CN109734793B - ZnT8 recombinant protein and preparation method and application thereof - Google Patents

Abstract

The invention provides a ZnT8 recombinant protein, a preparation method and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO.1, the nucleotide sequence of the protein is shown as SEQ ID NO.2, the invention transfects Expi293 eukaryotic cells by analyzing and screening target fragments, optimizing coding sequences and vectors, and stably expresses the protein with antigen activity, thereby providing a preparation method which can not only retain the biological activity of natural protein, but also reduce the production cost, and having wide application prospect and huge market value.

Description

ZnT8 recombinant protein and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a ZnT8 recombinant protein, and a preparation method and application thereof.

Background

Zinc transporter 8 (Zinc transporter 8, znt 8), is a member of the SLC30 family (ZnTs proteins), the SLC30 family being responsible for the transport of intracellular Zinc ions into the extracellular matrix or intracellular vesicles. ZnT8 is specifically expressed in pancreatic tissue and mainly located in islet beta cells, and over-expression of ZnT8 can further increase insulin secretion by the beta cells in response to glucose stimulation by increasing the density of insulin secretion channels, thereby regulating the processes of insulin synthesis, storage and secretion. The ZnT8 autoantibody can be an important type I diabetes marker and becomes an important supplement of the current diagnostic reagent, the specificity of the ZnT8 in the TIDM detection is as high as 98%, and 37.5% of positive rate still exists in a negative sample of the diabetes detection, so that the occurrence of missed diagnosis is effectively avoided.

The ZnT8 protein, in dimeric form, localizes on the β -cell insulin secretion/storage vesicle membrane regulating cytosolic and vesicle-reduced zinc transport. The ZnT8 protein monomer consists of 369 amino acids, the coding gene SLC30A8 is 1100bp in full length, the protein has a relative molecular weight of 40kD, and contains 6 transmembrane alpha helices (I-VI), wherein a loop between an IV structural domain and a V structural domain is rich in histidine. The C end and the N end of the protein are both positioned in cytoplasm, the C end is composed of two alpha helices (aa 284-292, aa 327-341) and three beta folds (aa 299-308, aa313-321 and aa 345-352), wherein the first two beta folds form a hairpin structure. In 2004, the scholars clone the SLC30A8 gene from the human pancreatic island cell for the first time and express the ZnT8 protein in the HeLa cell, but because the ZnT8 molecular structure is complex and is difficult to express in a large amount in a prokaryotic system, the main method for preparing the antigen protein by utilizing the reticulocyte in-vitro cell-free expression system is adopted at present, but the ZnT8 protein prepared by the method has low content and high cost, and the large-scale clinical application is restricted. CN102250242A discloses a ZnT-8 protein specifically expressed in pancreatic islet beta cells, polynucleotides encoding the above proteins related to insulin maturation and efflux, and their use, for example, in sorting and studying beta cells and screening drugs for treating diabetes and hyperinsulinemia. However, the total amount of protein expression in the prior art is low, and the preparation process is complicated.

The systems currently used for in vitro protein synthesis are mainly prokaryotic expression systems, yeast expression systems, mammalian cell and insect cell expression systems. Prokaryotic expression systems are the most commonly used expression systems, and are also the most economical protein expression systems. The prokaryotic expression system has the advantages of clear genetic background, low cost, high expression quantity, relatively simple separation and purification of expression products and the like, and has the defects of lack of processing mechanisms after protein translation, such as formation of disulfide bonds, protein glycosylation and correct folding, and small probability of obtaining the protein with biological activity. The yeast protein expression system has the advantages of high expression quantity, inductivity, glycosylation mechanism close to that of higher eukaryotes, easy purification of secreted protein, easy realization of high-density fermentation and the like, but protein products are easy to degrade, and the expression quantity is uncontrollable. The main advantages of mammalian cell and insect cell expression systems are that the post-translational processing mechanism of proteins is closest to the natural form in vivo, the biological activity is most easily retained, and the disadvantages are that the expression amount is generally low and the production cost is high.

The activity of the antigenic protein used to capture autoantibodies depends directly on the conformation of the antigenic protein, and low concentrations of ZnT8 autoantibodies can only be captured when the activity of the ZnT8 antigenic protein conforms to the conformation of the native ZnT8 protein in humans. The natural ZnT8 protein is an animal-derived transmembrane protein, the molecular structure of the protein contains a histidine-rich region, namely a binding site of zinc ions, and the protein comprises 6 transmembrane regions and a histidine loop between the IV region and the V region, wherein 50 percent of molecules are embedded in a phospholipid bilayer, the molecular structure is complex, and heterologous expression is difficult.

Therefore, the search and exploration for the preparation of protein expression methods which not only have the biological activity of natural proteins, but also can reduce the production cost is a technical problem to be solved at present.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a ZnT8 recombinant protein and a preparation method and application thereof, the invention transfects Expi293 eukaryotic cells by analyzing and screening target fragments, optimizing coding sequences and vectors and stably expressing proteins with antigen activity, provides a preparation method which not only has the biological activity of natural proteins but also can reduce the production cost, and has wide application prospect and great market value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a ZnT8 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO. 1.

SEQ ID NO.1 is as follows:

MALWMRLLPLLALLALWGPDPAAAHQRCLGHNHKEVQANASVRAAFVHALGDLFQSISVLISALIIYFKPEYKIADPICTFIFSILVLASTITILKDFSILLMEGVPKSLNYSGVKELILAVDGVLSVHSLHIWSLTMNQVILSAHVATAASRDSQVVRREIAKALSKSFTMHSLTIQMESPVDQDPDCLFCEDPCDGGSHHHHHHDYKDHDGDYKDHDIDYKDDDDK.

preferably, the nucleotide sequence of the protein is shown as SEQ ID NO. 2.

SEQ ID NO.2 is as follows:

ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGAACCTGACCCAGCCGCAGCCCACCAGCGGTGCCTGGGCCACAATCACAAAGAGGTGCAGGCCAACGCTTCTGTGCGGGCTGCTTTTGTTCACGCCCTGGGCGATCTGTTCCAGTCCATCTCCGTGCTGATCTCTGCCCTGATCATCTACTTCAAGCCCGAGTACAAGATCGCTGACCCCATCTGCACCTTCATCTTCTCCATCCTGGTGCTGGCCTCTACCATCACAATCCTGAAGGACTTCAGCATCCTGCTGATGGAAGGCGTGCCCAAGAGCCTGAACTACTCCGGCGTGAAAGAACTGATCCTGGCCGTGGATGGCGTGCTGTCTGTGCACTCTCTGCACATCTGGTCCCTGACCATGAATCAAGTGATCCTGAGCGCCCACGTGGCCACCGCTGCTTCTAGAGATTCTCAGGTCGTGCGGAGAGAGATCGCCAAGGCTCTGTCCAAGAGCTTCACCATGCACAGCCTGACCATCCAGATGGAAAGCCCCGTGGATCAGGACCCCGACTGTCTGTTTTGCGAGGACCCTTGCGACGGCGGCTCTCACCACCACCATCACCACGACTACAAGGACCACGACGGCGATTACAAGGATCATGACATCGACTATAAGGACGATGACGACAAGTGA.

in the invention, in the long-term scientific research and practice process, in order to research and develop a protein which has the biological activity of natural protein and can also obviously reduce the production cost and a preparation method, deeply research the physicochemical property of target protein and a technical method for protein expression, widely analyze and screen effective means and ways, through analyzing and screening target fragments, optimizing a coding sequence and a carrier, selecting and transfecting specific Expi293 eukaryotic cells, and under the synergistic interaction and mutual cooperation of all the steps, the stable expression of the protein with the antigen activity is finally realized, and the preparation method which has the biological activity of the natural protein and can also reduce the production cost is provided, so that the preparation method has wide application prospect and great market value.

Since the ZnT8 protein is an animal-derived transmembrane protein and comprises 6 transmembrane regions and a histidine loop between the IV region and the V region, 50 percent of molecules are embedded in a phospholipid bilayer, the molecular structure is complex, so that the protein is difficult to express in a large quantity and is difficult to discharge to a culture medium after expression, the purification and the application of the protein are not facilitated, and the improvement of a protein expression system is needed. Firstly, the protein can not be discharged and dissolved in a culture medium, and the inventor connects a section of signal peptide coding sequence in series at the front end of the coding sequence of the target protein, so that the protein can pass through a cell membrane to be discharged to cell sap after being expressed, and the purification and collection are convenient; secondly, because the expression yield of the protein is low, the expression can be realized by optimizing an expression vector, and the inventor adds a CAG enhancer element at the downstream of a CMV promoter and at the downstream of a multiple cloning site respectively so as to enhance the transcription level of the target protein.

In a second aspect, the present invention provides a recombinant plasmid comprising an expression vector and a gene of interest, said gene of interest comprising the nucleotide sequence of the protein of the first aspect.

Preferably, the nucleotide sequence of the expression vector of the recombinant plasmid is shown as SEQ ID NO. 3;

the original pcDNA3.1 (+) plasmid is 5428bp, and after two enhancer sequences are inserted, it is 6004bp

SEQ ID NO.3 is as follows:

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGA CGTC.

preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 4.

SEQ ID NO.4 is as follows:

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCCGGCCACCATGATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGAACCTGACCCAGCCGCAGCCCACCAGCGGTGCCTGGGCCACAATCACAAAGAGGTGCAGGCCAACGCTTCTGTGCGGGCTGCTTTTGTTCACGCCCTGGGCGATCTGTTCCAGTCCATCTCCGTGCTGATCTCTGCCCTGATCATCTACTTCAAGCCCGAGTACAAGATCGCTGACCCCATCTGCACCTTCATCTTCTCCATCCTGGTGCTGGCCTCTACCATCACAATCCTGAAGGACTTCAGCATCCTGCTGATGGAAGGCGTGCCCAAGAGCCTGAACTACTCCGGCGTGAAAGAACTGATCCTGGCCGTGGATGGCGTGCTGTCTGTGCACTCTCTGCACATCTGGTCCCTGACCATGAATCAAGTGATCCTGAGCGCCCACGTGGCCACCGCTGCTTCTAGAGATTCTCAGGTCGTGCGGAGAGAGATCGCCAAGGCTCTGTCCAAGAGCTTCACCATGCACAGCCTGACCATCCAGATGGAAAGCCCCGTGGATCAGGACCCCGACTGTCTGTTTTGCGAGGACCCTTGCGACGGCGGCTCTCACCACCACCATCACCACGACTACAAGGACCACGACGGCGATTACAAGGATCATGACATCGACTATAAGGACGATGACGACAAGTGACCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC.

in a third aspect, the present invention provides a recombinant strain comprising the recombinant plasmid of the second aspect.

In a fourth aspect, the invention provides an Expi293 cell expression system comprising the recombinant plasmid of the second aspect.

In a fifth aspect, the present invention provides a use of a recombinant protein according to the first aspect for the preparation of an immunoblotting kit and/or a medicament.

In a sixth aspect, the present invention provides a method for preparing the ZnT8 recombinant protein of the first aspect, comprising the steps of:

(1) Analyzing and screening an initial target gene sequence from a ZnT8 full-length gene coding sequence;

(2) Fusing a signal peptide sequence and a C-terminal fusion tag sequence at the N terminal and the C terminal of the target gene in the step (1) to obtain an optimized target gene;

(3) Adding a CAG enhancer sequence at each of two ends of a multiple cloning site at the downstream of a CMV promoter of an expression vector pcDNA3.1 (+) to obtain an optimized expression vector;

(4) Synthesizing the target gene sequence optimized in the step (2) and the expression vector sequence optimized in the step (3), constructing a recombinant plasmid, and transforming the recombinant plasmid to prokaryotic cells for storage;

(5) And (5) transfecting the recombinant plasmid obtained in the step (4) into an Expi293 eukaryotic expression system for expression to obtain the ZnT8 recombinant protein.

Preferably, the nucleotide sequence of the initial target gene in the step (1) is shown as SEQ ID NO.5;

SEQ ID NO.5 is as follows:

nucleotide sequence of the original gene of interest:

CACCAGCGGTGCCTGGGCCACAATCACAAAGAGGTGCAGGCCAACGCTTCTGTGCGGGCTGCTTTTGTTCACGCCCTGGGCGATCTGTTCCAGTCCATCTCCGTGCTGATCTCTGCCCTGATCATCTACTTCAAGCCCGAGTACAAGATCGCTGACCCCATCTGCACCTTCATCTTCTCCATCCTGGTGCTGGCCTCTACCATCACAATCCTGAAGGACTTCAGCATCCTGCTGATGGAAGGCGTGCCCAAGAGCCTGAACTACTCCGGCGTGAAAGAACTGATCCTGGCCGTGGATGGCGTGCTGTCTGTGCACTCTCTGCACATCTGGTCCCTGACCATGAATCAAGTGATCCTGAGCGCCCACGTGGCCACCGCTGCTTCTAGAGATTCTCAGGTCGTGCGGAGAGAGATCGCCAAGGCTCTGTCCAAGAGCTTCACCATGCACAGCCTGACCATCCAGATGGAAAGCCCCGTGGATCAGGACCCCGACTGTCTGTTTTGCGAGGACCCTTGCGAC.

preferably, the nucleotide sequence of the signal peptide in the step (2) is shown as SEQ ID NO. 6;

SEQ ID NO.6 is as follows:

ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGAACCTGACCCAGCCGCAGCC.

preferably, the tag of step (2) comprises any one of a 6 × his tag, a GST tag, a c-Myc tag, or a fluorescent protein tag, or a combination of at least two thereof, preferably a 6 × his tag.

Commonly used protein tags include: 6 x his tag, GST tag, c-Myc tag, fluorescent protein tag and the like, and the 6 x his tag is widely applied to the related technical fields of protein expression, purification, identification, functional research and the like.

Preferably, the nucleotide sequence of the optimized target gene in the step (2) is shown in SEQ ID NO. 2.

Preferably, the enhancer sequence of step (3) is inserted at the following position: a CAG enhancer was added downstream of the CMV promoter, 3bp upstream of the T7 promoter and after the BGH poly (A) site downstream of the multiple cloning site.

Site of enhancer insertion: a CAG enhancer is added at the downstream of the CMV promoter, 3bp at the upstream of the T7 promoter and after a BGH poly (A) site at the downstream of the multiple cloning site so as to enhance the transcription level of the target protein.

Preferably, the nucleotide sequence of the optimized expression vector in the step (3) is shown as SEQ ID NO. 3.

Preferably, the nucleotide sequence of the recombinant plasmid in the step (4) is shown as SEQ ID NO. 4.

Preferably, the prokaryotic cell of step (4) comprises any one of DH5 α, BL21, TOP10 or Rosetta, preferably e.

The commonly used prokaryotic host bacteria include DH5 alpha, BL21, TOP10, rosetta, etc., while the prokaryotic strain in this study is mainly used for preservation and cloning of recombinant plasmid, and DH5 alpha is usually selected.

As a preferred technical scheme, the method for preparing the ZnT8 recombinant protein comprises the following steps:

(1) Analyzing and screening an initial target gene sequence shown as SEQ ID NO.5 from a ZnT8 full-length gene coding sequence;

(2) Fusing a signal peptide sequence shown as SEQ ID NO.6 and a C-terminal fusion tag sequence at the N terminal of the target gene in the step (1) to obtain an optimized target gene shown as SEQ ID NO. 2;

(3) Adding a CAG enhancer sequence at each of two ends of a multiple cloning site at the downstream of a CMV promoter of an expression vector pcDNA3.1 (+) to obtain an optimized expression vector with a nucleotide sequence shown as SEQ ID NO. 3;

(4) Synthesizing the target gene sequence optimized in the step (2) and the expression vector sequence optimized in the step (3), constructing a recombinant plasmid with a nucleotide sequence shown in SEQ ID No.4, and transforming the recombinant plasmid into a prokaryotic cell E.coli DH5 alpha strain;

(5) And (5) transfecting the recombinant plasmid obtained in the step (4) into an Expi293 eukaryotic expression system for expression to obtain the ZnT8 recombinant protein.

The technical scheme is as follows:

1) Analyzing the ZnT8 full-length gene coding sequence by software, and selecting a gene fragment with good immunogenicity;

2) Optimizing a coding sequence, a fusion marker sequence and a signal peptide sequence;

3) Optimizing the vector and adding an enhancer repetitive sequence;

4) Synthesizing a target gene by using a genetic engineering technology, and constructing a recombinant expression plasmid;

5) Extracting plasmids, transfecting into Expi293 eukaryotic cells for expression;

6) Detecting the expression of the target protein product;

7) Affinity purifying to obtain target protein;

8) And (5) detecting the antigenicity.

ZnT8 protein is a member of a zinc ion transporter family, is mainly responsible for transporting zinc ions in cells to extracellular matrixes, znT8 is specifically expressed in pancreatic tissues, is a membrane structural protein of islet beta cells, contains a histidine-rich region, namely a binding site of zinc ions, in a molecular structure, comprises 6 transmembrane regions and a histidine loop between the IV region and the V region, wherein 50 percent of molecules are embedded in a phospholipid bilayer, the molecular structure is complex, the expression amount is low, heterologous expression is difficult, znT8 protein does not belong to an exocrine protein, and a signal peptide sequence is not contained in a ZnT8 protein gene, so that the expressed protein molecules are difficult to be discharged into a cell culture matrix through cell membranes, the burden of the cells is increased undoubtedly, and the protein expression of the cells is not facilitated.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention optimizes the original expression vector pcDNA3.1 (+), adds a CAG enhancer sequence at two ends of the multiple cloning site at the downstream of the CMV promoter respectively, improves the transcription level and increases the subsequent protein expression quantity;

(2) The invention fuses the signal peptide sequence of the immunoglobulin gene to the N end of the target gene, promotes the cell efflux efficiency of the expressed target protein, reduces the cell burden and is beneficial to the subsequent collection and purification of the target protein;

(3) The Expi293 eukaryotic expression system adopted by the invention can be used for expressing under the condition of high cell concentration, the Expi293 eukaryotic expression system is used, the operation method is simple and convenient, the expression period is short, and the expressed target protein can be ensured to have the natural conformation and antigenicity of the natural antigen protein.

Drawings

FIG. 1 is a technical flow chart of the present invention;

FIG. 2 is a view showing the result of agarose gel electrophoresis in the context of enzyme digestion of the ZnT8 recombinant plasmid of the present invention;

FIGS. 3 (A) -3 (B) are SDS-PAGE electrophoresis Coomassie brilliant blue staining patterns of recombinant ZnT8 protein expression of the present invention, FIG. 3 (A) is culture medium cell supernatant, and FIG. 3 (B) is cell;

FIGS. 3 (C) -3 (D) are SDS-PAGE electrophoresis Western Blot assays for recombinant ZnT8 protein expression of the invention, wherein FIG. 3 (C) is the culture medium cell supernatant and FIG. 3 (D) is the cells;

FIG. 4 is an SDS-PAGE electrophoresis of the purified recombinant ZnT8 protein of the present invention;

FIG. 5 is a detection chart of a diabetic patient serum immunoblotting membrane strip of the present invention.

Detailed Description

In order to further illustrate the technical means and effects of the present invention, the following detailed description is provided to further illustrate the technical solutions of the present invention with reference to the accompanying drawings, but the present invention is not limited to the scope of the embodiments.

Examples

1) Screening for Gene sequences of interest

Analyzing the ZnT8 full-length gene coding sequence published by GenBank, carrying out comprehensive analysis and prediction on the secondary structure, hydrophilicity, flexibility, surface accessibility and antigenicity of the ZnT8 protein through bioinformatics analysis software, and screening out a coding sequence with strong immunogenicity from a prediction result, wherein aa 197-369 at the C end is shown in SEQ ID NO.5;

2) Optimized coding sequences

Through software SignalP4.0 prediction analysis, a ZnT8 gene sequence does not contain a signal peptide coding sequence, in order to ensure that the expressed protein can reach the outside of a cell through a membrane, an insulin signal peptide coding sequence (shown as SEQ ID NO. 6) with the length of 72bp is fused at the N end of the selected coding sequence, a 6 x his tag sequence is fused at the C end, and then protective bases and EcoR I and Xho I enzyme cutting site sequences are added at the two ends of a target gene sequence;

3) Optimized vectors

The expression vector used by the method is pcDNA3.1 (+), the vector contains a CMV promoter and an upstream enhancer, and a CAG enhancer sequence is additionally arranged at two ends of a multiple cloning site at the downstream of the CMV promoter respectively in order to improve the transcription level of nucleic acid to a greater extent.

Enhancers greatly increase the activity of a promoter, and the position of an enhancer relative to a promoter is not fixed but can vary widely, and they can interact in two directions. An enhancer is not limited to promoting transcription from a particular promoter, and it can stimulate any promoter in its vicinity. The pcDNA3.1 (+) plasmid itself contains a CMV enhancer, upstream of the CMV promoter, and to increase transcription levels, a CAG enhancer sequence is added upstream of the T7 promoter and after the BGH poly (A) site downstream of the multiple cloning site.

4) Construction of recombinant plasmids

Sending the optimized target gene and vector to a biological company, synthesizing the target gene and vector sequence, digesting the vector pcDNA3.1 (+) and the same ZnT8 target gene fragment with endonuclease EcoR I and Xho I respectively to generate the same cohesive end, connecting the vector and the target gene to construct a recombinant plasmid, sending the recombinant plasmid to Huada gene for sequencing, and transforming the ZnT8 recombinant plasmid with the sequencing result being correct into E.coli DH5 alpha strain for storage;

the synthesized recombinant plasmids are respectively subjected to enzyme digestion, as shown in FIG. 2, the result shows that the target gene fragment with the size of 723bp is visible, and the sequencing proves that the result is correct, which indicates that the construction of the recombinant expression plasmids is successful.

5) Preparation and identification of ZnT8 recombinant protein

Extracting recombinant plasmids by using an endotoxin-free plasmid extraction kit, transfecting the recombinant plasmids into an Expi293 eukaryotic expression system for expression, centrifuging cells after 72h of expression, separating a cell supernatant culture medium from the cells, respectively performing electrophoresis, performing SDS-PAGE electrophoresis, and detecting the expression condition of the antigen protein by using Coomassie brilliant blue staining and Western Blot, wherein the expression condition is shown in a picture 3 (A) -a picture 3 (D);

SDS-PAGE shows that the ZnT8 protein expressed by the optimized plasmid is greatly enriched in the cell culture supernatant, the molecular weight of the target protein is about 25kD (FIG. 3 (A)), and the protein is expressed only in the cell by the non-optimized plasmid and the expression amount is very small (FIG. 3 (B)).

The anti-his antibody (marked by HRP) is used for detecting the target protein, and the result shows that the protein expressed in the cell culture medium supernatant can hardly detect a hybrid protein band, the content of the protein which can be detected in the cell culture medium supernatant by an unoptimized group is very small, the protein band which is obvious can be detected by the optimized group, the difference is larger compared with the unoptimized group (figure 3 (C)), the detection diagram of the cell (figure 3 (D)) also shows that the protein is expressed in the optimized group and the unoptimized group, but the expression amount is very small, and a large amount of hybrid protein bands exist in the cell, which can generate great interference on the subsequent protein purification.

6) Purification of ZnT8 proteins

As the recombinant DNA of ZnT8 is fused and expresses an amino acid sequence of 6 XHis-tag at the C end, the purification is carried out by adopting a conventional Ni ion affinity chromatographic column, the cell supernatant is collected, the Ni ion affinity purification is carried out after the filtration by a needle filter with the pore diameter of 0.45 mu m, the eluted target protein is collected and dialyzed by PBS solution overnight, and the silver staining result of SDS-PAGE electrophoresis is shown in figure 4, which shows that the purity of the recombinant CRP protein is higher.

7) Identification of antigenic Activity by immunoblotting

7.1 Carrying out SDS-PAGE electrophoresis on the purified recombinant antigen protein, transferring the protein on PAGE gel to a PVDF membrane, sealing with 10% skimmed milk powder at 4 ℃ overnight, and cutting the membrane into membrane strips with the width of about 0.5cm under a humid condition;

7.2 ) fetal calf serum is used as negative control, 15 sera of diabetics are randomly selected as an experimental group, the sera are respectively diluted by 100 times by using sample diluent and then added into corresponding membrane strip incubation grooves for incubation at room temperature for 30min, the membrane is washed by washing liquid for 1min, and the washing is repeated for 3 times;

7.3 Diluting anti-human IgG with the sample diluent, adding the diluted anti-human IgG into a corresponding membrane strip incubation groove, incubating for 30min at room temperature, washing the membrane for 1min with a washing solution, and repeatedly washing for 3 times;

7.4 Adding a color development solution, incubating for 15min at room temperature, washing with deionized water, stopping reaction, taking out and airing the membrane strip, and observing whether a specific strip appears on the membrane strip;

the randomly selected 15 sera of the diabetics are detected by the immunoblotting membrane strips (figure 5), the fetal calf serum is negative, 9 of the 15 random sera present negative reactions, 2 weak positive reactions and 4 strong positive reactions, and the antigenicity identification result proves that the expressed recombinant antigen protein has the antigen activity.

In conclusion, the recombinant protein and the preparation method thereof provided by the invention have the advantages that the protein has antigen activity and stable expression, and the preparation method is simple and efficient, and has popularization and application values.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Shenzhen City medicine inspection institute (Shenzhen City medical instrument inspection center)

<120> ZnT8 recombinant protein, preparation method and application thereof

<130> 2019

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 228

<212> PRT

<213> Artificial Synthesis

<400> 1

Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu

1 5 10 15

Trp Gly Pro Asp Pro Ala Ala Ala His Gln Arg Cys Leu Gly His Asn

20 25 30

His Lys Glu Val Gln Ala Asn Ala Ser Val Arg Ala Ala Phe Val His

35 40 45

Ala Leu Gly Asp Leu Phe Gln Ser Ile Ser Val Leu Ile Ser Ala Leu

50 55 60

Ile Ile Tyr Phe Lys Pro Glu Tyr Lys Ile Ala Asp Pro Ile Cys Thr

65 70 75 80

Phe Ile Phe Ser Ile Leu Val Leu Ala Ser Thr Ile Thr Ile Leu Lys

85 90 95

Asp Phe Ser Ile Leu Leu Met Glu Gly Val Pro Lys Ser Leu Asn Tyr

100 105 110

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

115 120 125

His Ser Leu His Ile Trp Ser Leu Thr Met Asn Gln Val Ile Leu Ser

130 135 140

Ala His Val Ala Thr Ala Ala Ser Arg Asp Ser Gln Val Val Arg Arg

145 150 155 160

Glu Ile Ala Lys Ala Leu Ser Lys Ser Phe Thr Met His Ser Leu Thr

165 170 175

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

180 185 190

Glu Asp Pro Cys Asp Gly Gly Ser His His His His His His Asp Tyr

195 200 205

Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp

210 215 220

Asp Asp Asp Lys

225

<210> 2

<211> 687

<212> DNA

<213> Artificial Synthesis

<400> 2

atggccctgt ggatgcgcct cctgcccctg ctggcgctgc tggccctctg ggaacctgac 60

ccagccgcag cccaccagcg gtgcctgggc cacaatcaca aagaggtgca ggccaacgct 120

tctgtgcggg ctgcttttgt tcacgccctg ggcgatctgt tccagtccat ctccgtgctg 180

atctctgccc tgatcatcta cttcaagccc gagtacaaga tcgctgaccc catctgcacc 240

ttcatcttct ccatcctggt gctggcctct accatcacaa tcctgaagga cttcagcatc 300

ctgctgatgg aaggcgtgcc caagagcctg aactactccg gcgtgaaaga actgatcctg 360

gccgtggatg gcgtgctgtc tgtgcactct ctgcacatct ggtccctgac catgaatcaa 420

gtgatcctga gcgcccacgt ggccaccgct gcttctagag attctcaggt cgtgcggaga 480

gagatcgcca aggctctgtc caagagcttc accatgcaca gcctgaccat ccagatggaa 540

agccccgtgg atcaggaccc cgactgtctg ttttgcgagg acccttgcga cggcggctct 600

caccaccacc atcaccacga ctacaaggac cacgacggcg attacaagga tcatgacatc 660

gactataagg acgatgacga caagtga 687

<210> 3

<211> 6004

<212> DNA

<213> Artificial Synthesis

<400> 3

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgac ataacttacg gtaaatggcc cgcctggctg accgcccaac 900

gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 960

ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 1020

gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 1080

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 1140

gtcatcaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa 1200

gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca 1260

cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg 1320

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 1380

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 1440

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 1500

gacaatagca ggcatgctgg ggatgcggtg ggctctatga cataacttac ggtaaatggc 1560

ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc 1620

atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact 1680

gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat 1740

gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact 1800

tggcagtaca tctacgtatt agtcatcgct tctgaggcgg aaagaaccag ctggggctct 1860

agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 1920

cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 1980

tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 2040

gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 2100

tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 2160

ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 2220

tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 2280

taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 2340

ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 2400

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 2460

agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 2520

ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 2580

cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 2640

gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag 2700

gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 2760

agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 2820

tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 2880

tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 2940

gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 3000

tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 3060

ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 3120

cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 3180

atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc 3240

gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 3300

tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 3360

gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 3420

ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 3480

atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc 3540

gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg 3600

cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 3660

ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa 3720

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 3780

caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc 3840

gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 3900

catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 3960

attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 4020

ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 4080

ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4140

aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 4200

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4260

gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 4320

gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 4380

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 4440

ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4500

ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4560

tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4620

tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4680

ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4740

aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg 4800

caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4860

ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 4920

aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 4980

tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 5040

agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 5100

gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 5160

accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 5220

tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 5280

tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 5340

acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 5400

atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5460

aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5520

tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5580

agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 5640

gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 5700

ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 5760

atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 5820

tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 5880

tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5940

tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 6000

cgtc 6004

<210> 4

<211> 6679

<212> DNA

<213> Artificial Synthesis

<400> 4

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgac ataacttacg gtaaatggcc cgcctggctg accgcccaac 900

gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 960

ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 1020

gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 1080

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 1140

gtcatcaaat taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa 1200

gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattcccggc caccatgatg 1260

gccctgtgga tgcgcctcct gcccctgctg gcgctgctgg ccctctggga acctgaccca 1320

gccgcagccc accagcggtg cctgggccac aatcacaaag aggtgcaggc caacgcttct 1380

gtgcgggctg cttttgttca cgccctgggc gatctgttcc agtccatctc cgtgctgatc 1440

tctgccctga tcatctactt caagcccgag tacaagatcg ctgaccccat ctgcaccttc 1500

atcttctcca tcctggtgct ggcctctacc atcacaatcc tgaaggactt cagcatcctg 1560

ctgatggaag gcgtgcccaa gagcctgaac tactccggcg tgaaagaact gatcctggcc 1620

gtggatggcg tgctgtctgt gcactctctg cacatctggt ccctgaccat gaatcaagtg 1680

atcctgagcg cccacgtggc caccgctgct tctagagatt ctcaggtcgt gcggagagag 1740

atcgccaagg ctctgtccaa gagcttcacc atgcacagcc tgaccatcca gatggaaagc 1800

cccgtggatc aggaccccga ctgtctgttt tgcgaggacc cttgcgacgg cggctctcac 1860

caccaccatc accacgacta caaggaccac gacggcgatt acaaggatca tgacatcgac 1920

tataaggacg atgacgacaa gtgaccgctc gagtctagag ggcccgttta aacccgctga 1980

tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 2040

tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 2100

tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 2160

ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgacataa 2220

cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 2280

atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 2340

tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 2400

cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 2460

tgggactttc ctacttggca gtacatctac gtattagtca tcgcttctga ggcggaaaga 2520

accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg 2580

ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 2640

ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 2700

cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 2760

gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 2820

acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 2880

cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 2940

aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt 3000

tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 3060

attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 3120

gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 3180

taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 3240

cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg 3300

gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc 3360

aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 3420

cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 3480

ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 3540

acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 3600

cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 3660

tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 3720

aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 3780

cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 3840

ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 3900

ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3960

gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 4020

tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 4080

ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 4140

agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 4200

aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 4260

ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 4320

cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 4380

ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 4440

tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 4500

tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 4560

cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4620

agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 4680

gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4740

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4800

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4860

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4920

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4980

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 5040

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5100

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5160

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5220

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5280

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5340

gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5400

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5460

cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 5520

tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 5580

ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 5640

taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 5700

tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 5760

cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 5820

gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 5880

cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 5940

ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 6000

aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 6060

atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 6120

tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 6180

gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 6240

aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 6300

acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 6360

ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 6420

tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 6480

aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 6540

catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 6600

atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 6660

aaaagtgcca cctgacgtc 6679

<210> 5

<211> 519

<212> DNA

<213> Artificial Synthesis

<400> 5

caccagcggt gcctgggcca caatcacaaa gaggtgcagg ccaacgcttc tgtgcgggct 60

gcttttgttc acgccctggg cgatctgttc cagtccatct ccgtgctgat ctctgccctg 120

atcatctact tcaagcccga gtacaagatc gctgacccca tctgcacctt catcttctcc 180

atcctggtgc tggcctctac catcacaatc ctgaaggact tcagcatcct gctgatggaa 240

ggcgtgccca agagcctgaa ctactccggc gtgaaagaac tgatcctggc cgtggatggc 300

gtgctgtctg tgcactctct gcacatctgg tccctgacca tgaatcaagt gatcctgagc 360

gcccacgtgg ccaccgctgc ttctagagat tctcaggtcg tgcggagaga gatcgccaag 420

gctctgtcca agagcttcac catgcacagc ctgaccatcc agatggaaag ccccgtggat 480

caggaccccg actgtctgtt ttgcgaggac ccttgcgac 519

<210> 6

<211> 72

<212> DNA

<213> Artificial Synthesis

<400> 6

atggccctgt ggatgcgcct cctgcccctg ctggcgctgc tggccctctg ggaacctgac 60

ccagccgcag cc 72

Claims (4)

1. A method for preparing ZnT8 recombinant protein, comprising the steps of:

(1) Analyzing and screening an initial target gene sequence from a ZnT8 full-length gene coding sequence;

(2) Fusing a signal peptide sequence and a C-terminal fusion tag sequence at the N terminal and the C terminal of the target gene in the step (1) to obtain an optimized target gene;

(3) Adding a CAG enhancer sequence at each of two ends of a multiple cloning site at the downstream of a CMV promoter of an expression vector pcDNA3.1 (+) to obtain an optimized expression vector;

(4) Synthesizing the target gene sequence optimized in the step (2) and the expression vector sequence optimized in the step (3), performing double enzyme digestion by using endonucleases EcoR I and Xho I respectively to generate the same cohesive ends, connecting the vector and the target gene to construct a recombinant plasmid, and transforming the recombinant plasmid to a prokaryotic cell for storage;

(5) Transfecting the recombinant plasmid obtained in the step (4) into an Expi293 eukaryotic expression system for expression to obtain the ZnT8 recombinant protein;

the nucleotide sequence of the initial target gene in the step (1) is shown as SEQ ID NO.5;

the amino acid sequence coded by the optimized target gene in the step (2) is shown as SEQ ID NO. 1;

the label in the step (2) is a 6 × his tag;

the nucleotide sequence of the optimized expression vector in the step (3) is shown as SEQ ID NO. 3.

2. The method of claim 1, wherein the prokaryotic cell of step (4) comprises any one of DH5 a, BL21, TOP10 or Rosetta.

3. The method of claim 1, wherein the prokaryotic cell of step (4) is E.coli DH5 alpha strain.

4. The method according to claim 1, characterized in that it comprises in particular the steps of:

(1) Analyzing and screening an initial target gene sequence shown as SEQ ID NO.5 from the ZnT8 full-length gene coding sequence;

(2) Fusing a signal peptide sequence and a C-terminal fusion tag sequence at the N terminal and the C terminal of the target gene in the step (1) to obtain an optimized target gene, wherein an amino acid sequence coded by the optimized target gene is shown as SEQ ID NO. 1;

(3) Adding a CAG enhancer sequence at each of two ends of a multiple cloning site at the downstream of a CMV promoter of an expression vector pcDNA3.1 (+) to obtain an optimized expression vector with a nucleotide sequence shown as SEQ ID NO. 3;

(4) Synthesizing the target gene sequence optimized in the step (2) and the expression vector sequence optimized in the step (3), performing double enzyme digestion by using endonucleases EcoR I and Xho I respectively to generate the same cohesive ends, connecting the vector and the target gene to construct a recombinant plasmid, and transforming the recombinant plasmid to a prokaryotic cell E.coli DH5 alpha strain;

(5) And (5) transfecting the recombinant plasmid obtained in the step (4) into an Expi293 eukaryotic expression system for expression to obtain the ZnT8 recombinant protein.

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