CN111269323B

CN111269323B - Fusion protein of monomer streptavidin and gauss luciferase and application thereof

Info

Publication number: CN111269323B
Application number: CN201811473698.3A
Authority: CN
Inventors: 李静; 王佑富; 郑越; 董宇亮; 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: 2022-02-22
Anticipated expiration: 2038-12-04
Also published as: CN111269323A

Abstract

The fusion protein comprises a monomer streptavidin full-length protein and a gauss luciferase full-length protein or a continuous luminescence mutant, wherein the monomer streptavidin full-length protein is connected with the gauss luciferase full-length protein or the continuous luminescence mutant through a flexible peptide. The fusion protein can conveniently and specifically identify biotin molecules, has the function of catalyzing substrate oxidation luminescence, and is convenient to detect. Compared with the detection of biotin by combining SA and anti-SA antibodies, the fusion protein is convenient and quick, does not cause substrate aggregation, can be obtained by only once expression and purification, has simple production process and is beneficial to large-scale production. The fusion protein has wide application prospect in the fields of DNA hybridization, immunodetection, biochemical diagnosis and the like.

Description

Fusion protein of monomer streptavidin and gauss luciferase and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a fusion protein of monomer streptavidin and Gaussian luciferase and application thereof.

Background

Streptavidin (SA) was found by Chaiet in 1963 as a product secreted by Streptomyces avidinii during culture. One SA peptide chain has 159 amino acid residues and molecular weight of 16.5 kDa. The complete SA protein consists of 4 SA polypeptide chains, forming a tetrameric structure. Each polypeptide chain can specifically bind to one Biotin (Biotin) molecule, and thus 1 molecule of SA can bind to 4 molecules of Biotin. The interaction between the two exists in a non-covalent binding form, and the affinity reaches 10^-15the/M, several orders of magnitude higher than the binding constant of antigen-antibody, is the most stable protein-small molecule interaction occurring in nature. The strong binding between the two results in biotin and SA being frequently used to develop labeling and detection tools. When the kit is used, biotin is usually labeled on a substrate to be detected, the biotin labeled on the substrate is combined by SA, then an antibody of the SA is combined with the SA, meanwhile, a luminescent group is connected to the antibody in advance, and the antibody can be excited to emit light, and finally, the substrate is detected by detecting the light emission.

1 between SA and biotin: the binding ratio of 4 may cause aggregation of the biotin-modified substrate, with unwanted side effects in certain applications. Therefore, researchers have hybridized the sequences of streptavidin and rhizopus avidin (another protein that has a similar structure to streptavidin and can efficiently bind biotin), and developed a monomeric streptavidin (MonoSA) formed from one peptide chain by means of genetic engineering, which can be represented by 1: a ratio of 1 binds biotin. MonoSA still maintains higher binding activity to biotin, and reaches 10^-9and/M, and simultaneously avoids side effects caused by substrate aggregation.

Luciferase is a generic term for a class of enzymes that catalyze the oxidative luminescence of luciferin or fatty aldehydes in vivo. Is usually found in lower animals. The luciferase commonly used at present comprises firefly luciferase, renilla luciferase and gauss luciferase. Firefly luciferase requires ATP and Mg for luminescence²⁺Assisted by Renilla luciferase, although ATP and Mg-independent²⁺And the like, but the luminous intensity is weaker, and more sensitive detection is needed in application.

Gaussia luciferase (Gluc) well makes up the defects of firefly luciferase and renilla luciferase, and ATP and Mg are not required during luminescence²⁺The cofactors also have the luminous efficiency 100 times higher than that of renilla luciferase. The gauss luciferase is the luciferase secreted by a marine flexo-phalangeal animal, is the luciferase with the minimum molecular weight discovered at present, has a signal peptide, can be secreted to the outside of cells, and is convenient for activity detection. In the presence of oxygen, the Gauss luciferase can spontaneously catalyze the oxidation luminescence of a substrate coelenterazine. The luminous intensity is the highest among the luciferases discovered at present, and the detection is easy. Due to the advantages, the Gauss luciferase is widely applied to the fields of living cell detection, protein-protein interaction, protein positioning, small interference RNA silencing technology, high-throughput drug screening and the like.

Currently, the recognition of biotin labeling using MonoSA is mainly performed by a combination of MonoSA and anti-MonoSA antibodies. Specifically recognizing biotin with MonoSA, then recognizing MonoSA with anti-MonoSA antibody with luminous group, and finally detecting biotin through detecting the luminescence of the luminous group on the antibody. The method is complicated and takes a long time. And also may cause substrate aggregation if SA and anti-SA antibodies are used.

Disclosure of Invention

The invention provides a fusion protein of monomer streptavidin and Gaussian luciferase and application thereof, wherein the fusion protein can be specifically combined with biotin, has high-intensity self-luminous capability and can be used for conveniently and rapidly detecting a biotin-labeled substrate.

According to a first aspect, in one embodiment there is provided a monomeric streptavidin and gauss luciferase fusion protein comprising a monomeric streptavidin full-length protein and a gauss luciferase full-length protein or a persistent luminescent mutant, said monomeric streptavidin full-length protein being linked to said gauss luciferase full-length protein or persistent luminescent mutant by a flexible peptide.

In a preferred embodiment, the monomeric streptavidin full-length protein is represented by SEQ ID NO: 1 is shown.

In a preferred embodiment, the sequence of the full-length protein of the gauss luciferase is shown as SEQ ID NO: 2, respectively.

In a preferred embodiment, the above-mentioned continuous light-emitting mutant has a sequence shown in SEQ ID NO: 3, respectively.

In a preferred embodiment, the flexible peptide sequence is as set forth in SEQ ID NO: 4, respectively.

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: 5, respectively.

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 sequence of the fusion protein is shown in SEQ ID NO: 6 or SEQ ID NO: shown at 7.

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 coding sequence for a monomeric streptavidin full-length protein and a coding sequence for a gauss luciferase full-length protein or a coding sequence for a persistent luminescent mutant, wherein the coding sequence for the monomeric streptavidin full-length protein is linked to the coding sequence for the gauss luciferase full-length protein or the coding sequence for the persistent luminescent mutant by a linker sequence.

In a preferred embodiment, the coding sequence of the monomeric streptavidin full-length protein is shown in SEQ ID NO: shown in fig. 8.

In a preferred embodiment, the coding sequence of the full-length protein of the gauss luciferase is shown as SEQ ID NO: shown at 9.

In a preferred embodiment, the coding sequence of the above-mentioned continuous light-emitting mutant is shown in SEQ ID NO: shown at 10.

In a preferred embodiment, the linker sequence is as set forth in SEQ ID NO: shown at 11.

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 12.

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

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

In a preferred embodiment, the isolated nucleic acid sequence is as set forth in SEQ ID NO: 13 or SEQ ID NO: as shown at 14.

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 a biotin molecule.

The invention carries out fusion expression on MonoSA and Gaussian luciferase genes by a gene engineering technology to form fusion protein. The fusion protein can be specifically combined with biotin, has high-intensity self-luminous capability and can be used for conveniently and rapidly detecting a biotin-labeled substrate. Compared with the detection of biotin by combining SA and anti-SA antibodies, the fusion protein is convenient and quick, does not cause substrate aggregation, can be obtained by only once expression and purification, has simple production process and is beneficial to large-scale production. The fusion protein has wide application prospect in the fields of DNA hybridization, immunodetection, biochemical diagnosis and the like.

Drawings

FIG. 1 is a schematic diagram showing the construction of a fusion vector in an embodiment of the present invention, in which MonoSA represents a monomer streptavidin full-length protein coding sequence, Gluc represents a Gaussian luciferase full-length protein coding sequence or a continuous light-emitting mutant coding sequence, Linker represents a Linker sequence, Signal peptide represents a Signal peptide coding sequence, and Overlap PCR represents a bridge PCR.

FIG. 2 is a SDS-PAGE detection result of the fusion protein in the example of the present invention, wherein lane 1: MonoSA-Gluc fusion protein, lane 2: marker.

FIG. 3 is a graph showing the results of bioluminescence detection of a wild-type MonoSA-Gluc fusion protein and a mutant MonoSA-G2L fusion protein in examples of the present invention, wherein MonoSA-Gluc represents the wild-type fusion protein, MonoSA-G2L represents the mutant fusion protein, Gluc represents Gaussian luciferase, abscissa represents Time (Time), and ordinate represents luminescence intensity (CL values).

FIG. 4 is a graph showing the results of measurement of binding force of wild-type MonoSA-Gluc fusion protein and mutant MonoSA-G2L fusion protein to biotin by the Elisa method in examples of the present invention, in which MonoSA-Gluc represents the wild-type fusion protein, MonoSA-G2L represents the mutant fusion protein, the abscissa represents the Concentration (Concentration) value, and the ordinate represents the OD450nm absorbance value.

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 invention aims to provide a fusion protein of monomer streptavidin and Gaussian luciferase. The protein can be specifically combined with Biotin and can catalyze substrate coelenterazine to emit light, so that the aim of conveniently and quickly detecting any fusion protein with Biotin (Biotin) labeled molecules is fulfilled, and the protein has the characteristics of simple preparation method, easiness in production and difficulty in causing substrate aggregation.

The invention belongs to the field of genetic engineering, and particularly relates to preparation and application of a fusion protein of Gaussian luciferase and monomer streptavidin (MonoSA). The invention uses gene engineering technology to connect the Gaussian luciferase full-length protein (or continuous luminous mutant G2L) and the monomer streptavidin (MonoSA) full-length protein through flexible peptide (linker) to obtain the fusion protein. The C end of the fusion protein is connected with a polyhistidine tag, expression and nickel column purification are carried out through CHO cells, and the obtained fusion protein can specifically bind to biotin on one hand and oxidize a substrate coelenterazine to emit light on the other hand. The self-luminescence can be detected by instruments such as an enzyme-labeling instrument and the like, can be observed by naked eyes under certain conditions, and can conveniently detect any molecule with a biotin label without the participation of any antibody and other luminescent molecules.

The method is characterized in that a PCR (polymerase chain reaction) and double enzyme digestion technology are utilized to construct a coding gauss luciferase full-length gene (or a continuous luminous mutant G2L gene) and a monomer streptavidin (MonoSA) full-length gene into a eukaryotic recombinant expression vector, a signal peptide is added at the N end of a target gene, a 6 × histidine (6 × His) tag is added at the C end, and a coding gene of a flexible peptide is added between the gauss luciferase and the monomer streptavidin. The constructed expression vector plasmid was transformed into CHO cells by the liposome method for transient expression. After the protein is expressed in the cell, the protein is secreted into the cell culture fluid under the action of the signal peptide, and the signal peptide is cut off in the secretion process. After the fermentation was completed, the cell culture fluid was collected by centrifugation. And purifying the collected culture solution by a Ni column to obtain the fusion protein. The fusion protein has the specific binding capacity with biotin and the capacity of catalyzing the oxidization and luminescence of the coelenterazine as a substrate.

The invention provides a fusion protein of monomer streptavidin and gauss luciferase, which comprises monomer streptavidin full-length protein and gauss luciferase full-length protein or continuous luminescence mutant, wherein the monomer streptavidin full-length protein and the gauss luciferase full-length protein or the continuous luminescence mutant are connected through flexible peptide.

In the fusion protein of the present invention, the connection mode (i.e. the sequence of the monomer streptavidin full-length protein and the gauss luciferase full-length protein or the continuous luminescent mutant) of the monomer streptavidin full-length protein and the gauss luciferase full-length protein or the continuous luminescent mutant can be adjusted, for example, the monomer streptavidin full-length protein is at the N-terminal of the fusion protein, and the gauss luciferase full-length protein or the continuous luminescent mutant is at the C-terminal of the fusion protein; or the Gaussian luciferase full-length protein or the continuous luminescence mutant is arranged at the N end of the fusion protein, and the monomer streptavidin full-length protein is arranged at the C end of the fusion protein. In a preferred embodiment, the monomeric streptavidin full-length protein is at the N-terminus of the fusion protein and the gauss luciferase full-length protein or the persistent light mutant is at the C-terminus of the fusion protein.

In the fusion protein of the present invention, the persistent luminescent mutant refers to a persistent luminescent mutant of the full-length protein of the gauss luciferase, and can refer to any mutant which has one or more amino acid mutations relative to the full-length protein of the wild-type gauss luciferase and still maintains the luminescent properties of the full-length protein of the gauss luciferase. In a preferred embodiment, the persistent luminescent mutant is persistent luminescent mutant G2L, described in more detail below.

In a preferred embodiment, the monomeric streptavidin full-length protein is as follows SEQ ID NO: 1, and the following components: EFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAAS (SEQ ID NO: 1).

In a preferred embodiment, the full-length protein sequence of the gauss luciferase is as follows SEQ ID NO: 2, as shown in the figure: KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD (SEQ ID NO: 2).

In a preferred embodiment, the persistent luminescent mutant is a G2L mutant having the sequence set forth in SEQ ID NO: 3, showing: KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD (SEQ ID NO: 3). Among them, one with underlining lines "L"refers to the mutated amino acid positions relative to the full-length protein sequence of the wild-type gauss luciferase, namely, the mutations M43L and M110L.

It is noted that the fusion protein of the present invention includes, in addition to the fusion protein using SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 1 and SEQ ID NO: 3, and also includes a sequence form having the same function although the sequence has a certain difference. For example, for the monomeric full-length streptavidin protein, the amino acid sequence can be the same as that of SEQ ID NO: 1, but also has the affinity activity for the corresponding monomeric streptavidin full-length protein, as shown in SEQ ID NO: 1 sequence with several amino acids added or reduced at both ends but with the same affinity activity; for the full-length protein sequence of the gauss luciferase, the sequence can be the same as that of SEQ ID NO: 2, but also has the activity of the full-length protein sequence of the gauss luciferase, as shown in SEQ ID NO: 2, the sequence with several amino acids increased or decreased at both ends of the sequence but the activity of the full-length protein sequence of the same Gauss luciferase is maintained; for the persistent light mutant sequence, it can be a nucleotide sequence similar to that of SEQ ID NO: 3, but also has the sequence activity of a persistent light-emitting mutant, as shown in SEQ ID NO: 3 sequence with several amino acids added or reduced at both ends but maintaining the activity of the same continuous light-emitting mutant sequence.

In the fusion protein of the present invention, the flexible peptide having a linking function may be selected from the following sequences:

GGGGSGGGGS(SEQ ID NO：4)；

GGGGGS(SEQ ID NO：15)；

GQGQGQGQGQG(SEQ ID NO：16)；

GSTSGSGKSSEKGKG(SEQ ID NO：17)；

VPGVGVPGVG(SEQ ID NO：18)；

SAPGTPSR(SEQ ID NO：19)；

EGKSSGSGSESKEF(SEQ ID NO：20)；

GSGGSG(SEQ ID NO：21)；

GSGGSGGG(SEQ ID NO：22)。

in a preferred embodiment, the flexible peptide sequence is as set forth in SEQ ID NO: 4, and (2) is as follows:

GGGGSGGGGS(SEQ ID NO：4)。

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: and 5, as follows:

MGVKVLFALICIAVAEA(SEQ ID NO：5)。

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 affinity 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 sequence SEQ ID NO: 6 or SEQ ID NO: 7, and:

MGVKVLFALICIAVAEAEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGGGG SGGGGSKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDHHHHHH(SEQ ID NO：6)；

MGVKVLFALICIAVAEAEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGGGG SGGGGSKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDHHHHHH(SEQ ID NO：7)。

wherein the underlined sequence (MGVKVLFALICIAVAEA) Represents a signal peptide; underlined sequence (GGGGSGGGGS) Indicates a flexible peptide; underlined sequence (HHHHHH) Represents the 6 × histidine tag sequence.

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

In a preferred embodiment, the isolated nucleic acid comprises a monomeric streptavidin full-length protein coding sequence and a gauss luciferase full-length protein coding sequence or a persistent light-emitting mutant coding sequence, the monomeric streptavidin full-length protein coding sequence and the gauss luciferase full-length protein coding sequence or the persistent light-emitting mutant coding sequence being linked by a linker sequence.

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 monomeric streptavidin full-length protein coding sequence is as follows SEQ ID NO: 8, showing: GAGTTTGCTTCCGCCGAGGCTGGCATCACCGGCACATGGTACAACCAGCACGGAAGCACCTTCACAGTGACCGCTGGAGCTGATGGAAACCTGACAGGACAGTATGAGAATAGGGCTCAGGGCACCGGCTGCCAGAACTCTCCCTACACACTGACCGGCAGGTATAATGGCACCAAGCTGGAGTGGCGGGTGGAGTGGAACAATAGCACAGAGAATTGTCATTCTAGGACCGAGTGGAGGGGACAGTACCAGGGAGGAGCTGAGGCTAGGATCAACACACAGTGGAATCTGACCTATGAGGGAGGATCCGGACCAGCTACCGAGCAGGGCCAGGATACCTTCACCAAGGTGAAGCCTTCCGCCGCTAGC (SEQ ID NO: 8).

In a preferred embodiment, the full-length protein coding sequence for gauss luciferase is as follows SEQ ID NO: 9 is as follows: AAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGATGGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCATGGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGAC (SEQ ID NO: 9).

In a preferred embodiment, the persistent light mutant coding sequence is as set forth in SEQ ID NO: 10, and: AAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGCTCGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCCTCGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGAC (SEQ ID NO: 10). Wherein the underlined codons represent mutated bases relative to the coding sequence of the full-length protein of gauss luciferase.

In a preferred embodiment, the linker sequence is as set forth in SEQ ID NO: 11, and:

GGAGGAGGAGGATCTGGAGGAGGAGGATCC(SEQ ID NO：11)。

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: 12, and:

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCC(SEQ ID NO：12)。

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 expression of the affinity and light-emitting active portions of the fusion protein.

In a most preferred embodiment, the isolated nucleic acid sequence of the invention is as set forth in SEQ ID NO: 13 or SEQ ID NO: 14, in the following:

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCCGAGTTTGCTTCCGCCGAGGCTGGCATCACCGGCACATGGTACAACCAGCACGGAAGCACCTTCACAGTGACCGCTGGAGCTGATGGAAACCTGACAGGACAGTATGAGAATAGGGCTCAGGGCACCGGCTGCCAGAACTCTCCCTACACACTGACCGGCAGGTATAATGGCACCAAGCTGGAGTGGCGGGTGGAGTGGAACAATAGCACAGAGAATTGTCATTCTAGGACCGAGTGGAGGGGACAGTACCAGGGAGGAGCTGAGGCTAGGATCAACACACAGTGGAATCTGACCTATGAGGGAGGATCCGGACCAGCTACCGAGCAGGGCCAGGATACCTTCACCAAGGTGAAGCCTTCCGCCGCTAGCGGAGGAGGAGGATCTGGAGGAGGAGGAT CCAAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGATGGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCATGGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGACCACCATCACCATCACCAT(SEQ ID NO：13)；

ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCTGAGGCCGAGTTTGCTTCCGCCGAGGCTGGCATCACCGGCACATGGTACAACCAGCACGGAAGCACCTTCACAGTGACCGCTGGAGCTGATGGAAACCTGACAGGACAGTATGAGAATAGGGCTCAGGGCACCGGCTGCCAGAACTCTCCCTACACACTGACCGGCAGGTATAATGGCACCAAGCTGGAGTGGCGGGTGGAGTGGAACAATAGCACAGAGAATTGTCATTCTAGGACCGAGTGGAGGGGACAGTACCAGGGAGGAGCTGAGGCTAGGATCAACACACAGTGGAATCTGACCTATGAGGGAGGATCCGGACCAGCTACCGAGCAGGGCCAGGATACCTTCACCAAGGTGAAGCCTTCCGCCGCTAGCGGAGGAGGAGGATCTGGAGGAGGAGGAT CCAAGCCAACAGAGAACAATGAGGATTTCAACATCGTGGCTGTGGCCAGCAATTTTGCTACCACAGACCTGGATGCCGACAGAGGCAAGCTGCCAGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGCTCGAGGCTAACGCTAGGAAGGCTGGATGTACCAGGGGATGCCTGATCTGTCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCAGGCCGCTGTCATACCTACGAGGGCGATAAGGAGTCCGCTCAGGGAGGAATCGGAGAGGCCATCGTGGATATCCCCGAGATCCCTGGCTTCAAGGACCTGGAGCCCCTCGAGCAGTTTATCGCTCAGGTGGATCTGTGCGTGGACTGTACCACAGGCTGCCTGAAGGGCCTGGCCAATGTGCAGTGTTCCGACCTGCTGAAGAAGTGGCTGCCTCAGAGGTGCGCTACCTTTGCCAGCAAGATCCAGGGCCAGGTGGATAAGATCAAGGGAGCTGGAGGCGACCACCATCACCATCACCAT(SEQ ID NO：14)。

wherein, SEQ ID NO: 13 or SEQ ID NO: among the 14 sequences, underlined sequences from the 5 'end to the 3' end are a signal peptide coding sequence, a flexible peptide coding sequence (linker 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 a biotin molecule.

The invention is useful for detecting biotin molecules, which can be in free form, independently, or any molecule bearing a biotin label, such as a biotin-labeled protein. In some embodiments, the biotin is labeled on a substrate to be detected, and the substrate can be detected by detecting a biotin molecule on the substrate. The invention has wide application in the fields of DNA hybridization, immunodetection, biochemical diagnosis and the like.

The fusion protein can conveniently and specifically identify biotin molecules, has the function of catalyzing substrate oxidation luminescence, and is convenient to detect. Compared with the coupling protein of the Gaussian luciferase and the streptavidin in the prior art, the fusion protein has the advantages of simple process and convenient production.

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 fusion expression vectors

As shown in FIG. 1, MonoSA gene and Gauss luciferase gene are used as templates, synthetic primers are designed, PCR amplification is carried out, and amplification products of the MonoSA and Gauss luciferase genes are obtained respectively. Based on the PCR product, a second round of bypass PCR is carried out to obtain MonoSA and Gaussian luciferase fusion gene amplification product (the sequence is shown as SEQ ID NO: 13). And (3) carrying out electrophoresis detection on the PCR amplification product by using 1% agarose gel, and recovering the product by using a gel recovery kit. The recovered product and the vector plasmid pcDNA3.1(+) are respectively subjected to double enzyme digestion reaction by EcoR I and Hind III, electrophoresis detection is carried out by 1% agarose gel after the reaction is finished, and the product is recovered by a gel recovery kit. The PCR-digested product and the plasmid-digested product were mixed at a molar ratio of 3:1 and ligated with T4 DNA ligase. The ligation products were transformed into TOP10 competent, 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 was performed to ensure correct sequence after PCR was identified as positive.

The mutant G2L fusion expression vector is based on the wild type fusion protein expression vector which is successfully prepared, plasmid PCR (wherein, the fusion gene sequences of MonoSA and the continuous luminous mutant are shown in SEQ ID NO: 14) is carried out by designing a primer containing a mutant gene, then the PCR product is treated by Dpn I enzyme, and the reaction is carried out 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

25 mu g of the constructed pcDNA3.1-MonoSA-Gluc fusion expression plasmid is uniformly mixed with 1ml of cell culture medium. Another 920. mu.L of cell culture medium and 80. mu.L of lip were takeno2000 transfection reagent (invitrogen) was mixed well. 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 prepared⁶For the cells of the/ml, the mixed mixture of pcDNA3.1-MonoSA-Gluc fusion expression plasmid and transfection reagent is added into CHO cells and is shaken up gently. 37 ℃ and 8% CO₂Cultured under the conditions for 3 days.

The cell culture fluid was collected, centrifuged at 6000rpm for 10min, and the supernatant was collected. The supernatant was filtered and transferred to a nickel column and 10ml of PBS was equilibrated. Then 10ml was washed with a buffer (20mM Tris, pH 8.0, 150mM NaCl, 20mM imidazole) to remove the hetero-protein, and finally eluted with a buffer (20mM Tris, pH 8.0, 150mM NaCl, 500mM imidazole). The eluted protein was detected by 12% SDS-PAGE, and the result of the detection by SDS-PAGE of the MonoSA-Gluc fusion protein is shown in FIG. 2. Shows that the MonoSA-Gluc fusion protein is successfully expressed.

The same procedure was used for the pcDNA3.1-MonoSA-G2L fusion expression plasmid.

Example 3: fusion protein luciferase bioluminescent assay

MonoSA-Gluc fusion protein and MonoSA-G2L fusion protein were diluted to 3nM with substrate diluent (50mM Tris, pH 8.0, 100mM NaCl), respectively, and 10. mu.L of each was added to a 96-well microplate. Then, 90. mu.L of coelenterazine diluted to 10. mu.M with the same solution was added, and the luminescence intensity was read with a luminescence module of a microplate reader, with the result shown in FIG. 3, where the luminescence intensity of the wild-type MonoSA-Gluc fusion protein was equivalent to that of the Gluc protein of the same concentration. The mutant fusion protein MonoSA-G2L fusion protein has the luminous intensity equivalent to that of Gluc, but obviously shows the continuous luminous characteristic, and the luminescence is not obviously weakened after 30 s.

Example 4: elisa test experiment for binding capacity of fusion protein and biotin

Preparing a solution:

PBS: 138mM NaCl, 2.6mM KCl (in 10mM potassium phosphate solution, pH 7.4);

PBST: PBS plus 0.05% Tween 20;

sealing liquid: PBS added with 40 mug/mL BSA;

diluting the solution: PBS was added with 0.15. mu.g/mL BSA.

(1) A96-well plate was used, 100. mu.L of biotin-labeled BSA (10 ng/. mu.g) was added to each well, and the plate was coated at 37 ℃ for 1 hour.

(2) The coating solution was decanted off and washed 3 times with 100. mu.L PBST for 5 minutes each.

(3) Add 100. mu.L of blocking solution to each well and block for 1 hour at 37 ℃.

(4) The blocking solution was decanted off and washed 3 times with 100. mu.L of PBST for 5 minutes each.

(5) MonoSA-Gluc fusion protein and MonoSA-G2L fusion protein were diluted to 500nM with diluent, and then diluted to 100nM,20nM,4nM,0.8nM,0.16nM and 0.032nM in 5-fold gradients, and the dilutions of each gradient were added to 96-well plates in sequence, with 3 replicate wells per gradient. The diluent without the fusion protein was used as a negative control. Binding was carried out at 37 ℃ for 1 hour.

(6) The plate was decanted and washed 3 times with 100. mu.L of PBST for 5 minutes each.

(7) The horseradish peroxidase-labeled SA antibody was diluted with a diluent and added to a 96-well plate at 100. mu.L per well, and bound at 37 ℃ for 1 hour.

(8) The plate was decanted and washed 3 times with 100. mu.L of PBST for 5 minutes each. The cells were washed 3 times with 100. mu.L PBS by gentle shaking for 5 minutes each.

(9) The luminescence was developed using a DMB luminescence kit (Biotech). The kit is operated according to the kit instructions, and after color development, the absorbance value of 450nM is read by a microplate reader.

(10) And (4) counting the light absorption values of all concentrations, taking the average of 3 repeated holes, making a corresponding curve of the concentrations and the light absorption values, and calculating the binding force constant.

The results are shown in FIG. 4, which shows that the binding force of the wild-type MonoSA-Gluc fusion protein and the sustained luminescent mutant MonoSA-G2L to biotin are both 10^-9An order of magnitude.

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> monomer streptavidin and Gaussian luciferase and application thereof

<130> 18I27211

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 123

<212> PRT

<213> Artificial sequence

<400> 1

Glu Phe Ala Ser Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln

1 5 10 15

His Gly Ser Thr Phe Thr Val Thr Ala Gly Ala Asp Gly Asn Leu Thr

20 25 30

Gly Gln Tyr Glu Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser Pro

35 40 45

Tyr Thr Leu Thr Gly Arg Tyr Asn Gly Thr Lys Leu Glu Trp Arg Val

50 55 60

Glu Trp Asn Asn Ser Thr Glu Asn Cys His Ser Arg Thr Glu Trp Arg

65 70 75 80

Gly Gln Tyr Gln Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Asn

85 90 95

Leu Thr Tyr Glu Gly Gly Ser Gly Pro Ala Thr Glu Gln Gly Gln Asp

100 105 110

Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120

<210> 2

<211> 168

<212> PRT

<213> Artificial sequence

<400> 2

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> 3

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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

<210> 4

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 5

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<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

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<211> 324

<212> PRT

<213> Artificial sequence

<400> 6

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

1 5 10 15

Ala Glu Phe Ala Ser Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn

20 25 30

Gln His Gly Ser Thr Phe Thr Val Thr Ala Gly Ala Asp Gly Asn Leu

35 40 45

Thr Gly Gln Tyr Glu Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser

50 55 60

Pro Tyr Thr Leu Thr Gly Arg Tyr Asn Gly Thr Lys Leu Glu Trp Arg

65 70 75 80

Val Glu Trp Asn Asn Ser Thr Glu Asn Cys His Ser Arg Thr Glu Trp

85 90 95

Arg Gly Gln Tyr Gln Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp

100 105 110

Asn Leu Thr Tyr Glu Gly Gly Ser Gly Pro Ala Thr Glu Gln Gly Gln

115 120 125

Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn

145 150 155 160

Ile Val Ala Val Ala Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp

165 170 175

Arg Gly Lys Leu Pro Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu

180 185 190

Met Glu Ala Asn Ala Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile

195 200 205

Cys Leu Ser His Ile Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro

210 215 220

Gly Arg Cys His Thr Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly

225 230 235 240

Ile Gly Glu Ala Ile Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp

245 250 255

Leu Glu Pro Met Glu Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp

260 265 270

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

275 280 285

Leu Leu Lys Lys Trp Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys

290 295 300

Ile Gln Gly Gln Val Asp Lys Ile Lys Gly Ala Gly Gly Asp His His

305 310 315 320

His His His His

<210> 7

<211> 324

<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 Glu Phe Ala Ser Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn

20 25 30

Gln His Gly Ser Thr Phe Thr Val Thr Ala Gly Ala Asp Gly Asn Leu

35 40 45

Thr Gly Gln Tyr Glu Asn Arg Ala Gln Gly Thr Gly Cys Gln Asn Ser

50 55 60

Pro Tyr Thr Leu Thr Gly Arg Tyr Asn Gly Thr Lys Leu Glu Trp Arg

65 70 75 80

Val Glu Trp Asn Asn Ser Thr Glu Asn Cys His Ser Arg Thr Glu Trp

85 90 95

Arg Gly Gln Tyr Gln Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp

100 105 110

Asn Leu Thr Tyr Glu Gly Gly Ser Gly Pro Ala Thr Glu Gln Gly Gln

115 120 125

Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn

145 150 155 160

Ile Val Ala Val Ala Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp

165 170 175

Arg Gly Lys Leu Pro Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu

180 185 190

Leu Glu Ala Asn Ala Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile

195 200 205

Cys Leu Ser His Ile Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro

210 215 220

Gly Arg Cys His Thr Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly

225 230 235 240

Ile Gly Glu Ala Ile Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp

245 250 255

Leu Glu Pro Leu Glu Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp

260 265 270

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

275 280 285

Leu Leu Lys Lys Trp Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys

290 295 300

Ile Gln Gly Gln Val Asp Lys Ile Lys Gly Ala Gly Gly Asp His His

305 310 315 320

His His His His

<210> 8

<211> 369

<212> DNA

<213> Artificial sequence

<400> 8

gagtttgctt ccgccgaggc tggcatcacc ggcacatggt acaaccagca cggaagcacc 60

ttcacagtga ccgctggagc tgatggaaac ctgacaggac agtatgagaa tagggctcag 120

ggcaccggct gccagaactc tccctacaca ctgaccggca ggtataatgg caccaagctg 180

gagtggcggg tggagtggaa caatagcaca gagaattgtc attctaggac cgagtggagg 240

ggacagtacc agggaggagc tgaggctagg atcaacacac agtggaatct gacctatgag 300

ggaggatccg gaccagctac cgagcagggc caggatacct tcaccaaggt gaagccttcc 360

gccgctagc 369

<210> 9

<211> 504

<212> DNA

<213> Artificial sequence

<400> 9

aagccaacag agaacaatga ggatttcaac atcgtggctg tggccagcaa ttttgctacc 60

acagacctgg atgccgacag aggcaagctg ccaggcaaga agctgcccct ggaggtgctg 120

aaggagatgg aggctaacgc taggaaggct ggatgtacca ggggatgcct gatctgtctg 180

tctcacatca agtgcacacc taagatgaag aagttcatcc caggccgctg tcatacctac 240

gagggcgata aggagtccgc tcagggagga atcggagagg ccatcgtgga tatccccgag 300

atccctggct tcaaggacct ggagcccatg gagcagttta tcgctcaggt ggatctgtgc 360

gtggactgta ccacaggctg cctgaagggc ctggccaatg tgcagtgttc cgacctgctg 420

aagaagtggc tgcctcagag gtgcgctacc tttgccagca agatccaggg ccaggtggat 480

aagatcaagg gagctggagg cgac 504

<210> 10

<211> 504

<212> DNA

<213> Artificial sequence

<400> 10

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> 11

<211> 30

<212> DNA

<213> Artificial sequence

<400> 11

ggaggaggag gatctggagg aggaggatcc 30

<210> 12

<211> 51

<212> DNA

<213> Artificial sequence

<400> 12

atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggctgaggc c 51

<210> 13

<211> 972

<212> DNA

<213> Artificial sequence

<400> 13

atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggctgaggc cgagtttgct 60

tccgccgagg ctggcatcac cggcacatgg tacaaccagc acggaagcac cttcacagtg 120

accgctggag ctgatggaaa cctgacagga cagtatgaga atagggctca gggcaccggc 180

tgccagaact ctccctacac actgaccggc aggtataatg gcaccaagct ggagtggcgg 240

gtggagtgga acaatagcac agagaattgt cattctagga ccgagtggag gggacagtac 300

cagggaggag ctgaggctag gatcaacaca cagtggaatc tgacctatga gggaggatcc 360

ggaccagcta ccgagcaggg ccaggatacc ttcaccaagg tgaagccttc cgccgctagc 420

ggaggaggag gatctggagg aggaggatcc aagccaacag agaacaatga ggatttcaac 480

atcgtggctg tggccagcaa ttttgctacc acagacctgg atgccgacag aggcaagctg 540

ccaggcaaga agctgcccct ggaggtgctg aaggagatgg aggctaacgc taggaaggct 600

ggatgtacca ggggatgcct gatctgtctg tctcacatca agtgcacacc taagatgaag 660

aagttcatcc caggccgctg tcatacctac gagggcgata aggagtccgc tcagggagga 720

atcggagagg ccatcgtgga tatccccgag atccctggct tcaaggacct ggagcccatg 780

gagcagttta tcgctcaggt ggatctgtgc gtggactgta ccacaggctg cctgaagggc 840

ctggccaatg tgcagtgttc cgacctgctg aagaagtggc tgcctcagag gtgcgctacc 900

tttgccagca agatccaggg ccaggtggat aagatcaagg gagctggagg cgaccaccat 960

caccatcacc at 972

<210> 14

<211> 972

<212> DNA

<213> Artificial sequence

<400> 14

atgggcgtga aggtgctgtt cgccctgatc tgcatcgccg tggctgaggc cgagtttgct 60

tccgccgagg ctggcatcac cggcacatgg tacaaccagc acggaagcac cttcacagtg 120

accgctggag ctgatggaaa cctgacagga cagtatgaga atagggctca gggcaccggc 180

tgccagaact ctccctacac actgaccggc aggtataatg gcaccaagct ggagtggcgg 240

gtggagtgga acaatagcac agagaattgt cattctagga ccgagtggag gggacagtac 300

cagggaggag ctgaggctag gatcaacaca cagtggaatc tgacctatga gggaggatcc 360

ggaccagcta ccgagcaggg ccaggatacc ttcaccaagg tgaagccttc cgccgctagc 420

ggaggaggag gatctggagg aggaggatcc aagccaacag agaacaatga ggatttcaac 480

atcgtggctg tggccagcaa ttttgctacc acagacctgg atgccgacag aggcaagctg 540

ccaggcaaga agctgcccct ggaggtgctg aaggagctcg aggctaacgc taggaaggct 600

ggatgtacca ggggatgcct gatctgtctg tctcacatca agtgcacacc taagatgaag 660

aagttcatcc caggccgctg tcatacctac gagggcgata aggagtccgc tcagggagga 720

atcggagagg ccatcgtgga tatccccgag atccctggct tcaaggacct ggagcccctc 780

gagcagttta tcgctcaggt ggatctgtgc gtggactgta ccacaggctg cctgaagggc 840

ctggccaatg tgcagtgttc cgacctgctg aagaagtggc tgcctcagag gtgcgctacc 900

tttgccagca agatccaggg ccaggtggat aagatcaagg gagctggagg cgaccaccat 960

caccatcacc at 972

<210> 15

<211> 6

<212> PRT

<213> Artificial sequence

<400> 15

Gly Gly Gly Gly Gly Ser

1 5

<210> 16

<211> 11

<212> PRT

<213> Artificial sequence

<400> 16

Gly Gln Gly Gln Gly Gln Gly Gln Gly Gln Gly

1 5 10

<210> 17

<211> 15

<212> PRT

<213> Artificial sequence

<400> 17

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

1 5 10 15

<210> 18

<211> 10

<212> PRT

<213> Artificial sequence

<400> 18

Val Pro Gly Val Gly Val Pro Gly Val Gly

1 5 10

<210> 19

<211> 8

<212> PRT

<213> Artificial sequence

<400> 19

Ser Ala Pro Gly Thr Pro Ser Arg

1 5

<210> 20

<211> 14

<212> PRT

<213> Artificial sequence

<400> 20

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

1 5 10

<210> 21

<211> 6

<212> PRT

<213> Artificial sequence

<400> 21

Gly Ser Gly Gly Ser Gly

1 5

<210> 22

<211> 8

<212> PRT

<213> Artificial sequence

<400> 22

Gly Ser Gly Gly Ser Gly Gly Gly

1 5

Claims

1. A fusion protein of monomer streptavidin and Gaussian luciferase, which is characterized by comprising a full-length monomer streptavidin protein and a continuous luminescence mutant of Gaussian luciferase, wherein the continuous luminescence mutant has a sequence shown in SEQ ID NO: 3, the monomeric streptavidin full-length protein is connected with the Gaussian luciferase continuous luminescence mutant through flexible peptide.

2. The fusion protein of claim 1, wherein the monomeric streptavidin full-length protein has a sequence as set forth in SEQ ID NO: 1 is shown.

3. The fusion protein of claim 1, wherein the flexible peptide sequence is as set forth in SEQ ID NO: 4, respectively.

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

5. The fusion protein of claim 4, wherein the signal peptide is located at the N-terminus of the fusion protein.

6. The fusion protein of claim 4, wherein the signal peptide sequence is as set forth in SEQ ID NO: 5, respectively.

7. The fusion protein of claim 1, further comprising: a fusion tag for affinity purification of the fusion protein.

8. The fusion protein of claim 7, wherein the fusion tag is a 6 × histidine tag.

9. The fusion protein of claim 1, wherein the fusion protein has the sequence set forth in SEQ ID NO: shown at 7.

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

11. The isolated nucleic acid of claim 10, wherein the isolated nucleic acid comprises a monomeric streptavidin full-length protein coding sequence and a gaussian luciferase persistent light mutant coding sequence as set forth in SEQ ID NO: 10 is shown in the figure; the coding sequence of the monomer streptavidin full-length protein is connected with the coding sequence of the continuous luminous mutant of the Gaussian luciferase through a connecting sequence.

12. The isolated nucleic acid of claim 11, wherein the monomeric streptavidin full-length protein coding sequence is set forth in SEQ ID NO: shown in fig. 8.

13. The isolated nucleic acid of claim 11, wherein the linker sequence is set forth in SEQ ID NO: shown at 11.

14. The isolated nucleic acid of claim 11, further comprising: a signal peptide coding sequence which encodes a signal peptide for directing secretion of the fusion protein from the expressing cell to the outside.

15. The isolated nucleic acid of claim 14, wherein the signal peptide coding sequence is located 5' to the isolated nucleic acid.

16. The isolated nucleic acid of claim 14, wherein the signal peptide coding sequence is set forth in SEQ ID NO: shown at 12.

17. The isolated nucleic acid of claim 11, further comprising: a fusion tag coding sequence encoding a fusion tag for affinity purification of the fusion protein.

18. The isolated nucleic acid of claim 17, wherein the fusion tag coding sequence is a sequence encoding a 6 x histidine tag.

19. The isolated nucleic acid of claim 10, wherein the isolated nucleic acid sequence is as set forth in SEQ ID NO: as shown at 14.

20. An expression vector comprising a nucleic acid sequence encoding the fusion protein of any one of claims 1-9, and a vector backbone sequence.

21. A recombinant host cell comprising the expression vector of claim 20.

22. Use of the fusion protein of any one of claims 1 to 9, the isolated nucleic acid of any one of claims 10-19, the expression vector of claim 20, or the recombinant host cell of claim 21 in a biotin molecule assay.