CN111269324B

CN111269324B - Fusion protein of Gauss luciferase and digoxin single-chain antibody and application thereof

Info

Publication number: CN111269324B
Application number: CN201811475418.2A
Authority: CN
Inventors: 董宇亮; 王佑富; 李静; 马旭妍; 郑越; 章文蔚; 徐崇钧
Original assignee: Qingdao Huada Zhizao Technology Co ltd
Current assignee: Qingdao Huada Zhizao Technology Co ltd
Priority date: 2018-12-04
Filing date: 2018-12-04
Publication date: 2023-10-20
Anticipated expiration: 2038-12-04
Also published as: CN111269324A

Abstract

A fusion protein of a Gaussian luciferase and a digoxin single-chain antibody and application thereof. The fusion protein comprises a heavy chain variable region, a light chain variable region and a Gaussian luciferase or persistent luminescence mutant of a digoxin single-chain antibody, wherein the heavy chain variable region and the light chain variable region are connected through a first flexible peptide, and the Gaussian luciferase or the persistent luminescence mutant is connected with the heavy chain variable region or the light chain variable region through a second flexible peptide. The fusion protein disclosed by the invention retains the activity of two parts of proteins, can be specifically combined with digoxin on one hand, can play the self-luminescence advantage of luciferase on the other hand, realizes self-luminescence detection while specifically combining with target digoxin, and can be applied to clinical detection or related scientific research.

Description

Fusion protein of Gauss luciferase and digoxin single-chain antibody and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a fusion protein of a Gaussian luciferase and a digoxin single-chain antibody and application thereof.

Background

Luciferase (Luciferase) is a generic term for enzymes capable of producing bioluminescence in an organism. Are widely used as reporter genes due to their self-luminescent effect. Applications in detection and the like are also becoming more and more widespread. The classes of luciferases include: bacterial luciferases (Bacterial Luciferase, GLuc), firefly luciferases (Firefly Luciferase, FLuc), renilla luciferases (Renilla Luciferase, RLuc) and gaussian luciferases (Gaussia Luciferase, GLuc). Wherein the luminescence activity of the Gaussian luciferase is more than 100 times higher than that of other types of luciferases. Gauss luciferase is a luciferase derived from marine radial organisms (Gaussia princps) and has a size of 19.9kDa, and only 185 amino acids, which can be secreted by cells to the outside, and only coelenterazine and oxygen are needed for reaction. Currently, gaussian luciferase is widely used as a reporter gene. Through coexpression of Gaussian luciferase and target proteins, research such as positioning tracing and the like can be performed on the target proteins. Since it is found later than other luciferases, it has less development and application. Particularly in detection applications. Therefore, the method has wide application prospect for development and application.

Digoxin is widely used as a cardiac glycoside drug for clinically treating heart failure and arrhythmia. Meanwhile, digoxin is widely applied to detection research as a molecular target. Especially in clinical diagnosis and scientific research detection experiments, digoxin is used as a target or a marker, and further research and application in molecular diagnosis or molecular positioning are more and more performed by utilizing digoxin antibody specificity to recognize the target.

Currently, digoxin antibody is mostly used as a diagnosis or detection means, digoxin target is identified by the digoxin antibody, then the digoxin antibody is identified by a secondary antibody for amplification, and finally a color reaction is carried out by using a label carried on the secondary antibody. Such that the detection requires two or more steps to complete. Yet another way is to add a fluorescent tag to the digoxin antibody. The fluorescence label can emit fluorescence to be received by the detector after absorbing excitation light, and the detection process requires hardware facilities such as a fluorescence microscope, so that the application is limited. Currently, there is no product on the market with fluorescent labels on digoxin antibodies. But may have similar functions by combination. After modifying the digoxin antibody by Biotin (Biotin), the digoxin antibody can be specifically identified and self-luminous detection can be realized by amplifying by using an SA-GLU coupling product. However, this antibody combination method is cumbersome and time-consuming.

Disclosure of Invention

The invention provides a fusion protein of a Gaussian luciferase and a digoxin single-chain antibody and application thereof, and the fusion protein disclosed by the invention keeps the activities of two parts of proteins, can be specifically combined with digoxin on one hand, can exert the self-luminous advantage of the luciferase on the other hand, realizes self-luminous detection while specifically combining with a target digoxin, and can be applied to clinical detection or related scientific research.

According to a first aspect, in one embodiment there is provided a fusion protein of a gaussian luciferase and a digoxin single chain antibody, the fusion protein comprising a heavy chain variable region of a digoxin single chain antibody, a light chain variable region, and a gaussian luciferase or persistent luminescent mutant, the heavy chain variable region and the light chain variable region being linked by a first flexible peptide, the gaussian luciferase or persistent luminescent mutant and the heavy chain variable region or the light chain variable region being linked by a second flexible peptide.

In a preferred embodiment, the heavy chain variable region sequence is as set forth in SEQ ID NO: 1.

In a preferred embodiment, the light chain variable region sequence is as set forth in SEQ ID NO: 2.

In a preferred embodiment, the above gaussian luciferase has the sequence shown in SEQ ID NO: 3.

In a preferred embodiment, the sequence of the persistent luminescence mutant is as shown in SEQ ID NO: 4.

In a preferred embodiment, each of said first flexible peptide and said second flexible peptide is independently selected from the group consisting of SEQ ID NOs: 5 and SEQ ID NO: 6.

In a preferred embodiment, the first flexible peptide is SEQ ID NO:5, a sequence shown in seq id no; the second flexible peptide is seq id NO: 6.

In a preferred embodiment, the fusion protein further comprises: a signal peptide for directing the secretion of the above fusion protein from the inside of the expressing cell.

In a preferred embodiment, the signal peptide is located at the N-terminus of the fusion protein.

In a preferred embodiment, the signal peptide sequence is as set forth in SEQ ID NO: shown at 7.

In a preferred embodiment, the fusion protein further comprises: fusion tags for affinity purification of the fusion proteins.

In a preferred embodiment, the fusion tag is a 6×histidine tag.

In a preferred embodiment, the fusion tag is located at the C-terminus of the fusion protein.

In a preferred embodiment, the sequence of the fusion protein is as set forth in SEQ ID NO:8 or SEQ ID NO: shown at 9.

According to a second aspect, in one embodiment there is provided an isolated nucleic acid encoding the fusion protein of the first aspect.

In a preferred embodiment, the isolated nucleic acid comprises a heavy chain variable region coding sequence, a light chain variable region coding sequence, and a gaussian luciferase coding sequence or a persistent light emitting mutant coding sequence of a digoxin single chain antibody, wherein the heavy chain variable region coding sequence and the light chain variable region coding sequence are linked by a first linker sequence, and wherein the gaussian luciferase coding sequence or the persistent light emitting mutant coding sequence and the heavy chain variable region coding sequence or the light chain variable region coding sequence are linked by a second linker sequence.

In a preferred embodiment, the heavy chain variable region coding sequence is as set forth in SEQ ID NO: shown at 10.

In a preferred embodiment, the light chain variable region coding sequence is as set forth in SEQ ID NO: 11.

In a preferred embodiment, the coding sequence of the Gaussian luciferase is shown as SEQ ID NO: shown at 12.

In a preferred embodiment, the above-mentioned continuous luminescence mutant has a coding sequence as shown in SEQ ID NO: shown at 13.

In a preferred embodiment, each of said first and said second linker sequences is independently selected from the group consisting of SEQ ID NOs: 14 and SEQ ID NO: 15.

In a preferred embodiment, the first linking sequence is SEQ ID NO:14, a sequence shown in seq id no; the second connecting sequence is SEQ ID NO: 15.

In a preferred embodiment, the isolated nucleic acid further comprises: a signal peptide coding sequence encoding a signal peptide for directing secretion of the fusion protein from within the expressing cell.

In a preferred embodiment, the signal peptide coding sequence is located 5' to the isolated nucleic acid.

In a preferred embodiment, the signal peptide has a coding sequence as set forth in SEQ ID NO: shown at 16.

In a preferred embodiment, the isolated nucleic acid further comprises a fusion tag coding sequence for encoding a fusion tag for affinity purification of the fusion protein.

In a preferred embodiment, the fusion tag coding sequence is a sequence encoding a 6×histidine tag.

In a preferred embodiment, the fusion tag coding sequence is located at the 3' end of the isolated nucleic acid.

In a preferred embodiment, the isolated nucleic acid sequence is set forth in SEQ ID NO:17 or SEQ ID NO: shown at 18.

According to a third aspect, in one embodiment there is provided an expression vector comprising a nucleic acid sequence encoding the fusion protein of the first aspect, and a vector backbone sequence.

According to a fourth aspect, in one embodiment there is provided a recombinant host cell comprising the expression vector of the third aspect.

According to a fifth aspect, there is provided in an embodiment the use of the fusion protein of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect or the recombinant host cell of the fourth aspect in digoxin molecule detection.

The fusion protein of the Gaussian luciferase and the digoxin single-chain antibody can specifically identify digoxin on one hand and has a self-luminous function on the other hand, and is convenient to detect. Compared with the existing mode of detecting digoxin through coupling amplification, the fusion protein has the advantages of simple process, convenience in production, simplicity and convenience in use and shorter time consumption.

Drawings

FIG. 1 is a schematic flow chart of the construction principle of a Fusion Protein expression vector in the embodiment of the invention, wherein a heavy chain (H chain) variable region coding sequence of a digoxin single-chain antibody and a light chain (L chain) variable region coding sequence (comprising a Signal peptide coding sequence) are connected through a first connecting sequence (Linker 1), and the heavy chain (H chain) variable region coding sequence of the digoxin single-chain antibody and a Gaussian luciferase (Gluc) coding sequence are connected through a second connecting sequence (Linker 2) to obtain a Fusion Protein (Fusion Protein) coding sequence, and then the Fusion Protein coding sequence is integrally connected into a vector skeleton sequence pcDNA3.1 (+) to obtain a recombinant Fusion Protein expression vector.

FIG. 2 is a SDS-PAGE identification and Western blot identification chart of anti DIG scFv-GLuc fusion proteins in the embodiment of the invention, lane 1: anti DIG scFv-GLuc fusion protein, lane M: a marker.

FIG. 3 is a diagram showing experimental results of ELISA detection of digoxin binding force of an anti-DIG scFv-GLuc fusion protein antibody part in the embodiment of the invention, wherein anti-DIG scFv-GLuc represents a wild-type fusion protein, anti-DIG scFv-G2L represents a mutant fusion protein, anti-DIG scFv represents a digoxin single-chain antibody, GLuc represents a Gaussian luciferase, G2L represents a continuous luminescence mutant of the Gaussian luciferase, and the abscissa represents a Concentration (Concentration) value and the ordinate represents an OD450nm absorbance value.

FIG. 4 is a diagram showing the results of bioluminescence assay of an anti DIG scFv-GLuc fusion protein in the example of the present invention, wherein anti DIG scFv-GLuc represents the fusion protein, anti DIG scFv-G2L represents the mutant fusion protein, anti DIG scFv represents the digoxin single chain antibody, GLuc represents the Gaussian luciferase, G2L represents the Gaussian luciferase continuous luminescence mutant, the abscissa represents Time (Time), and the ordinate represents luminescence intensity (CL values).

FIG. 5 is a diagram showing the results of an experiment for detecting the bifunctional activity of an anti DIG scFv-GLuc fusion protein in the embodiment of the invention, wherein anti DIG scFv-GLuc represents the fusion protein, anti DIG scFv-G2L represents the mutant fusion protein, anti DIG scFv represents the digoxin single-chain antibody, GLuc represents the Gaussian luciferase, G2L represents the continuous luminescence mutant of the Gaussian luciferase, the abscissa represents the Concentration (Concentration) value, and the ordinate represents the luminescence intensity (CL values).

Detailed Description

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

Herein, "first," "second," etc. are used merely to distinguish between the described objects, and do not have any sequential or technical meaning.

The invention provides a fusion protein of a single-chain antibody of gauss luciferase and digoxin, which is an antibody fusion protein of targeting digoxin, and is based on the single-chain antibody of digoxin and gauss luciferase or a continuous luminescence mutant, and the single-chain antibody of digoxin and the gauss luciferase or the continuous luminescence mutant are connected through a flexible peptide through gene reconstruction.

Specifically, the fusion protein comprises a heavy chain (H chain) variable region, a light chain (L chain) variable region and a Gaussian luciferase (Gluc) or persistent luminescence mutant of the digoxin single-chain antibody, wherein the heavy chain variable region and the light chain variable region are connected through a first flexible peptide, and the Gaussian luciferase or the persistent luminescence mutant is connected with the heavy chain variable region or the light chain variable region through a second flexible peptide.

In the fusion protein of the present invention, there are various connection modes of the heavy chain (H chain) variable region, the light chain (L chain) variable region and Gauss luciferase (Gluc) or a persistent luminescence mutant of the digoxin single-chain antibody, for example, four connection modes of H+L+gluc, L+H+gluc, gluc+L+H or gluc+H+L may be sequentially arranged from the N end to the C end of the protein. In a preferred embodiment, an H+L+gluc linkage is used.

In a preferred embodiment, the heavy chain variable region sequence is as set forth in SEQ ID NO:1 is shown as follows:

QVQLLQSAAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASPSGFDYWGQGTLVTVSS(SEQID NO：1)。

in a preferred embodiment, the light chain variable region sequence is as set forth in SEQ ID NO:2 is shown as follows:

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRAVVFGGGTKLTVLGG(SEQ ID NO：2)。

in a preferred embodiment, the gaussian luciferase sequence is as follows SEQ ID NO:3, shown in the following:

KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD(SEQ ID NO：3)。

in a preferred embodiment, the persistent luminescent mutant sequence is as follows SEQ ID NO:4, as follows:

KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD(SEQ ID NO：4)。

it should be noted that, in addition to the fusion protein of the present invention using the above-mentioned SEQ ID NO: 1-3 or SEQ ID NO: 1-2 and SEQ ID NO:4, and a sequence form having the same function although the sequences are different from each other. For example, for the heavy chain variable region, it may be a sequence identical to SEQ ID NO:1 but also has the corresponding epitope properties, as in SEQ ID NO:1, adding or reducing a plurality of amino acids at two ends of the sequence but maintaining the same epitope characteristics; for the light chain variable region, it may be a sequence identical to SEQ ID NO:2 but also has the corresponding epitope properties, as in SEQ ID NO:2, adding or subtracting a plurality of amino acids at two ends of the sequence but maintaining the same epitope characteristic; for gaussian luciferase, it may be as defined in SEQ id no:3 but also has a sequence of gaussian luciferase activity as shown in SEQ ID NO:3, but maintains the same gaussian luciferase activity.

In the fusion protein of the present invention, the first flexible peptide and the second flexible peptide having a linking effect may be each independently selected from the following sequences:

GGGGSGGGGSGGS(SEQ ID NO：5)；

GGGGSGGGGS(SEQ ID NO：6)；

GQGQGQGQGQG(SEQ ID NO：19)；

GSTSGSGKSSEKGKG(SEQ ID NO：20)；

VPGVGVPGVG(SEQ ID NO：21)；

SAPGTPSR(SEQ ID NO：22)；

EGKSSGSGSESKEF(SEQ ID NO：23)；

GSGGSG(SEQ ID NO：24)。

the first flexible peptide and the second flexible peptide may be any one of the above sequences, and may be the same sequence or different sequences.

In a preferred embodiment, the first flexible peptide and the second flexible peptide are each independently selected from the following sequences:

GGGGSGGGGSGGS(SEQ ID NO：5)；GGGGSGGGGS(SEQ ID NO：6)。

the first flexible peptide and the second flexible peptide may be the same sequence or different sequences.

In a preferred embodiment, the fusion protein further comprises: a signal peptide for directing secretion of the fusion protein from within the expressing cell.

The signal peptide may be located at the N-terminus or the C-terminus of the fusion protein of the present invention, and in a preferred embodiment, the signal peptide is located at the N-terminus of the fusion protein without affecting the affinity activity and the luminescence activity in the fusion protein of the present invention.

In a preferred embodiment, the signal peptide sequence is as set forth in SEQ ID NO:7, as follows:

MGVKVLFALICIAVAEA(SEQ ID NO：7)。

in the present invention, methods useful for purifying fusion proteins include, but are not limited to: nickel column purification, ion exchange chromatography (Q column, P column), hydrophobic chromatography, heparin column chromatography, ammonium sulfate precipitation, etc.

In a preferred embodiment, the fusion protein of the invention further comprises: a fusion tag for affinity purification of the fusion protein. Specifically, the fusion tag is affinity-bound to a purification device (e.g., a purification column) used in various purification methods, and then the purified fusion protein is obtained by an appropriate method. The fusion tag may be located at the N-terminus or C-terminus, preferably at the C-terminus, of the fusion protein of the invention without affecting the antibody activity and the luminescent activity in the fusion protein of the invention.

The fusion tag can be a 6 XHis tag (6 XHis tag) or other fusion tags such as GST, MBP, SUMO, and the corresponding affinity purification method of the fusion tag is selected according to different fusion tags. In a preferred embodiment, the fusion tag is a 6 x histidine tag.

In a most preferred embodiment, the fusion protein of the invention has the following SEQ ID NO:8 or SEQ ID NO:9 is shown as follows:

MGVKVLFALICIAVAEAQVQLLQSAAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASPSGFDYWGQGTLVTVSSGGGGSGGGGS GGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRAVVFGGGTKLTVLGGGGGGSGGGGSKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDHHHHHH(SEQ ID NO：8)。

MGVKVLFALICIAVAEAQVQLLQSAAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASPSGFDYWGQGTLVTVSSGGGGSGGGGS GGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRAVVFGGGTKLTVLGGGGGGSGGGGSKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDHHHHHH(SEQ ID NO：9)。

wherein the underlined sequence [ ]MGVKVLFALICIAVAEA) Representing a signal peptide; underlined sequence [ ]GGGGSGGGGSGGS) A first flexible peptide representing a heavy chain (H chain) variable region and a light chain (L chain) variable region of a digoxin single-chain antibody; underlined sequence [ ]GGGGSGGGGS) A second flexible peptide representing a linkage of a light chain (L chain) variable region and gaussian luciferase (Gluc); underlined sequence [ ]HHHHHH) Represents a 6 Xhistidine tag sequence.

The fusion protein of the invention can be obtained by the following method: cloning and recombining the screened Gaussian luciferase or continuous luminous mutant gene and the digoxin single-chain antibody gene by utilizing a PCR technology, constructing a digoxin single-chain antibody/Gaussian luciferase or continuous luminous mutant fusion protein recombinant vector, electrically transferring the recombinant vector into a host expression cell (such as a CHO cell), integrating the fusion protein gene into a chromosome, and screening a stable cell strain of the high-expression fusion antibody by utilizing neomycin G418; expanding culture of stable cell strain, centrifuging at low temperature to obtain supernatant, and separating and purifying the supernatant with nickel column; then Western Blot identification, separation and purification are carried out to obtain fusion protein; ELISA experiments identify the affinity activity of the fusion protein antibody portion; enzyme activity experiments identified the self-luminescent activity of the luciferase fraction. The fusion proteins all well retain the activity of both protein portions.

An embodiment of the invention provides an isolated nucleic acid encoding a fusion protein of the invention.

In a preferred embodiment, the isolated nucleic acid comprises a heavy chain variable region coding sequence, a light chain variable region coding sequence, and a gaussian luciferase coding sequence or a persistent luminescent mutant coding sequence of a digoxin single chain antibody, the heavy chain variable region coding sequence and the light chain variable region coding sequence being linked by a first linker sequence, and the gaussian luciferase coding sequence or the persistent luminescent mutant coding sequence and the heavy chain variable region coding sequence or the light chain variable region coding sequence being linked by a second linker sequence.

The nucleotide sequences encoding the same polypeptide or protein may be different due to the degeneracy of the codons, so long as the isolated nucleic acids of the invention are capable of encoding the fusion proteins of the invention. The sequence is not particularly limited.

In a preferred embodiment, the heavy chain variable region coding sequence is as set forth in SEQ ID NO:10, as shown in:

CAGGTGCAGCTGCTGCAGAGCGCCGCTGAGGTGAAGAAGCCAGGCGAGTCCCTGAAGATCAGCTGTAAGGGCTCCGGCTATAGCTTCACATCTTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTATCCCGGCGACTCTGATACCCGCTACAGCCCTTCTTTTCAGGGCCAGGTGACCATCTCCGCTGATAAGTCCATCAGCACAGCCTATCTGCAGTGGTCCAGCCTGAAGGCTTCTGATACAGCCGTGTACTATTGCGCTAGGGCCTCTCCCTCCGGCTTTGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCC(SEQ ID NO：10)。

in a preferred embodiment, the light chain variable region coding sequence is as set forth in SEQ ID NO:11, as shown in:

CAGTCTGTGCTGACCCAGCCACCTTCCGCTAGCGGAACCCCTGGACAGAGGGTGACAATCTCTTGTTCCGGCAGCTCTTCCAACATCGGCTCTAATTACGTGTATTGGTACCAGCAGCTGCCTGGCACAGCCCCAAAGCTGCTGATCTATAGGAACAATCAGAGGCCATCCGGAGTGCCTGATCGGTTCAGCGGATCTAAGTCCGGCACCTCCGCCTCCCTGGCTATCTCTGGCCTGAGGTCCGAGGATGAGGCTGACTACTATTGCGCTGCTTGGGACGATTCCCTGAGGGCTGTGGTGTTTGGAGGAGGAACCAAGCTGACAGTGCTGGGCGGA(SEQ ID NO：11)。

in a preferred embodiment, the gaussian luciferase coding sequence is as set forth in SEQ ID NO:12, as shown in:

AAGCCAACAGAGAACAATGAGGACTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGATGGAGGCTAACGCCAGAAAGGCTGGCTGTACCCGCGGCTGCCTGATCTGTCTGTCCCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCCCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCAGAGATCCCCGGCTTCAAGGACCTGGAGCCTATGGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTAGCGACCTGCTGAAGAAGTGGCTGCCACAGCGGTGCGCTACCTTTGCCTCTAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGAC(SEQ ID NO：12)。

in a preferred embodiment, the bioluminescent mutant encodes the sequence set forth in SEQ ID NO:13, as shown in: AAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGCTCGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCCTCGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGAC(SEQ ID NO：13)。

In a preferred embodiment, the first and second linker sequences are each independently selected from the following sequences:

GGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGAGGAAGC(SEQ ID NO：14)；GGAGGAGGAGGATCCGGCGGAGGTGGAAGT(SEQ ID NO：15)。

the first and second linker sequences may be the same sequence or different sequences.

In a preferred embodiment, the first linking sequence is SEQ ID NO:14, a sequence shown in seq id no; the second linking sequence is SEQ id no: 15.

Since the signal peptide in the fusion protein of the present invention may be located at the N-terminus or the C-terminus of the fusion protein, the signal peptide coding sequence may be located at the 5 '-terminus or the 3' -terminus of the isolated nucleic acid. The signal peptide is located at the N-terminus of the fusion protein of the invention without affecting the affinity and luminescent activity of the fusion protein, and thus in a preferred embodiment, the signal peptide coding sequence is located at the 5' end of the isolated nucleic acid.

In a preferred embodiment, the signal peptide coding sequence is as set forth in SEQ ID NO:16, as shown in:

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCC(SEQ ID NO：16)。

in a preferred embodiment, the isolated nucleic acid further comprises a fusion tag coding sequence for encoding a fusion tag for affinity purification of the fusion protein described above.

In a preferred embodiment, the fusion tag coding sequence is a sequence encoding a 6 x histidine tag.

The fusion tag coding sequence may be located at the 3' end or the 5' end of the isolated nucleic acid, and in preferred embodiments, the fusion tag coding sequence is located at the 3' end of the isolated nucleic acid without affecting the antibody activity and expression of the light-emitting active moiety protein.

In a most preferred embodiment, the isolated nucleic acid sequence of the invention is as set forth in SEQ ID NO:17 or SEQ ID NO:18, as shown in:

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCCCAGGTGCAGCTGCTGCAGAGCGCCGCTGAGGTGAAGAAGCCAGGCGAGTCCCTGAAGATCAGCTGTAAGGGCTCCGGCTATAGCTTCACATCTTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTATCCCGGCGACTCTGATACCCGCTACAGCCCTTCTTTTCAGGGCCAGGTGACCATCTCCGCTGATAAGTCCATCAGCACAGCCTATCTGCAGTGGTCCAGCCTGAAGGCTTCTGATACAGCCGTGTACTATTGCGCTAGGGCCTCTCCCTCCGGCTTTGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCCGGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGAGGAAGCCAGTCTGTGCTGACCCAGCCACCTTCCGCTAGCGGAACCCCTGGACAGAGGGTGACAATCTCTTGTTCCGGCAGCTCTTCCAACATCGGCTCTAATTACGTGTATTGGTACCAGCAGCTGCCTGGCACAGCCCCAAAGCTGCTGATCTATAGGAACAATCAGAGGCCATCCGGAGTGCCTGATCGGTTCAGCGGATCTAAGTCCGGCACCTCCGCCTCCCTGGCTATCTCTGGCCTGAGGTCCGAGGATGAGGCTGACTACTATTGCGCTGCTTGGGACGATTCCCTGAGGGCTGTGGTGTTTGGAGGAGGAACCAAGCTGACAGTGCTGGGCGGAGGAGGAGGAGGATCCGGCGGAGGTGGAAGTAAGCCAACAGAGAACAATGAGGACTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGATGGAGGCTAACGCCAGAAAGGCTGGCTGTACCCGCGGCTGCCTGATCTGTCTGTCCCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCCCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCAGAGATCCCCGGCTTCAAGGACCTGGAGCCTATGGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTAGCGACCTGCTGAAGAAGTGGCTGCCACAGCGGTGCGCTACCTTTGCCTCTAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGACCACCATCACCATCACCAT(SEQ ID NO：17)。

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCCCAGGTGCAGCTGCTGCAGAGCGCCGCTGAGGTGAAGAAGCCAGGCGAGTCCCTGAAGATCAGCTGTAAGGGCTCCGGCTATAGCTTCACATCTTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTATCCCGGCGACTCTGATACCCGCTACAGCCCTTCTTTTCAGGGCCAGGTGACCATCTCCGCTGATAAGTCCATCAGCACAGCCTATCTGCAGTGGTCCAGCCTGAAGGCTTCTGATACAGCCGTGTACTATTGCGCTAGGGCCTCTCCCTCCGGCTTTGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCCGGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGAGGAAGCCAGTCTGTGCTGACCCAGCCACCTTCCGCTAGCGGAACCCCTGGACAGAGGGTGACAATCTCTTGTTCCGGCAGCTCTTCCAACATCGGCTCTAATTACGTGTATTGGTACCAGCAGCTGCCTGGCACAGCCCCAAAGCTGCTGATCTATAGGAACAATCAGAGGCCATCCGGAGTGCCTGATCGGTTCAGCGGATCTAAGTCCGGCACCTCCGCCTCCCTGGCTATCTCTGGCCTGAGGTCCGAGGATGAGGCTGACTACTATTGCGCTGCTTGGGACGATTCCCTGAGGGCTGTGGTGTTTGGAGGAGGAACCAAGCTGACAGTGCTGGGCGGAGGAGGAGGAGGATCCGGCGGAGGTGGAAGTAAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGCTCGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCCTCGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGACCACCATCACCATCACCAT(SEQ ID NO：18)。

wherein, the underlined sequences from the 5 'end to the 3' end in the sequences are signal peptide coding sequences, respectively, a first linker sequence for linking the heavy chain variable region coding sequence and the light chain variable region coding sequence of the digoxin single-chain antibody, a second linker sequence for linking the light chain variable region coding sequence and the gaussian luciferase coding sequence, and a sequence for encoding a 6×histidine tag.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding a fusion protein of the invention, and a vector backbone sequence. The vector backbone sequence may be any suitable expression vector sequence, for example, in one embodiment, the vector backbone sequence is pcDNA3.1 (+).

One embodiment of the present invention provides a recombinant host cell comprising an expression vector of the present invention. The host cell may be any host cell capable of expressing the fusion proteins of the invention, e.g., in one embodiment, CHO cells.

The invention also provides the use of fusion proteins, isolated nucleic acids, expression vectors or recombinant host cells in the detection of digoxin molecules.

The present invention is directed to the detection of digoxin molecules, which may be free digoxin molecules that are present independently, or may be in a form that binds to a suitable protein or the like, and the detection of such protein can be indirectly detected by detecting digoxin molecules that bind to such protein.

The following detailed description of the present invention is provided by way of example only, and should not be construed as limiting the scope of the invention.

Example 1 construction of Fusion Protein (Fusion Protein) recombinant expression vector pcDNA3.1-anti DIG scFv-GLuc

As shown in FIG. 1, PCR amplification was performed by designing and synthesizing primers using heavy and light chain genes of digoxin antibody, gaussian luciferase gene and pCDNA3.1 plasmid as templates, respectively. The construction of the fusion protein anti DIG scFv-GLuc is based on a heavy chain gene (H chain) and a light chain gene (L chain) of a digoxin antibody, and the fusion protein anti DIG scFv-GLuc is connected by a first connecting sequence (Linker 1) (GGGGSGGGGSGGGGS) to carry out overlapping PCR extension amplification to obtain a complete scFv gene. The PCR product was detected by 1.0% agarose gel electrophoresis, and the target gene was recovered by agarose gel recovery kit. And based on the product and GLuc gene, connecting by using a second connecting sequence (Linker 2) (GGGGS), overlapping, performing PCR extension amplification to obtain a complete anti DIG scFv-GLuc gene (the sequence is shown as SEQ ID NO: 17), finally performing double enzyme digestion on a PCR amplification final product and a plasmid pCDNA3.1 by using restriction enzyme, and connecting overnight at 16 ℃ by using T4 ligase after the enzyme digestion product is recovered by using restriction enzyme. After ligation, E.coli HB2151 was transformed to be competent, plated, and the next day was picked up for double digestion and sequencing identification.

The mutant G2L fusion expression vector is based on a wild fusion protein expression vector which is successfully prepared, plasmid PCR is carried out by designing a primer containing a mutant gene (wherein the sequence of the anti DIG scFv-G2L gene is shown as SEQ ID NO: 18), and then the PCR product is treated by Dpn I enzyme and reacts for 2h at 37 ℃. The product was transformed into TOP10 competent cells and plated on LB plates containing 100 ng/. Mu.L ampicillin. The next day, the single clone was picked up on the plate, the plasmid was extracted after the expansion culture, and sequencing was performed to ensure the correct mutation site.

Example 2 transfection, expression and purification of fusion expression vectors

Mu.g of the constructed pcDNA3.1-anti DIG scFv-GLuc fusion protein expression plasmid was uniformly mixed with 1ml of cell culture medium. mu.L of the cell culture medium was mixed with 80. Mu.L of lipo2000 transfection reagent (Invitrogen). Mixing the diluted expression vector and the diluted transfection reagent, and standing at room temperature for 20 minutes. 25ml of a concentration of 2X 10 was prepared ⁶ Per ml of CHO cells, the mixed fusion protein expression plasmid and transfection reagent mixture was added to the cells and gently shaken. 37 ℃,8% CO ₂ Culturing under the condition for 10 days.

Cell culture broth was collected and centrifuged at 8000rpm/min for 10min, and the supernatant was collected. The supernatant was filtered and applied to a nickel column, and washed with 10ml of PBS. 10ml were washed with buffer (20mM Tris,pH 8.0, 150mM NaCl,20mM imidazole) to remove the contaminating proteins and finally eluted with buffer (20mM Tris,pH 8.0, 150mM NaCl,500mM imidazole).

The eluted proteins were then detected by 10% SDS-PAGE. Western Blot identification was performed on the high purity protein samples. Constant current transfer at 250mA at 4℃for 1.5h, transfer proteins to PVDF membrane (from Millipore); after the transfer printing is finished, sealing the film in 5% skimmed milk for 2 hours at room temperature; washing 3 times with PBS, adding anti-H+ L, anti-Fc and anti-MICA antibody at a ratio of 1:2000, incubating at 37deg.C for 1H, washing 3 times with PBS (TPBS) containing 0.05% Tween, adding HRP-coupled goat anti-mouse IgG polyclonal antibody at a ratio of 1:5000, incubating at 37deg.C for 1H, washing with TBS for 3 times, dripping ECL luminescent color development liquid, and exposing and photographing with a gel imager. The final purified and identified samples were dialyzed or ultrafiltered into PBS, snap frozen in liquid nitrogen and stored at-70 ℃. FIG. 2 shows SDS-PAGE and Western Blot detection results, and shows that the eluted protein has clear bands and higher purity.

The pcDNA3.1-anti DIG scFv-G2L fusion protein expression plasmid was similarly constructed as described above.

Example 3 fusion protein antibody portion Elisa test affinity experiments

(1) Antigen 100. Mu.L BSA-DIG protein was coated on the enzyme strips at a concentration of 1. Mu.g/ml overnight. 200. Mu.L of PBST was added and washed with shaking for 10min, 3 times in total and 3 times in PBS.

(2) 200. Mu.L of 1% BSA-PBS was added thereto and the mixture was blocked at 37℃for 2 hours. PBST 200. Mu.L, washed 10min, washed 3 times, PBS 200. Mu.L, washed 10min, washed 3 times.

(3) 100 mu L of a protein solution to be detected with concentration gradient dilution is added, and the mixture is incubated for 2 hours at 37 ℃. PBST 200. Mu.L, washed 10min, washed 3 times, PBS 200. Mu.L, washed 10min, washed 3 times.

(4) Diluted commercial horseradish peroxidase-conjugated murine anti-6 x his antibody was added 100 μl and incubated for 1h at 37 ℃. PBST 200. Mu.L, washed 10min, washed 3 times, PBS 200. Mu.L, washed 10min, washed 3 times.

(5) The color development was performed for 10min using tetramethyl benzidine microporous peroxidase substrate (TMB) as a substrate.

(6) After the reaction was terminated by adding 50. Mu.L of sulfuric acid at a concentration of 0.1mol/L, the absorbance at 450nm was measured.

(7) The absorbance and concentration were analyzed, and the result of the effect analysis is shown in fig. 3.

The results show that: the binding force to digoxin of the fusion proteins anti DIG scFv-GLuc and anti DIG scFv-G2L is measured by an antibody part Elisa method, which shows that the fusion protein retains the affinity activity of the antibody to digoxin.

Example 4 detection of luciferase partial Activity of fusion proteins

The fusion protein was diluted to 3nM with substrate dilution (50 mM Tris, pH 8.0, 100mM NaCl) and 10. Mu.L was added to a 96-well ELISA plate. The substrate coelenterazine 90. Mu.L diluted to 10. Mu.M with the same solution was then added, and the luminescence intensity (CL values) was read from the luminescence module using a microplate reader, and the results are shown in FIG. 4.

The results show that: the luciferase parts of the fusion protein anti DIG scFv-GLuc and the anti DIG scFv-G2L both catalyze self-luminescence activity, and the fusion protein retains the activity of the luciferase for catalyzing the substrate to emit light.

Example 5 detection of bifunctional Activity of fusion proteins ELISA detection binding and bioluminescence experiments

(1) Antigen 100. Mu.L BSA-DIG protein was coated on the enzyme strips at a concentration of 1. Mu.g/ml overnight. 200. Mu.L of PBST was added and washed with shaking for 10min, 3 times, and 3 times with PBS.

(4) The substrate coelenterazine 90. Mu.L diluted to 10. Mu.M with the solution was added and the luminescence intensity (CL values) read from the luminescence module using a microplate reader.

(5) The absorbance and concentration were analyzed, and the result of the effect analysis is shown in fig. 5.

The results show that: the fusion proteins anti DIG scFv-GLuc and anti DIG scFv-G2L have the dual-function activity of an antibody and luciferase, and can catalyze the substrate to bioluminescence after combining with target digoxin.

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

SEQUENCE LISTING

<110> Shenzhen Hua institute of great life science

<120> fusion protein of Gauss luciferase and digoxin single-chain antibody and application thereof

<130> 18I27209

<160> 24

<170> PatentIn version 3.3

<210> 1

<211> 117

<212> PRT

<213> artificial sequence

<400> 1

Gln Val Gln Leu Leu Gln Ser Ala Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ser Pro Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 2

<211> 112

<212> PRT

<213> artificial sequence

<400> 2

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

100 105 110

<210> 3

<211> 168

<212> PRT

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<400> 3

Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala Ser

1 5 10 15

Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro Gly

20 25 30

Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala Arg

35 40 45

Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile Lys

50 55 60

Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr Tyr

65 70 75 80

Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile Val

85 90 95

Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu Gln

100 105 110

Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys Leu

115 120 125

Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp Leu

130 135 140

Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val Asp

145 150 155 160

Lys Ile Lys Gly Ala Gly Gly Asp

165

<210> 4

<211> 168

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<213> artificial sequence

<400> 4

Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala Ser

1 5 10 15

Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro Gly

20 25 30

Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala Arg

35 40 45

Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile Lys

50 55 60

Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr Tyr

65 70 75 80

Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile Val

85 90 95

Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Leu Glu Gln

100 105 110

Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys Leu

115 120 125

Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp Leu

130 135 140

Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val Asp

145 150 155 160

Lys Ile Lys Gly Ala Gly Gly Asp

165

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser

1 5 10

<210> 6

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<400> 6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 7

<211> 17

<212> PRT

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Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala

<210> 8

<211> 443

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<400> 8

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Gln Val Gln Leu Leu Gln Ser Ala Ala Glu Val Lys Lys Pro Gly

20 25 30

Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser

35 40 45

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

50 55 60

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser

65 70 75 80

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

85 90 95

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val Tyr Tyr

100 105 110

Cys Ala Arg Ala Ser Pro Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr

145 150 155 160

Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile

165 170 175

Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro

180 185 190

Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp

195 200 205

Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser

210 215 220

Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp

225 230 235 240

Asp Ser Leu Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

245 250 255

Leu Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Pro Thr

260 265 270

Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala Ser Asn Phe Ala

275 280 285

Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro Gly Lys Lys Leu

290 295 300

Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala Arg Lys Ala Gly

305 310 315 320

Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile Lys Cys Thr Pro

325 330 335

Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr Tyr Glu Gly Asp

340 345 350

Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile Val Asp Ile Pro

355 360 365

Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu Gln Phe Ile Ala

370 375 380

Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys Leu Lys Gly Leu

385 390 395 400

Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp Leu Pro Gln Arg

405 410 415

Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val Asp Lys Ile Lys

420 425 430

Gly Ala Gly Gly Asp His His His His His His

435 440

<210> 9

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<400> 9

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Gln Val Gln Leu Leu Gln Ser Ala Ala Glu Val Lys Lys Pro Gly

20 25 30

Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser

35 40 45

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

50 55 60

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser

65 70 75 80

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

85 90 95

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val Tyr Tyr

100 105 110

Cys Ala Arg Ala Ser Pro Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr

145 150 155 160

Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile

165 170 175

Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro

180 185 190

Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp

195 200 205

Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser

210 215 220

Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp

225 230 235 240

Asp Ser Leu Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

245 250 255

Leu Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Pro Thr

260 265 270

Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala Ser Asn Phe Ala

275 280 285

Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro Gly Lys Lys Leu

290 295 300

Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala Arg Lys Ala Gly

305 310 315 320

Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile Lys Cys Thr Pro

325 330 335

Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr Tyr Glu Gly Asp

340 345 350

Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile Val Asp Ile Pro

355 360 365

Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Leu Glu Gln Phe Ile Ala

370 375 380

Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys Leu Lys Gly Leu

385 390 395 400

Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp Leu Pro Gln Arg

405 410 415

Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val Asp Lys Ile Lys

420 425 430

Gly Ala Gly Gly Asp His His His His His His

435 440

<210> 10

<211> 351

<212> DNA

<213> artificial sequence

<400> 10

caggtgcagc tgctgcagag cgccgctgag gtgaagaagc caggcgagtc cctgaagatc 60

agctgtaagg gctccggcta tagcttcaca tcttactgga tcggctgggt gagacagatg 120

cccggcaagg gcctggagtg gatgggcatc atctatcccg gcgactctga tacccgctac 180

agcccttctt ttcagggcca ggtgaccatc tccgctgata agtccatcag cacagcctat 240

ctgcagtggt ccagcctgaa ggcttctgat acagccgtgt actattgcgc tagggcctct 300

ccctccggct ttgactactg gggccagggc accctggtga cagtgtcttc c 351

<210> 11

<211> 336

<212> DNA

<213> artificial sequence

<400> 11

cagtctgtgc tgacccagcc accttccgct agcggaaccc ctggacagag ggtgacaatc 60

tcttgttccg gcagctcttc caacatcggc tctaattacg tgtattggta ccagcagctg 120

cctggcacag ccccaaagct gctgatctat aggaacaatc agaggccatc cggagtgcct 180

gatcggttca gcggatctaa gtccggcacc tccgcctccc tggctatctc tggcctgagg 240

tccgaggatg aggctgacta ctattgcgct gcttgggacg attccctgag ggctgtggtg 300

tttggaggag gaaccaagct gacagtgctg ggcgga 336

<210> 12

<211> 504

<212> DNA

<213> artificial sequence

<400> 12

aagccaacag agaacaatga ggacttcaac atcgtggctg tggccagcaa ttttgctacc 60

acagacctgg atgccgacag aggcaagctg ccaggcaaga agctgcccct ggaggtgctg 120

aaggagatgg aggctaacgc cagaaaggct ggctgtaccc gcggctgcct gatctgtctg 180

tcccacatca agtgcacacc taagatgaag aagttcatcc caggccgctg tcatacctac 240

gagggcgata aggagtccgc ccagggagga atcggagagg ccatcgtgga tatcccagag 300

atccccggct tcaaggacct ggagcctatg gagcagttta tcgctcaggt ggatctgtgc 360

gtggactgta ccacaggctg cctgaagggc ctggccaatg tgcagtgtag cgacctgctg 420

aagaagtggc tgccacagcg gtgcgctacc tttgcctcta agatccaggg ccaggtggat 480

aagatcaagg gagctggagg cgac 504

<210> 13

<211> 504

<212> DNA

<213> artificial sequence

<400> 13

aagccaacag agaacaatga ggatttcaac atcgtggctg tggccagcaa ttttgctacc 60

acagacctgg atgccgacag aggcaagctg ccaggcaaga agctgcccct ggaggtgctg 120

aaggagctcg aggctaacgc taggaaggct ggatgtacca ggggatgcct gatctgtctg 180

tctcacatca agtgcacacc taagatgaag aagttcatcc caggccgctg tcatacctac 240

gagggcgata aggagtccgc tcagggagga atcggagagg ccatcgtgga tatccccgag 300

atccctggct tcaaggacct ggagcccctc gagcagttta tcgctcaggt ggatctgtgc 360

gtggactgta ccacaggctg cctgaagggc ctggccaatg tgcagtgttc cgacctgctg 420

aagaagtggc tgcctcagag gtgcgctacc tttgccagca agatccaggg ccaggtggat 480

aagatcaagg gagctggagg cgac 504

<210> 14

<211> 39

<212> DNA

<213> artificial sequence

<400> 14

ggaggaggag gaagcggagg aggaggatct ggaggaagc 39

<210> 15

<211> 30

<212> DNA

<213> artificial sequence

<400> 15

ggaggaggag gatccggcgg aggtggaagt 30

<210> 16

<211> 51

<212> DNA

<213> artificial sequence

<400> 16

atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggctgaggc c 51

<210> 17

<211> 1329

<212> DNA

<213> artificial sequence

<400> 17

atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggctgaggc ccaggtgcag 60

ctgctgcaga gcgccgctga ggtgaagaag ccaggcgagt ccctgaagat cagctgtaag 120

ggctccggct atagcttcac atcttactgg atcggctggg tgagacagat gcccggcaag 180

ggcctggagt ggatgggcat catctatccc ggcgactctg atacccgcta cagcccttct 240

tttcagggcc aggtgaccat ctccgctgat aagtccatca gcacagccta tctgcagtgg 300

tccagcctga aggcttctga tacagccgtg tactattgcg ctagggcctc tccctccggc 360

tttgactact ggggccaggg caccctggtg acagtgtctt ccggaggagg aggaagcgga 420

ggaggaggat ctggaggaag ccagtctgtg ctgacccagc caccttccgc tagcggaacc 480

cctggacaga gggtgacaat ctcttgttcc ggcagctctt ccaacatcgg ctctaattac 540

gtgtattggt accagcagct gcctggcaca gccccaaagc tgctgatcta taggaacaat 600

cagaggccat ccggagtgcc tgatcggttc agcggatcta agtccggcac ctccgcctcc 660

ctggctatct ctggcctgag gtccgaggat gaggctgact actattgcgc tgcttgggac 720

gattccctga gggctgtggt gtttggagga ggaaccaagc tgacagtgct gggcggagga 780

ggaggaggat ccggcggagg tggaagtaag ccaacagaga acaatgagga cttcaacatc 840

gtggctgtgg ccagcaattt tgctaccaca gacctggatg ccgacagagg caagctgcca 900

ggcaagaagc tgcccctgga ggtgctgaag gagatggagg ctaacgccag aaaggctggc 960

tgtacccgcg gctgcctgat ctgtctgtcc cacatcaagt gcacacctaa gatgaagaag 1020

ttcatcccag gccgctgtca tacctacgag ggcgataagg agtccgccca gggaggaatc 1080

ggagaggcca tcgtggatat cccagagatc cccggcttca aggacctgga gcctatggag 1140

cagtttatcg ctcaggtgga tctgtgcgtg gactgtacca caggctgcct gaagggcctg 1200

gccaatgtgc agtgtagcga cctgctgaag aagtggctgc cacagcggtg cgctaccttt 1260

gcctctaaga tccagggcca ggtggataag atcaagggag ctggaggcga ccaccatcac 1320

catcaccat 1329

<210> 18

<211> 1329

<212> DNA

<213> artificial sequence

<400> 18

atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggctgaggc ccaggtgcag 60

ctgctgcaga gcgccgctga ggtgaagaag ccaggcgagt ccctgaagat cagctgtaag 120

ggctccggct atagcttcac atcttactgg atcggctggg tgagacagat gcccggcaag 180

ggcctggagt ggatgggcat catctatccc ggcgactctg atacccgcta cagcccttct 240

tttcagggcc aggtgaccat ctccgctgat aagtccatca gcacagccta tctgcagtgg 300

tccagcctga aggcttctga tacagccgtg tactattgcg ctagggcctc tccctccggc 360

tttgactact ggggccaggg caccctggtg acagtgtctt ccggaggagg aggaagcgga 420

ggaggaggat ctggaggaag ccagtctgtg ctgacccagc caccttccgc tagcggaacc 480

cctggacaga gggtgacaat ctcttgttcc ggcagctctt ccaacatcgg ctctaattac 540

gtgtattggt accagcagct gcctggcaca gccccaaagc tgctgatcta taggaacaat 600

cagaggccat ccggagtgcc tgatcggttc agcggatcta agtccggcac ctccgcctcc 660

ctggctatct ctggcctgag gtccgaggat gaggctgact actattgcgc tgcttgggac 720

gattccctga gggctgtggt gtttggagga ggaaccaagc tgacagtgct gggcggagga 780

ggaggaggat ccggcggagg tggaagtaag ccaacagaga acaatgagga tttcaacatc 840

gtggctgtgg ccagcaattt tgctaccaca gacctggatg ccgacagagg caagctgcca 900

ggcaagaagc tgcccctgga ggtgctgaag gagctcgagg ctaacgctag gaaggctgga 960

tgtaccaggg gatgcctgat ctgtctgtct cacatcaagt gcacacctaa gatgaagaag 1020

ttcatcccag gccgctgtca tacctacgag ggcgataagg agtccgctca gggaggaatc 1080

ggagaggcca tcgtggatat ccccgagatc cctggcttca aggacctgga gcccctcgag 1140

cagtttatcg ctcaggtgga tctgtgcgtg gactgtacca caggctgcct gaagggcctg 1200

gccaatgtgc agtgttccga cctgctgaag aagtggctgc ctcagaggtg cgctaccttt 1260

gccagcaaga tccagggcca ggtggataag atcaagggag ctggaggcga ccaccatcac 1320

catcaccat 1329

<210> 19

<211> 11

<212> PRT

<213> artificial sequence

<400> 19

Gly Gln Gly Gln Gly Gln Gly Gln Gly Gln Gly

1 5 10

<210> 20

<211> 15

<212> PRT

<213> artificial sequence

<400> 20

Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Lys Gly Lys Gly

1 5 10 15

<210> 21

<211> 10

<212> PRT

<213> artificial sequence

<400> 21

Val Pro Gly Val Gly Val Pro Gly Val Gly

1 5 10

<210> 22

<211> 8

<212> PRT

<213> artificial sequence

<400> 22

Ser Ala Pro Gly Thr Pro Ser Arg

1 5

<210> 23

<211> 14

<212> PRT

<213> artificial sequence

<400> 23

Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Glu Phe

1 5 10

<210> 24

<211> 6

<212> PRT

<213> artificial sequence

<400> 24

Gly Ser Gly Gly Ser Gly

1 5

Claims

1. A fusion protein of a gauss luciferase and a digoxin single-chain antibody, characterized in that the fusion protein comprises a heavy chain variable region, a light chain variable region and a gauss luciferase or a continuously-emitting mutant of the digoxin single-chain antibody, wherein the heavy chain variable region and the light chain variable region are connected through a first flexible peptide, and the gauss luciferase or the continuously-emitting mutant is connected with the heavy chain variable region or the light chain variable region through a second flexible peptide;

the sequence of the fusion protein is shown as SEQ ID NO:8 or SEQ ID NO: shown at 9.

2. An isolated nucleic acid encoding the fusion protein of claim 1.

3. The isolated nucleic acid of claim 2, wherein the isolated nucleic acid sequence is set forth in SEQ ID NO:17 or SEQ ID NO: shown at 18.

4. An expression vector comprising a nucleic acid sequence encoding the fusion protein of claim 1, and a vector backbone sequence.

5. A recombinant host cell comprising the expression vector of claim 4.

6. Use of the fusion protein of claim 1, the isolated nucleic acid of claim 2 or 3, the expression vector of claim 4 or the recombinant host cell of claim 5 in the detection of digoxin molecules for non-diagnostic and therapeutic purposes.