CN111269324A

CN111269324A - Fusion protein of Gauss luciferase and digoxin single-chain antibody and application thereof

Info

Publication number: CN111269324A
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: Shenzhen Huada Zhizaojichuang Technology Co Ltd
Current assignee: Qingdao Huada Zhizao Technology Co ltd
Priority date: 2018-12-04
Filing date: 2018-12-04
Publication date: 2020-06-12
Anticipated expiration: 2038-12-04
Also published as: CN111269324B

Abstract

A fusion protein of gauss luciferase and digoxin single-chain antibody and application thereof. The fusion protein comprises a heavy chain variable region and a light chain variable region of a digoxin single-chain antibody, and the gauss luciferase or the sustained luminescence mutant, wherein the heavy chain variable region is connected with the light chain variable region through a first flexible peptide, and the gauss luciferase or the sustained luminescence mutant is connected with the heavy chain variable region or the light chain variable region through a second flexible peptide. The fusion protein of the invention reserves the activity of two parts of proteins, can be specifically combined with digoxin on one hand, can exert the advantage of self luminescence of luciferase on the other hand, realizes the self luminescence detection while specifically combining with the 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 Gauss luciferase and digoxin single-chain antibody and application thereof.

Background

Luciferase (Luciferase) is a generic term for enzymes capable of producing bioluminescence in an organism. Due to its spontaneous light effect, it is widely used as a reporter gene. The application in the aspects of detection and the like is also more and more extensive. The kinds of luciferases include: bacterial Luciferase (Bacterial Luciferase, blu), Firefly Luciferase (Firefly Luciferase, FLuc), Renilla Luciferase (Renilla Luciferase, RLuc), and gaussian Luciferase (Gaussia Luciferase, GLuc). Wherein the luminous activity of the Gaussian luciferase is more than 100 times higher than that of other luciferase. The gauss luciferase is a luciferase which is derived from the inside of a marine copepod (Gaussia princeps), has the size of 19.9kDa and only 185 amino acids, can be secreted to the outside of cells by the cells, and only coelenterazine and oxygen are needed for reaction. At present, the gauss luciferase is widely used as a reporter gene. By coexpressing the Gaussian luciferase and the target protein, the target protein can be researched by positioning tracing and the like. Since it is found later than other luciferases, it is less developed and applied. In particular, the application in detection is particularly limited. Therefore, the method has wide application prospect in development and application.

Digoxin is widely used in clinical cardiac glycosides for treating heart failure and arrhythmia. Simultaneously, the digoxin is widely applied to detection research as a molecular target. In particular, in the aspects of clinical diagnosis and scientific research and detection experiments, the research and application of taking digoxin as a target or a marker, and further utilizing a digoxin antibody to specifically recognize the target to perform molecular diagnosis or molecular localization are more and more.

Most of the existing digoxin antibodies as a diagnosis or detection means are that after a digoxin target is recognized by a digoxin antibody, a second antibody is used for recognizing the digoxin antibody for amplification, and finally a color reaction is carried out by using a label carried on the second antibody. Thus, the detection requires two or more steps to be completed. Yet another way is to add a fluorescent label to the digoxigenin antibody. The fluorescent label can emit fluorescence to be received by the detector after absorbing the exciting light, and the detection process needs hardware facilities such as a fluorescent microscope, so the application is limited. At present, no product with a fluorescent label added on digoxin antibody exists in the market. But may have similar functionality through combination. After the digoxin antibody is modified by Biotin (Biotin), the digoxin antibody is amplified by using an SA-GLU coupling product, so that the digoxin can be specifically recognized and self-luminescence detection can be realized. However, the antibody combination method is complicated and time-consuming.

Disclosure of Invention

The fusion protein provided by the invention retains the activity of two parts of proteins, can be specifically combined with digoxin on one hand, can exert the advantage of self luminescence of luciferase on the other hand, realizes the self luminescence 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 gauss luciferase and a digoxin single chain antibody, the fusion protein comprising a heavy chain variable region, a light chain variable region and a gauss luciferase or a persistent light-emitting mutant of the digoxin single chain antibody, wherein the heavy chain variable region and the light chain variable region are linked by a first flexible peptide, and the gauss luciferase or the persistent light-emitting mutant and the heavy chain variable region or the light chain variable region are 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 is shown.

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

In a preferred embodiment, the sequence of the above-mentioned gaussian luciferase is as shown in SEQ ID NO: 3, respectively.

In a preferred embodiment, the above-mentioned continuous light-emitting mutant has a sequence shown in SEQ ID NO: 4, respectively.

In a preferred embodiment, the first flexible peptide and the second flexible peptide are each 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; the second flexible peptide is seq id NO: 6.

In a preferred embodiment, the above fusion protein further comprises: a signal peptide for directing secretion of the fusion protein from the inside to the outside of the expression 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 above fusion protein further comprises: a fusion tag for affinity purification of the above fusion protein.

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

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

In a preferred embodiment, the sequence of the fusion protein is as shown 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 gauss luciferase coding sequence or a persistent light emitting mutant coding sequence of a single chain digoxin antibody, wherein the heavy chain variable region coding sequence is linked to the light chain variable region coding sequence by a first linking sequence, and the gauss luciferase coding sequence or the persistent light emitting mutant coding sequence is linked to the heavy chain variable region coding sequence or the light chain variable region coding sequence by a second linking sequence.

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

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

In a preferred embodiment, the coding sequence of the above-mentioned gauss luciferase is as set forth in SEQ ID NO: shown at 12.

In a preferred embodiment, the coding sequence of the above-mentioned continuous light-emitting mutant is shown in SEQ ID NO: shown at 13.

In a preferred embodiment, said first linker sequence and said second linker sequence are each independently selected from the group consisting of seq id NO: 14 and SEQ ID NO: 15, or a sequence shown in figure 15.

In a preferred embodiment, the first linker sequence is SEQ ID NO: 14, or a sequence shown in fig. 14; the second connecting sequence is SEQ ID NO: 15, or a sequence shown in figure 15.

In a preferred embodiment, the above isolated nucleic acid further comprises: a signal peptide coding sequence which codes for a signal peptide for directing secretion of the above fusion protein from the inside to the outside of the expression 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 coding sequence is 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 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 above-described 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 as set forth in SEQ ID NO: 17 or SEQ ID NO: 18, respectively.

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

According to a fifth aspect, there is provided in one 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 the detection of digoxin molecules.

The fusion protein of the gauss luciferase and the digoxin single-chain antibody can specifically recognize digoxin on one hand, and has a self-luminous function on the other hand, so that the detection is convenient. Compared with the existing mode of detecting digoxin by coupling amplification, the fusion protein disclosed by the invention has the advantages of simple process, convenience in production, simplicity and convenience in use and shorter time consumption.

Drawings

FIG. 1 is a schematic diagram of a schematic construction principle flow of a Fusion Protein expression vector in an embodiment of the present invention, in which a heavy chain (H chain) variable region coding sequence of a digoxin single-chain antibody is connected with a light chain (L chain) variable region coding sequence (including a Signal peptide coding sequence) through a first connecting sequence (Linker 1), a heavy chain (Hchain) variable region coding sequence of the digoxin single-chain antibody is connected with a Gaussureae luciferase (Gluc) coding sequence through a second connecting sequence (Linker 2) to obtain a Fusion Protein coding sequence, and the Fusion Protein coding sequence is integrally connected into a vector backbone sequence pcDNA3.1(+) to obtain a recombinant Fusion Protein expression vector.

FIG. 2 is a SDS-PAGE identification and Westernblot identification of anti DIG scFv-GLuc fusion protein in the example of the present invention, lane 1: anti DIG scFv-GLuc fusion protein, lane M: marker.

FIG. 3 is a graph showing the results of experiments on binding force of anti DIG scFv-GLuc fusion protein antibody portion ELISA to digoxin in examples of the present invention, wherein anti DIG scFv-GLuc represents wild-type fusion protein, anti DIGscFv-G2L represents mutant fusion protein, anti DIG scFv represents digoxin single-chain antibody, GLuc represents Gauss luciferase, G2L represents Gauss luciferase continuous luminescence mutant, abscissa represents Concentration (Concentration) value, and ordinate represents OD450nm absorbance value.

FIG. 4 is a graph showing the results of bioluminescence assay experiments using anti DIG scFv-GLuc fusion protein according to the example of the present invention, wherein anti DIG scFv-GLuc represents fusion protein, anti DIG scFv-G2L represents mutant fusion protein, anti DIG scFv represents digoxin single chain antibody, GLuc represents Gauss luciferase, G2L represents continuous light emitting mutant of Gauss luciferase, abscissa represents Time (Time), and ordinate represents luminescence intensity (CL values).

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

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other materials, methods.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

The terms "first," "second," and the like, herein are used merely to distinguish one element from another, and do not have any sequential or technical meaning.

The invention provides a fusion protein of a gauss luciferase and a digoxin single-chain antibody, which is a digoxin-targeted antibody fusion protein, and is a fusion protein constructed by gene recombination based on a digoxin single-chain antibody and the gauss luciferase or a continuous luminous mutant, wherein the digoxin single-chain antibody and the gauss luciferase or the continuous luminous mutant are connected through a flexible peptide.

Specifically, the fusion protein comprises a heavy chain (H chain) variable region, a light chain (L chain) variable region and a Gauss luciferase (Gluc) or continuous light-emitting 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 Gauss luciferase or the continuous light-emitting mutant and the heavy chain variable region or the light chain variable region are connected 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 of the single-chain digoxin antibody and the gauss luciferase (Gluc) or the continuous light-emitting mutant, for example, four connection modes of H + L + Gluc, L + H + Gluc, Gluc + L + H, or Gluc + H + L may be sequentially performed from the N-terminus to the C-terminus of the protein. In a preferred embodiment, an H + L + Gluc connection is used.

In a preferred embodiment, the heavy chain variable region sequence is as follows SEQ ID NO: 1, and the following components:

QVQLLQSAAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASPSGFDYWGQGTLVTVSS(SEQID NO：1)。

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

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRAVVFGGGTKLTVLGG(SEQ ID NO：2)。

in a preferred embodiment, the gaussian luciferase sequence is as set forth in SEQ ID NO: 3, showing:

KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD(SEQ ID NO：3)。

in a preferred embodiment, the sequence of the persistent light mutant is as follows SEQ ID NO: 4, and (2) is as follows:

KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD(SEQ ID NO：4)。

it is noted that the fusion protein of the present invention employs, in addition to the above-mentioned SEQ ID NO: 1-3 or SEQ ID NO: 1-2 and SEQ ID NO: 4, and also includes a sequence form having the same function although the sequence has a certain difference. For example, for the heavy chain variable region, it may be a heavy chain variable region corresponding to SEQ ID NO: 1, but also has the corresponding epitope properties, as shown in SEQ ID NO: 1, a sequence with a plurality of amino acids added or reduced at two ends of the sequence and the same epitope characteristics are kept; for the light chain variable region, it may be a sequence similar to SEQ ID NO: 2, but also have corresponding epitope properties, as shown in SEQ ID NO: 2, the sequence with a plurality of amino acids added or reduced at both ends of the sequence but keeps the same epitope characteristics; for gauss luciferase, the luciferase can be a luciferase similar to seq id no: 3, but also has a gaussian luciferase activity, as shown in SEQ ID NO: 3 sequence with several amino acids added or reduced on both ends but maintaining the same gauss luciferase activity.

In the fusion protein of the present invention, the first flexible peptide and the second flexible peptide having a linking function may be 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 either of the above-mentioned 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 the expressing cell to the outside.

The signal peptide may be located at the N-terminus or 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 above 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, and:

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 and 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 apparatus (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 luminescence activity in the fusion protein of the invention.

The fusion tag can be a 6 × histidine tag (6 × His tag) or other fusion tags such as GST, MBP, SUMO and the like, and an affinity purification method corresponding to the fusion tag is selected according to the difference of the fusion tag. In a preferred embodiment, the fusion tag is a 6 × 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 as follows:

MGVKVLFALICIAVAEAQVQLLQSAAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASPSGFDYWGQGTLVTVSSGGGGSGGGGS GGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRAVVFGGGTKLTVLGGGGGGSGGGGSKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDHHHHHH(SEQID NO：8)。

MGVKVLFALICIAVAEAQVQLLQSAAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASPSGFDYWGQGTLVTVSSGGGGSGGGGS GGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRAVVFGGGTKLTVLGGGGGGSGGGGSKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDHHHHHH(SEQID NO：9)。

wherein the underlined sequence (MGVKVLFALICIAVAEA) Represents 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) Represents a second flexible peptide linking the light chain (L chain) variable region and gaussian luciferase (Gluc); underlined sequence (HHHHHH) Represents the 6 × histidine tag sequence.

The fusion protein of the invention can be obtained by the following method: cloning and recombining the screened Gauss luciferase or continuous luminous mutant gene and a digoxin single-chain antibody gene by utilizing a PCR (polymerase chain reaction) technology to construct a digoxin single-chain antibody/Gauss luciferase or continuous luminous mutant fusion protein recombinant vector, electrically transferring the recombinant vector into a host expression cell (such as a CHO (Chinese hamster ovary) 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; performing enlarged culture on the stable cell strain, centrifuging at low temperature to obtain a supernatant, and separating and purifying the supernatant by a nickel column; then, Western Blot identifies, separates and purifies the obtained fusion protein; the ELISA experiment identifies the affinity activity of the antibody part of the fusion protein; enzyme activity experiments identify the spontaneous optical activity of the luciferase moiety. The fusion protein well retains the activity of the two-part protein.

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

It is to be noted that, due to the degeneracy of codons, nucleotide sequences encoding the same polypeptide or protein may differ, and therefore, an isolated nucleic acid of the present invention may be any nucleic acid capable of encoding a fusion protein of the present invention. The sequence is not particularly limited.

In a preferred embodiment, the heavy chain variable region encoding sequence is as follows SEQ ID NO: 10, and:

CAGGTGCAGCTGCTGCAGAGCGCCGCTGAGGTGAAGAAGCCAGGCGAGTCCCTGAAGATCAGCTGTAAGGGCTCCGGCTATAGCTTCACATCTTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTATCCCGGCGACTCTGATACCCGCTACAGCCCTTCTTTTCAGGGCCAGGTGACCATCTCCGCTGATAAGTCCATCAGCACAGCCTATCTGCAGTGGTCCAGCCTGAAGGCTTCTGATACAGCCGTGTACTATTGCGCTAGGGCCTCTCCCTCCGGCTTTGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCC(SEQ ID NO：10)。

in a preferred embodiment, the light chain variable region encoding sequence is as follows SEQ ID NO: 11, and:

CAGTCTGTGCTGACCCAGCCACCTTCCGCTAGCGGAACCCCTGGACAGAGGGTGACAATCTCTTGTTCCGGCAGCTCTTCCAACATCGGCTCTAATTACGTGTATTGGTACCAGCAGCTGCCTGGCACAGCCCCAAAGCTGCTGATCTATAGGAACAATCAGAGGCCATCCGGAGTGCCTGATCGGTTCAGCGGATCTAAGTCCGGCACCTCCGCCTCCCTGGCTATCTCTGGCCTGAGGTCCGAGGATGAGGCTGACTACTATTGCGCTGCTTGGGACGATTCCCTGAGGGCTGTGGTGTTTGGAGGAGGAACCAAGCTGACAGTGCTGGGCGGA(SEQ ID NO：11)。

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

AAGCCAACAGAGAACAATGAGGACTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGATGGAGGCTAACGCCAGAAAGGCTGGCTGTACCCGCGGCTGCCTGATCTGTCTGTCCCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCCCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCAGAGATCCCCGGCTTCAAGGACCTGGAGCCTATGGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTAGCGACCTGCTGAAGAAGTGGCTGCCACAGCGGTGCGCTACCTTTGCCTCTAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGAC(SEQ ID NO：12)。

in a preferred embodiment, the persistent light mutant coding sequence is as set forth in SEQ ID NO: 13, and: AAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGCTCGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCCTCGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGAC(SEQ ID NO：13)。

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

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

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

In preferred embodiments, the isolated nucleic acid further comprises: a signal peptide coding sequence which encodes a signal peptide for directing secretion of the fusion protein from the expressing cell to the outside.

Since the signal peptide in the fusion protein of the present invention may be located at the N-terminus or C-terminus of the fusion protein, the signal peptide coding sequence may be located at the 5 'end or 3' end of the isolated nucleic acid. The signal peptide is located at the N-terminus of the above fusion protein without affecting the affinity and luminescence activity in the fusion protein of the present invention, 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 is as follows:

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCC(SEQ ID NO：16)。

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

The fusion tag coding sequence can be located at the 3 ' end or 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 and does not affect the antibody activity and expression of the light-emitting active portion 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, and:

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCCCAGGTGCAGCTGCTGCAGAGCGCCGCTGAGGTGAAGAAGCCAGGCGAGTCCCTGAAGATCAGCTGTAAGGGCTCCGGCTATAGCTTCACATCTTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTATCCCGGCGACTCTGATACCCGCTACAGCCCTTCTTTTCAGGGCCAGGTGACCATCTCCGCTGATAAGTCCATCAGCACAGCCTATCTGCAGTGGTCCAGCCTGAAGGCTTCTGATACAGCCGTGTACTATTGCGCTAGGGCCTCTCCCTCCGGCTTTGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCCGGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGAGGAAGCCAGTCTGTGCTGACCCAGCCACCTTCCGCTAGCGGAACCCCTGGACAGAGGGTGACAATCTCTTGTTCCGGCAGCTCTTCCAACATCGGCTCTAATTACGTGTATTGGTACCAGCAGCTGCCTGGCACAGCCCCAAAGCTGCTGATCTATAGGAACAATCAGAGGCCATCCGGAGTGCCTGATCGGTTCAGCGGATCTAAGTCCGGCACCTCCGCCTCCCTGGCTATCTCTGGCCTGAGGTCCGAGGATGAGGCTGACTACTATTGCGCTGCTTGGGACGATTCCCTGAGGGCTGTGGTGTTTGGAGGAGGAACCAAGCTGACAGTGCTGGGCGGAGGAGGAGGAGGATCCGGCGGAGGTGGAAGTAAGCCAACAGAGAACAATGAGGACTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGATGGAGGCTAACGCCAGAAAGGCTGGCTGTACCCGCGGCTGCCTGATCTGTCTGTCCCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCCCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCAGAGATCCCCGGCTTCAAGGACCTGGAGCCTATGGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTAGCGACCTGCTGAAGAAGTGGCTGCCACAGCGGTGCGCTACCTTTGCCTCTAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGACCACCATCACCATCACCAT(SEQ ID NO：17)。

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCCCAGGTGCAGCTGCTGCAGAGCGCCGCTGAGGTGAAGAAGCCAGGCGAGTCCCTGAAGATCAGCTGTAAGGGCTCCGGCTATAGCTTCACATCTTACTGGATCGGCTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTATCCCGGCGACTCTGATACCCGCTACAGCCCTTCTTTTCAGGGCCAGGTGACCATCTCCGCTGATAAGTCCATCAGCACAGCCTATCTGCAGTGGTCCAGCCTGAAGGCTTCTGATACAGCCGTGTACTATTGCGCTAGGGCCTCTCCCTCCGGCTTTGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCCGGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGAGGAAGCCAGTCTGTGCTGACCCAGCCACCTTCCGCTAGCGGAACCCCTGGACAGAGGGTGACAATCTCTTGTTCCGGCAGCTCTTCCAACATCGGCTCTAATTACGTGTATTGGTACCAGCAGCTGCCTGGCACAGCCCCAAAGCTGCTGATCTATAGGAACAATCAGAGGCCATCCGGAGTGCCTGATCGGTTCAGCGGATCTAAGTCCGGCACCTCCGCCTCCCTGGCTATCTCTGGCCTGAGGTCCGAGGATGAGGCTGACTACTATTGCGCTGCTTGGGACGATTCCCTGAGGGCTGTGGTGTTTGGAGGAGGAACCAAGCTGACAGTGCTGGGCGGAGGAGGAGGAGGATCCGGCGGAGGTGGAAGTAAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGCTCGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCCTCGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGACCACCATCACCATCACCAT(SEQ ID NO：18)。

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

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 protein of the invention, e.g., in one embodiment, a CHO cell.

The invention also provides the use of the fusion protein, the isolated nucleic acid, the expression vector or the recombinant host cell in the detection of digoxin molecules.

The present invention is useful for detecting a digoxin molecule, which may be a free digoxin molecule independently present or a form bound to an appropriate protein or the like, and the protein can be indirectly detected by detecting a digoxin molecule bound to the protein.

The technical solutions of the present invention are described in detail below by way of examples, and it should be understood that the examples are only illustrative and should not be construed as limiting the scope of the present invention.

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

As shown in FIG. 1, primers were designed and synthesized for PCR amplification using digoxin antibody heavy and light chain genes, Gaussian luciferase gene and pCDNA3.1 plasmid as templates, respectively. The construction of the fusion protein anti DIG scFv-GLuc takes a digoxin antibody heavy chain gene (H chain) and a light chain gene (L chain) as the basis, and uses a first connecting sequence (Linker 1) (GGGGSGGGGSGGGGS) for connection to carry out overlapped PCR extension amplification to obtain a complete scFv gene. The PCR product is detected by 1.0% agarose gel electrophoresis, and the target gene is recovered by an agarose gel recovery kit. And then on the basis of the product and the GLuc gene, connecting by using a second connecting sequence (Linker 2) (GGGGS), overlapping PCR extension amplification to obtain a complete anti DIGscFv-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, recovering the cut enzyme of the enzyme digestion product, and connecting by using T4 ligase at 16 ℃ overnight. After connection, Escherichia coli HB2151 is transformed, coated on a plate, and single-clone double-enzyme digestion and sequencing identification are selected the next day.

Mutant G2L fusion expression vector on the basis of the successfully prepared wild type fusion protein expression vector, plasmid PCR (wherein, the gene sequence of anti DIG scFv-G2L is shown in SEQ ID NO: 18) is carried out by designing a primer containing a mutant gene, and then the PCR product is treated by Dpn I enzyme and reacted 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, single clones were picked from the plates, plasmids were extracted after extensive culture, and sequencing ensured that the mutation sites were correct.

Example 2 transfection, expression and purification of fusion expression vectors

30 mu g of the constructed pcDNA3.1-anti DIG scFv-GLuc fusion protein expression plasmid was mixed with 1ml of cell culture medium. Another 920. mu.L of cell culture medium was mixed with 80. mu.L of lipo2000 transfection reagent (invitrogen). The diluted expression vector and the diluted transfection reagent were mixed well and left at room temperature for 20 minutes. 25ml of 2X 10 solution was prepared⁶In the case of CHO cells of ml, the mixed mixture of the fusion protein expression plasmid and the transfection reagent was added to the cells and shaken gently. 37 ℃ and 8% CO₂Culturing under the condition for 10 days.

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

Then, the eluted protein was detected by 10% SDS-PAGE. High purity protein samples were subjected to WesternBlot identification. Transfer of proteins to PVDF membrane (purchased from Millipore) at 4 ℃ for 1.5h with a constant current of 250 mA; after the transfer printing is finished, sealing the membrane in 5% skimmed milk for 2h at room temperature; washing with PBS for 3 times, adding anti-H + L, anti-Fc and anti-MICA antibodies according to the ratio of 1: 2000, incubating for 1H at 37 ℃, washing for 3 times with PBS (TPBS) containing 0.05% Tween, adding HRP-coupled goat anti-mouse IgG polyclonal antibody according to the ratio of 1: 5000, incubating for 1H at 37 ℃, washing for 3 times with TBS, dripping ECL luminescence developing solution, and exposing and photographing with a gel imager. The final purified and characterized samples were dialyzed or ultrafiltered into PBS, snap frozen in liquid nitrogen, and stored at-70 ℃. FIG. 2 shows the results of SDS-PAGE and Western Blot detection, and it can be seen that the eluted protein band is clear and has high purity.

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

Example 3 fusion protein antibody moiety Elisa test affinity assay

(1) Antigen 100. mu.L BSA-DIG protein was coated overnight onto the enzyme strips at a concentration of 1. mu.g/ml. Washing with 200 μ L PBST for 10min with shaking, 3 times total, and washing with PBS with shaking for 3 times.

(2) Add 1% BSA-PBS 200. mu.L, block for 2h at 37 ℃. PBST 200 u L, washing 10min, washing 3 times, PBS200 u L, washing 10min, washing 3 times.

(3) Add 100. mu.L of test protein solution diluted with concentration gradient and incubate for 2h at 37 ℃. PBST 200 u L, washing 10min, washing 3 times, PBS200 u L, washing 10min, washing 3 times.

(4) Diluted commercial horseradish peroxidase-conjugated mouse anti-6 × His antibody (100 μ L) was added and incubated at 37 ℃ for 1 h. PBST 200 u L, washing 10min, washing 3 times, PBS200 u L, washing 10min, washing 3 times.

(5) Color development was performed for 10min using tetramethylbenzidine microporous peroxidase substrate (TMB) as substrate.

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

(7) The absorbance and concentration were analyzed, and the results of the effect analysis are shown in FIG. 3.

The results show that: the binding force to digoxin was determined by the antibody portion Elisa method of the fusion proteins anti DIG scFv-GLuc and anti DIG scFv-G2L, showing that the fusion proteins retain the affinity activity of the antibody for digoxin.

Example 4 detection of luciferase partial Activity of fusion protein

The fusion protein was diluted to 3nM with substrate diluent (50mM Tris, pH 8.0, 100mM NaCl) and 10. mu.L was added to a 96-well microplate. Then, 90. mu.L of coelenterazine diluted to 10. mu.M with the same solution was added thereto, 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 proteins anti DIG scFv-GLuc and anti DIG scFv-G2L both catalyze the self-luminescence activity, and the fusion proteins retain the activity of luciferase for catalyzing substrate luminescence.

Example 5 fusion protein bifunctional Activity detection-ELISA detection binding and bioluminescence assay

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

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

(5) The results of the effect analysis by analyzing the absorbance and concentration are shown in FIG. 5.

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

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

SEQUENCE LISTING

<110> Shenzhen Huashengshengsciences institute

Fusion protein of <120> 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 IleTyr 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 SerAla 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

<213> Artificial sequence

<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

<212>PRT

<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 5560

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

<210>5

<211>13

<212>PRT

<213> Artificial sequence

<400>5

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

1 5 10

<210>6

<211>10

<212>PRT

<213> Artificial sequence

<400>6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210>7

<211>17

<212>PRT

<213> Artificial sequence

<400>7

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

<212>PRT

<213> Artificial sequence

<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

385390 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

<211>443

<212>PRT

<213> Artificial sequence

<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 gcctggcacagccccaaagc 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 Gaussian luciferase and a digoxin single-chain antibody is characterized by comprising a heavy chain variable region, a light chain variable region and the Gaussian luciferase or persistent light-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 Gaussian luciferase or persistent light-emitting mutant and the heavy chain variable region or the light chain variable region are connected through a second flexible peptide.

2. The fusion protein of claim 1, wherein the heavy chain variable region sequence is as set forth in SEQ ID NO: 1 is shown in the specification;

optionally, the light chain variable region sequence is as set forth in SEQ ID NO: 2 is shown in the specification;

optionally, the sequence of the gaussian luciferase is as set forth in SEQ ID NO: 3 is shown in the specification;

optionally, the sequence of the persistent luminescent mutant is as follows SEQ ID NO: 4, respectively.

3. The fusion protein of claim 1 or 2, wherein the first flexible peptide and the second flexible peptide are each independently selected from the sequences: SEQ ID NO: 5 or SEQ ID NO: 6;

preferably, the first flexible peptide is SEQ ID NO: 5; the second flexible peptide is SEQ ID NO: 6.

4. The fusion protein of claim 1 or 2, further comprising: a signal peptide for directing secretion of the fusion protein from the expressing cell to the outside;

preferably, the signal peptide is located at the N-terminus of the fusion protein;

preferably, the signal peptide sequence is as set forth in SEQ ID NO: 7 is shown in the specification;

preferably, the fusion protein further comprises: a fusion tag for affinity purification of the fusion protein;

preferably, the fusion tag is a 6 × histidine tag;

preferably, the fusion tag is located at the C-terminus of the fusion protein.

5. The fusion protein of claim 1 or 2, wherein the sequence of the fusion protein is as set forth in SEQ id no: 8 or SEQ ID NO: shown at 9.

6. An isolated nucleic acid encoding the fusion protein of any one of claims 1 to 5.

7. The isolated nucleic acid of claim 6, wherein the isolated nucleic acid comprises a heavy chain variable region encoding sequence, a light chain variable region encoding sequence, and a Gauss luciferase encoding sequence or a persistent light mutant encoding sequence of a single chain digoxin antibody, wherein the heavy chain variable region encoding sequence is linked to the light chain variable region encoding sequence by a first linking sequence, and the Gauss luciferase encoding sequence or the persistent light mutant encoding sequence is linked to the heavy chain variable region encoding sequence or the light chain variable region encoding sequence by a second linking sequence;

preferably, the heavy chain variable region encoding sequence is as set forth in SEQ ID NO: 10 is shown in the figure;

preferably, the light chain variable region encoding sequence is as set forth in SEQ ID NO: 11 is shown in the figure;

preferably, the coding sequence of the gaussian luciferase is as shown in SEQ ID NO: 12 is shown in the specification;

preferably, the persistent luminescent mutant coding sequence is as set forth in SEQ ID NO: 13 is shown in the figure;

preferably, the first and second joining sequences are each independently selected from the sequences: SEQ ID NO: 14 or SEQ ID NO: 15;

preferably, the first linker sequence is SEQ ID NO: 14, or a sequence shown in fig. 14; the second connecting sequence is SEQ ID NO: 15, or a sequence shown in seq id no;

preferably, the isolated nucleic acid further comprises: a signal peptide coding sequence which encodes a signal peptide for directing secretion of the fusion protein from the expressing cell to the outside;

preferably, the signal peptide coding sequence is located 5' to the isolated nucleic acid;

preferably, the signal peptide coding sequence is as set forth in SEQ ID NO: 16 is shown in the figure;

preferably, the isolated nucleic acid further comprises a fusion tag coding sequence encoding a fusion tag for affinity purification of the fusion protein;

preferably, the fusion tag coding sequence is a sequence encoding a 6 × histidine tag;

preferably, the fusion tag coding sequence is located at the 3' end of the isolated nucleic acid;

preferably, the isolated nucleic acid sequence is as set forth in SEQ ID NO: 17 or SEQ ID NO: 18, respectively.

8. An expression vector comprising a nucleic acid sequence encoding the fusion protein of any one of claims 1 to 5, and a vector backbone sequence.

9. A recombinant host cell comprising the expression vector of claim 8.

10. Use of the fusion protein of any one of claims 1 to 5, the isolated nucleic acid of claim 6 or 7, the expression vector of claim 8, or the recombinant host cell of claim 9 in the detection of digoxin molecules.