The circuit set of cancer cell status recognition genes of claim 7, wherein the first fluorescent protein sequence, the second fluorescent protein sequence, the third fluorescent protein sequence, and the mScarlet sequence are all different.

9. The method for preparing a circuit set for identifying a cancer cell state according to any one of claims 1 to 8, comprising the steps of:

10. The method for preparing a circuit set for identifying a cancer cell state according to claim 9, wherein in the step 1, the first promoter is an active promoter in the cancer cell, and the second promoter is an inactive promoter in the cancer cell.