Construction and application of mitochondrial fluorescent labeling tools

Abstract

The application relates to the technical field of biotechnology, and particularly discloses a pig-derived TXN2 mitochondrion fluorescence marking tool and a construction method of a split-type fluorescence interaction detection system. The application constructs and screens a TXN2-eGFP high-specificity mitochondrion marking plasmid suitable for a pig cell system, verifies imaging precision, protein stability and cell safety of the plasmid, and constructs a Split-eGFP split-type fluorescence reconstruction system based on the mitochondrion positioning property of TXN2, so that the interaction relationship between a virus protein and mitochondria can be detected in situ and with high sensitivity. The technical system formed by the above cell organ morphological observation and virus-host protein interaction detection can be widely applied to pig cell biology research and pig-derived virus infection and pathogenic mechanism analysis, and can make up for the technical defects of existing mitochondrion marking tools, such as fuzzy imaging, poor stability, low species adaptability and insufficient interaction detection sensitivity in pig-derived cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the construction and application of mitochondrial fluorescent labeling tools. Background Technology

[0002] Mitochondria, as important energy metabolism centers in eukaryotic cells, participate in various physiological and pathological processes such as apoptosis, signal transduction, and immune responses. During viral infection, mitochondrial structure often undergoes significant remodeling and abnormal interactions with other organelles such as the endoplasmic reticulum, making them important targets for elucidating viral pathogenic mechanisms. Therefore, constructing efficient and stable mitochondrial fluorescent labeling tools is of significant scientific value for conducting research on organelle morphology observation, organelle interaction detection, and virus-host protein interaction visualization.

[0003] Currently, commonly used mitochondrial fluorescent labeling tools are mostly constructed based on human or mouse genes, and used in conjunction with fluorescent proteins such as eGFP to achieve mitochondrial localization imaging. These tools perform well in human and mouse cells and have been widely used in basic cell biology research. However, pigs, as important agricultural economic animals and viral infection models, differ significantly from rodents and primates in gene sequences and protein functions. Cross-species mitochondrial labeling proteins in pig cells suffer from weak fluorescence signals, ambiguous localization, and poor protein stability. This not only results in poor two-dimensional imaging quality but also makes it difficult to support high-precision three-dimensional volume reconstruction and detailed analysis of organelle interactions, failing to meet the needs of fine morphological observation of pig cell mitochondria and detection of dynamic changes in organelles during viral infection.

[0004] Furthermore, porcine coronaviruses, such as porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV), are important pathogens that seriously threaten the pig industry. Their pathogenic mechanisms are closely related to host organelle structural remodeling and viral protein-mitochondrial interactions. However, the lack of efficient mitochondrial labeling tools adapted to porcine cell systems limits the visualization of organelle pathological changes in the pathogenic mechanisms of porcine coronaviruses. Traditional detection methods, such as immunoprecipitation and conventional colocalization imaging, are insufficient in terms of sensitivity and in-situ dynamic detection capabilities, making it difficult to capture transient and weak virus-mitochondrial interaction events.

[0005] Therefore, developing a mitochondrial fluorescent labeling tool that is specific to porcine cells, highly stable, has high imaging quality, and is biosafety-friendly, and establishing a cell organelle detection and virus-host interaction visualization technology system based on this tool for studying the pathogenic mechanism of porcine coronaviruses, has significant scientific research value and translational prospects. Summary of the Invention

[0006] In view of this, the present invention provides a method for constructing and applying a porcine TXN2 mitochondrial fluorescent labeling tool and a fission-type fluorescent interaction detection system.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for constructing a porcine TXN2 mitochondrial fluorescent labeling plasmid, characterized by comprising the following steps: Step A, Amplification TXN2 Porcine mitochondrial tag genes; Step B: Linearize the pcDNA4.0-eGFP vector by double digestion with EcoRI and XhoI to obtain a linearized vector; Step C: Recombining the... TXN2 The porcine mitochondrial tag gene was ligated into the linearized vector, transformed into DH5α competent cells, and verified by bacterial PCR and sequencing. The positive plasmid pcDNA4.0-TXN2-eGFP was extracted. The TXN2 The porcine mitochondrial tag gene has the nucleotide sequence shown in SEQ ID No. 4.

[0008] In some specific embodiments of the present invention, the... TXN2 The protein encoded by the porcine mitochondrial tag gene has the amino acid sequence shown in SEQ ID No. 8.

[0009] In some specific embodiments of the present invention, the linearized vector has a nucleotide sequence as shown in SEQ ID No. 9.

[0010] In some specific embodiments of the present invention, the linearized vector has an amino acid sequence as shown in SEQ ID No. 10.

[0011] Secondly, the present invention also provides a porcine TXN2 mitochondrial fluorescent labeling plasmid obtained by the construction method described above.

[0012] Thirdly, the present invention also provides a method for constructing a Split-eGFP splitting fluorescence interaction detection system, which, based on the porcine TXN2 mitochondrial fluorescent labeling plasmid obtained by the construction method, includes the following steps: Amplification was performed using the porcine TXN2 mitochondrial fluorescent labeling plasmid as a template. TXN2 Gene fragments; Using the Split-eGFP dual-fragment system with pcDNA4.0 as a unified backbone, TXN2-eGFP was constructed separately via overlap PCR. 1-10 PDCoV M-eGFP 11 PEDV M-eGFP11 Fusion recombinant plasmid; The TXN2-eGFP 1-10 have: (I) A nucleotide sequence as shown in SEQ ID No. 15; and / or (II) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (I), and having the same function as the nucleotide sequence shown in (I); and / or (III) A nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence shown in (I) or (II). The plurality is at least two; The TXN2-eGFP 1-10 have: (I) An amino acid sequence as shown in SEQ ID No. 18, or consisting essentially of an amino acid sequence as shown in SEQ ID No. 8, or containing an amino acid sequence as shown in SEQ ID No. 8; (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (III) Sequences that are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more homologous to the amino acid sequences shown in (I) or (II); The PDCoV M-eGFP 11 have: (I) A nucleotide sequence as shown in SEQ ID No. 16; and / or (II) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (I), and having the same function as the nucleotide sequence shown in (I); and / or (III) A nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence shown in (I) or (II). The plurality is at least two; The PDCoV M-eGFP 11 have: (I) An amino acid sequence as shown in SEQ ID No. 18, or consisting essentially of an amino acid sequence as shown in SEQ ID No. 8, or containing an amino acid sequence as shown in SEQ ID No. 8; (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (III) Sequences that are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more homologous to the amino acid sequences shown in (I) or (II); The PEDV M-eGFP 11 It has the nucleotide sequence shown in SEQ ID No. 15; The PEDV M-eGFP 11 It has the amino acid sequence shown in SEQ ID No. 18.

[0013] Thirdly, the present invention also provides a Split-eGFP splitting fluorescence interaction detection system obtained by the construction method described above.

[0014] Fourthly, the present invention also provides the application of the porcine TXN2 mitochondrial fluorescent labeling plasmid and / or the Split-eGFP splitting fluorescent interaction detection system in any of the following: (I) Observation of mitochondrial morphology in pig cells; (II) Detection of porcine organelle interactions; (III) Research on the pathogenic mechanism of porcine coronavirus; The detection of porcine organelle interactions includes visual observation and / or quantitative analysis of mitochondrial-endoplasmic reticulum spatial interactions; The study on the pathogenesis mechanism of porcine coronavirus includes in situ visualization detection of virus-mitochondrial interactions.

[0015] Fifthly, the present invention also provides a method for detecting mitochondrial-endoplasmic reticulum spatial interactions. The porcine TXN2 mitochondrial fluorescent labeling plasmid and the endoplasmic reticulum labeling molecule PDI-mCherry are co-transfected into porcine cells to establish a dual fluorescent labeling detection system, thereby enabling visual observation and quantitative analysis of mitochondrial-endoplasmic reticulum spatial interactions.

[0016] Sixthly, the present invention also provides an in-situ visualization detection method for virus-mitochondrial interactions, which uses mitochondrial-anchored TXN2-eGFP in the Split-eGFP splitting fluorescence interaction detection system. 1-10 eGFP, a plasmid fused with viral proteins 11Plasmids were co-transfected into porcine cells, and fluorescence signals were detected using laser confocal microscopy after culture. When the viral protein interacted spatially with the mitochondria and the distance reached a threshold, eGFP was activated. 1-10 With eGFP 11 The fragment spontaneously reconstructs into a complete fluorescent protein and restores green fluorescence, enabling in-situ visualization detection of virus-mitochondrial interactions.

[0017] This invention discloses a method for constructing a porcine TXN2 mitochondrial fluorescent labeling tool and a split-eGFP fluorescence interaction detection system, belonging to the field of biotechnology. The steps employed in this invention are as follows: construction of a eukaryotic recombinant expression vector; performance verification of the mitochondrial labeling tool; construction and verification of the Split-eGFP split-eGFP fluorescence interaction detection system. This invention constructs and screens a TXN2-eGFP highly specific mitochondrial labeling plasmid adapted to porcine cell systems, verifying its imaging accuracy, protein stability, and cell safety. Simultaneously, based on the mitochondrial localization characteristics of TXN2, a Split-eGFP split-eGFP fluorescence reconstruction system is constructed, enabling in situ, high-sensitivity detection of the interaction between viral proteins and mitochondria. The aforementioned system for observing organelle morphology and detecting virus-host protein interactions can be widely applied to porcine cell biology research and the elucidation of porcine viral infection and pathogenesis mechanisms, overcoming the technical shortcomings of existing mitochondrial labeling tools in porcine cells, such as blurred imaging, poor stability, low species adaptability, and insufficient sensitivity in interaction detection. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 This diagram illustrates the connection between the mitochondrial localization motif and the eGFP tag in this invention. Figure 2 The PCR amplification results of four recombinant plasmids in a preferred embodiment of the present invention are shown, wherein M represents DL2000 DNA Marker, Fis-eGFP represents pcDNA4.0-Fis1-eGFP, TOM20-eGFP represents pcDNA4.0-TOM20-eGFP, TXN2-eGFP represents pcDNA4.0-TXN2-eGFP, and COX8-eGFP represents pcDNA4.0-COX8-eGFP; Figure 3 This diagram illustrates the principle of the Split-eGFP fluorescence reconstruction system used in this invention; wherein, eGFP... 1-10 With eGFP 11 After the two green fluorescent protein fragments bind to each other, they can reassemble to form a complete eGFP and restore the green fluorescence signal; Figure 4 This image shows the immunoblotting results of four types of mitochondrial tag plasmid proteins in a preferred embodiment of the present invention. Figure 5 This invention presents a comparison of imaging effects of four types of mitochondrial tag plasmids in a preferred embodiment; Figure 6 This invention demonstrates a preferred embodiment of mitochondrial three-dimensional reconstruction imaging comparison based on Fis1-eGFP and TXN2-eGFP; Figure 7 This shows the results of establishing a detection system for porcine mitochondrial-endoplasmic reticulum interaction in a preferred embodiment of the present invention; Figure 8 This invention illustrates a preferred embodiment of the TXN2 gene sequence differential analysis across multiple species. Figure 9 This invention presents a comparison of the half-life detection results of four types of mitochondrial tag plasmid proteins in a preferred embodiment; Figure 10 This shows the cell safety test results of TXN2-eGFP in a preferred embodiment of the present invention; Figure 11 The present invention provides fluorescence imaging results of the interaction between PDCoV and PEDV coronavirus proteins and mitochondria based on the Split-eGFP splitting fluorescence complementary system in a preferred embodiment of the present invention. Detailed Implementation

[0020] This invention discloses the construction and application of a mitochondrial fluorescent labeling tool. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0021] This invention provides a method for constructing a porcine TXN2 mitochondrial fluorescent labeling tool and a split-eGFP fluorescence interaction detection system, comprising the following steps: (i) construction of a eukaryotic expression vector; (ii) performance verification of the mitochondrial labeling tool; and (iii) construction of a Split-eGFP split-eGFP fluorescence interaction detection system.

[0022] Further, step (i) involves the construction of the eukaryotic expression vector, and the steps are as follows: (1) Construction of mitochondrial tag vector: Four porcine mitochondrial tag genes, Fis1, COX8, TOM20, and TXN2, were amplified. Their gene sequences are shown in SEQ ID NO:1-4, and their amino acid sequences are shown in SEQ ID NO:5-8. The pcDNA4.0-eGFP vector was linearized by double enzyme digestion. The four tag genes were ligated into the linearized vector by homologous recombination. After transformation into DH5α, the vectors were verified by bacterial PCR and sequencing. Positive plasmids were extracted for later use.

[0023] (2) Construction of Split-eGFP series vectors: Using the Split-eGFP dual-fragment system with pcDNA4.0 as the backbone, mitochondrial-anchored TXN2-eGFP was constructed by overlap PCR. 1-10 Plasmids, and PDCoV M-eGFP 11 PEDV M-eGFP 11 Viral protein fusion plasmids; after all plasmids have been sequenced and verified to be correct, endotoxin-free plasmids are extracted for later use.

[0024] Further, step (ii) involves performance verification of the mitochondrial labeling tool, as follows: (1) Protein expression identification: LLC-PK1 cells were plated and cultured to 80% confluence. After transfection with plasmids in proportion, the expression of the target protein was verified by Western blotting.

[0025] (2) Laser confocal two-dimensional imaging screening: After transfecting, the cells were fixed and stained. The localization effect and imaging quality of the four mitochondrial tags were compared by using an inverted laser confocal microscope to screen the optimal labeling tool.

[0026] (3) Three-dimensional reconstruction imaging verification: Z-axis tomography and three-dimensional volume reconstruction were performed on the selected TXN2-eGFP and the control Fis-eGFP tag to compare the differences in mitochondrial spatial network structure and fine morphology presentation to verify the three-dimensional imaging capability.

[0027] (4) Sequence difference analysis and protein half-life detection: Homology comparison of TXN2 genes in multiple species was carried out, and the protein half-life of different tags was detected and compared by the cyclohexane inhibition experiment to verify the stability difference.

[0028] (5) Cell safety test: Cell viability after transfection with different doses of plasmid was tested by CCK-8 assay to evaluate the biosafety of the TXN2-eGFP labeling tool.

[0029] (6) Detection of mitochondrial-endoplasmic reticulum interaction: TXN2-eGFP and PDI-mCherry, an endoplasmic reticulum marker molecule, were co-transfected into cells to establish a dual fluorescent labeling mitochondrial-endoplasmic reticulum interaction detection system, so as to realize the visualization analysis of the interaction between organelles.

[0030] Further, step (iii) involves constructing the Split-eGFP splitting fluorescence interaction detection system, as follows: TXN2-eGFP... 1-10 Plasmid and viral protein-eGFP 11 Plasmids were co-transfected into porcine cells, and the fluorescence signal was detected by laser confocal microscopy using the fluorescent fragment reconstruction effect. The interaction between viral proteins and mitochondria was then visualized in situ.

[0031] This invention overcomes the technical shortcomings of existing commercial mitochondrial labeling tools, such as blurred imaging, poor stability, and low species adaptability in porcine cells, by independently constructing a porcine TXN2-eGFP mitochondrial fluorescent labeling tool and establishing a complete technical system for detecting porcine cell organelles and visualizing virus-host interactions. It fills the gap in dedicated tools for dynamic detection of organelles in the study of the pathogenesis mechanism of porcine coronaviruses.

[0032] This invention screened and verified that the porcine TXN2 protein, compared to traditional mitochondrial tags such as Fis1, COX8, and TOM20, exhibits higher fluorescence imaging accuracy, stronger protein stability, and a longer half-life in porcine cells. Specifically, TXN2-eGFP supports high-precision three-dimensional volume reconstruction of mitochondria, clearly revealing the spatial network structure and morphological details of mitochondria. The three-dimensional imaging quality is significantly superior to traditional tags, meeting the three-dimensional observation requirements for the fine morphology of porcine mitochondria. Furthermore, this tool can be coupled with the endoplasmic reticulum marker molecule PDI-mCherry to establish a stable visualization detection system for mitochondrial-endoplasmic reticulum interactions, accurately presenting the spatial co-localization and contact regions of the two organelles, expanding the application scenarios of this tool in organelle interaction research.

[0033] Furthermore, this invention, based on the porcine TXN2 protein, for the first time constructs a Split-eGFP fluorescence interaction detection system adapted to porcine cells. Employing a Split-eGFP dual-fragment system, it separates and expresses mitochondrial localization fragments and viral protein fragments, utilizing fluorescence reconstruction effects to achieve in-situ detection of virus-mitochondrial interactions. Compared to traditional immunoprecipitation and conventional co-localization imaging, this system offers higher sensitivity and specificity, capturing transient and weak virus-host organelle interaction events. It is also broadly compatible with various porcine coronaviruses such as PDCoV and PEDV, providing a high-resolution visualization tool for studying the pathogenic mechanisms of porcine coronaviruses.

[0034] 1. Fis1 base sequence (as shown in SEQ ID NO:1): ATGGAGGCCGTGCTGAACGAGTTGGTGTCTGTGGAGGATCTGCTGAAATTTGAAAGGAAATTTCAGTCTGAGAAGGCAGCAGGCTCGGTGTCCAAGAGCACGCAGTTTGAATATGCCTGGTGTCTGGTGCGAAGCAAGTACAACGATGACATCCGTAAAGGCCTTGCTCTGCTGGAGGAGCTGCTACCCAAAGGGAGCAAAGAGGAGCAGCGGGATTATGTCTTCTACCTGGCCGTGGGGAACTACCGGCTCAAGGAATATGAAAAGGCCCTAAAGTATGTGCGCGGGCTGCTGCAGACAGAGCCACAGAACAACCAGGCCAAGGAACTGGAAGGGCTCATTGACAAGGCCATGAAGAAAGATGGACTAGTGGGCATGGCCATTGTTGGAGGCATGGCCCTGGGCGTGGCCGGACTGGCTGGACTCATTGGACTTGCTGTATCCAAGTCCAAATCCTGA 2. COX8 base sequence (as shown in SEQ ID NO: 2): ATGTCCGCGCTGACTCCGCTGCTGCTCAGGGGCCTGACAGGCCCGGCCCGACGGCTCCTGGTGCCACGTGCCCAGGTTCATTCCAAGCCGCCGCGGGAGCAGCTCGGGTCCATGGATGTCACCATCGCGCTCACCGCCTGCTTCGTGAGTTTCCTCCTGCCCGCGGGCTGGGTCCTGTCACACCTGGACAGCTACAAGAAGCGGGAGTGA 3. TOM20 base sequence (as shown in SEQ ID NO: 3): ATGGTGGGCCGGAACAGCGCCATTGCCGCCGGCGTGTGCGGGGCCCTTTTCATCGGGTACTGCATTTACTTCGACCGCAAGAGACGAAGTGACCCCAACTTCAAGAACAGGCTGCGAGAACGAAGAAAGAAACAAAAGCTTGCTAAGGAGAGAGCTGGACTTTCCAAGTTACCTGACCTTAAAGATGCTGAGGCTGTTCAGAAATTCTTCCTCGAAGAAATTCAGCTTGGTGAAGAGTTACTAGCTCAAGGTGAATATGAGAAGGGTGTAGACCATCTGACAAATGCCATTGCTGTGTGTGGACAGCCACAGCAGTTACTGCAAGTGTTACAGCAAACTCTTCCACCACCAGTGTTCCAGATGCTTCTGACTAAGCTCCCAACAATTAGTCAGAGAATTGTAAGTGCTCAGAGCTTGGCTGAAGATGATGTGGAGTGA 4. The base sequence of TXN2 (as shown in SEQ ID NO: 4): ATGGTTCGAAGTGGTATCGCGGCTGAGGCGCCTCTGCAGATGGCTCAGCGTCTTCTCCTGAGGAGGTTCCTGGCCTCTGTAGTCTCCAGGAAGCCCTCTCATAGTCGGTGGGCACCCCTCGCTTCCAGAGCCCTGCAGACCCCACAGCTCAGCCTTAGTAACCTGACAGGAACACCCAGCCAAGCCCGGTCATTATACACTACCCAGGTCTGTACAACAACCTTTAACATCCAGGATGGACCTGACTTTCAAGACCGAGTTGTCAACAGTGAGACGCCAGTGGTTGTGGATTTCCATGCACAGTGGTGTGGTCCCTGCAAGATCCTGGGGCCAAGGTTAGAGAAGGTGGTGGCCAAGCAGCATGGAAAGGTGGTGATGGCCAAGGTGGATATCGACGACCACACAGACCTCGCCATCGAGTACGAGGTGTCGGCGGTGCCCACTGTGCTGGCCATCAAAAATGGGGACGTGGTGGACAAGTTTGTGGGCATCAAGGATGAGGACCAGCTGGAGGCCTTTCTGAAGAAGCTGATTGGCTGA 5. Fis1 amino acid sequence (as shown in SEQ ID NO: 5): MEAVLNELVSVEDLLKFERKFQSEKAAGSVSKSTQFEYAWCLVRSKYNDDIRKGLALLEELLPKGSKEEQRDYVFYLAVGNYRLKEYEKALKYVRGLLQTEPQNNQAKELEGLIDKAMKKDGLVGMAIVGGMALGVAGLAGLIGLAVSKSKS. 6. COX8 amino acid sequence (as shown in SEQ ID NO: 6): MSALTPLLLRGLTGPARRLLVPRAQVHSKPPREQLGSMDVTIALTACFVSFLLPAGWVLSHLDSYKKRE. 7. TOM20 amino acid sequence (as shown in SEQ ID NO: 7): MVGRNSAIAAGVCGALFIGYCIYFDRKRRSDPNFKNRLRERRKKQKLAKERAGLSKLPDLKDAEAVQKFFLEEIQLGEELLAQGEYEKGVDHLTNAIAVCGQPQQLLQVLQQTLPPPVFQMLLTKLPTISQRIVSAQSLAEDDVE. 8. Amino acid sequence of TXN2 (as shown in SEQ ID NO: 8): MVRSGIAAEAPLQMAQRLLLRRFLASVVSRKPSHSRWAPLASRALQTPQLSLSNLTGTPSQARSLYTTQVCTTTFNIQDGPDFQDRVVNSETPVVVDFHAQWCGPCKILGPRLEKVVAKQHGKVVMAKVDIDDHTDLAIEYEVSAVPTVLAIKNGDVVDKFVGIKDEDQLEAFLKKLIG. 9. Base sequence of vector PCDNA4.0-eGFP (as shown in SEQ ID NO: 9): 10. The amino acid sequence of the vector PCDNA4.0-eGFP (as shown in SEQ ID NO:10): DGSGDLPIPYGALSVQSALMPHS.ASICSLLVCWRSLSSARAKFKLQQGKA.PTIA.RICLGLGVLRCFAMYGPDIRVDIDY.LVINSNQLRGH.FIAHIWSSALHNLR.MARLADRPTTPAH. RQ..RMFP..RQ.GLSIDVNGWSIYGKLPTWQYIKCIICQVRPLLTSMTVNGPPGIMPST.PYGTFLLGSTSTY.SSLLPW.CGFGSTSMGVDSGLTHGDFQVSTPLTSMGVCFGTKINGTFQN VVTTPPH.RKWAVGVYGGRSI.AELSLSVIEISLSVIEIVDELV..TVRSPGDAIHAVLTSIEDTGTDPASGL.RLNLSLVPSSDPLVQCGGILQISSTVAAARDGEQGRGAVHRGGAHPGRAG RRRKRPQVQRVRRGRGRCHLRQADPEVHLHHRQAARALAHPRDHPDLRRAVLQPLPRPHEAARLLQVRHARRLRPGAHHLLQGRRQLQDPRRGEVRGRHPGEPHRAEGHRLQGGRQHPGAQAGVQ LQQPQRLYHGRQAEERHQGELQDPPQHRGRQRAARRPPAEHPHRRRPRAAARQPLPEHPVRPEQRPQREARSHGPAGVRDRRRDHSRHGRAVQVI.RARLNPLISLDCAF.LPAICCLPLPRA FLDPGRCHSHCPFLIK.GNCIALSE.VSFYSGGWGGAGQQGGGLGRQ.QACWGCGGLYGF.GGKNQLGL.GVSPRAL.RRIKRGGCGGYAQRDYTCQRPSARSFLPFLSRHVRRLSPSSSK SGAPFRVPI.CFTAPRPQKT.LG.WFT.WAIALIDGFSPFDVGVHVL..WTLVPNWNNTQPYLGLFF.FIRDFADFGLLVKK.ADLTKI.RELILWNVCQLGCGKSPGSPAGRSMQSMHLN.SA TRCGKSPGSPAGRSMQSMHLN.SATIVPPLTPPIPPLTPPSSAHSPPHG.LIFFIYAEAEAASASELFQK..GGFFGGLGFCKKLPGACISIFGSDQHVLTINHRHSISA.YNTTR.GTKPWPS.PVPFRCSPRATSPERSSSGPTGSGSPGTSWRTTSPVWSGTT.PCSSARSRTRWCRTTPWPGCGCAAWTSCTPSGRRSCPRTSGTPPGRP.PRSASSRGGGSSPCATRPATACTSWPRSRTDTCYEISIPPPPSMKGWASESFSGTPAG.SSSAGISCWSSSPTPTCLLQLIMVTNKAIASQISQIKHFFHCILVVVCPNSSMYLIMSVYRRPLARAWRNHGHSCFLCEIVIRSQFHTTYEPEA.SVKPGVPNE.ANSH.LRCAHCPLSSRETCRASCINESANARGEAVCVLGALPLPRSLTRCARSFGCGERYQLTQRR.YGYPQNQGITQERTCEQKASKRPGTVKRPRCWRFSIGSAPLTSITKIDAQVRGGETRQDYKDTRRFPLEAPSCALLFRPCRLPDTCPPFSLREAWRFLIAHAVGISVRCRSFAPSWAVCTNPPFSPTAAPYPVTIVLSPTR.DTTYRHWQQPLVTGLAERGM.AVLQSS.SGGLTTATLEEQYLVSALC.SQLPSEKELVALDPANKPPLVAVFLFASSRLRAEKKDLKKIL.SFLRGLTLSGTKTHVKGFWS.DYQKGSSPRSF.IKNEVLNQSKVYMSKLGLTVTNA.SVRHLSQRSVYFVHP.LPDSPSCR.LRYGRAYHLAPVLQ.YRETHAHRLQIYQQ.TSQPEGPSAEVVLQLYPPPSSLLIVAGKLE.VVRQLIVCATLLPLLQASWCHARRLVWLHSAPVPNDQGELHDPPCCAKKRLAPSVLRSLSEVSWPQCYHSWLWQHCIILLLSCHP.DAFL.LVSTQPSHSENSVCGDRVALARRQYGIIPRHIAEL.KCSSLENVLRGENSQGSYRC.DPVRCNPLVHPTDLQHLLLSPAFLGEQKQEGKMPQKRE.GRHGNVEYSYSSFFNIIEAFIRVIVS.ADTYLNVFRKINK.GFRAHFPEKCHLT. 11. The sequencing sequence of PCDNA4.0-eGFP-Fis1 gene (as shown in SEQ ID NO: 11): 12. PCDNA4.0-eGFP-COX8 gene sequencing sequence (as shown in SEQ ID NO: 12): ATGTCCGCGCTGACTCCGCTGCTGCTCAGGGGCCTGACAGGCCCGGCCCGACGGCTCCTGGTGCCACGTGCCCAGGTTCATTCCAAGCCGCCGCGGGAGCAGCTCGGGTCCATGGATGTCACCATCGCGCTCACCGCCTGCTTCGTGAGTTTCCTCCTGCCCGCGGGCTGGGTCCTGTCACACCTGGACAGCTACAAGAAGCGGGAGTGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGaccccaacgag aagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa 13. Sequencing sequence of PCDNA4.0-eGFP-TOM20 gene (as shown in SEQ ID NO:13): 14. Sequencing sequence of PCDNA4.0-eGFP-TXN2 gene (as shown in SEQ ID NO:14): 15. TXN2-eGFP 1-10 Gene sequencing sequence (as shown in SEQ ID NO:15): 16. PDCoV-eGFP 11 Gene sequencing sequence (as shown in SEQ ID NO: 16): ATGTCTGACGCAGAAGAGTGGCAAATTATTGTTTTCATTGCGATCATATGGGCGCTTGGCGTTATCCTCCAAGGAGGCTATGCCACGCGTAATCGTGTGATCTATGTTATTAAACTTATTCTGCTTTGGCTGCTCCAACCCTTCACCCTAGTGGTGACCATTTGGACCGCAGTTGACAGATCATCTAAGAAGGACGCAGTTTTCATTGTGTCCATAATTTTTGCCGTATTGACCTTTATATCCTGGGCCAAGTACTGGTATGACTCAATTCGTTTATTAATGAAAACCAGATCTGCATGGGCACTCTCACCTGAGAGTAGACTCCTTGCAGGGATTATGGATCCAATGGGTACATGGAGGTGCATTCCCATCGACCACATGGCTCCAATACTCACACCAGTCGTTAAGCATGGCAAGCTCAAGCTACATGGGCAAGAGCTGGCCAATGGCATATCAGTTAGAAATCCGCCACAGGATATGGTGATAGTGTCACCAAGTGACACCTTTCACTACACTTTTAAGAAACCTGTGGAATCAAACAACGATCCAGAATTCGCTGTTCTGATATACCAGGGTGACCGTGCTTCAAACGCTGGACTTCACACTATAACCACTTCAAAGGCCGGTGACGCTCGCCTGTATAAGTATATGCGTGACCACATGGTGCTGCATGAATATGTGAACGCGGCGGGCATCACG 17. PEDV-eGFP 11 Gene sequencing sequence (as shown in SEQ ID NO: 17): ATGTCTAACGGTTCTATTCCCGTTGATGAGGTGATTCAACACCTTAGAAACTGGAATTTCACATGGAATATCATACTGACGATACTACTTGTAGTGCTTCAGTATGGCCATTACAAGTACTCTGCGTTCTTGTATGGTGTCAAGATGGCTATTCTATGGATACTTTGGCCTCTTGTGTTAGCACTGTCACTTTTTGATGCATGGGCTAGCTTTCAGGTCAATTGGGTCTTTTTTGCTTTCAGCATCCTTATGGCTTGCATCACTCTTATGCTGTGGATAATGTACTTTGTCAATAGCATTCGGTTGTGGCGCAGGACACATTCTTGGTGGTCTTTCAATCCTGAAACAGACGCGCTTCTCACTACTTCTGTGATGGGCCGACAGGTCTGCATTCCAGTGCTTGGAGCACCAACTGGTGTAACGCTAACACTCCTTAGTGGTACATTGCTTGTAGAGGGCTATAAGGTTGCTACTGGCGTACAGGTAAGTCAATTACCTAATTTCGTCACAGTCGCCAAGGCCACTACAACAATTGTCTACGGACGTGTTGGTCGTTCAGTCAATGCTTCATCTGGCACTGGTTGGGCTTTCTATGTCCGGTCCAAACACGGCGACTACTCAGCTGTGAGTAATCCGAGTTCGGTTCTCACAGATAGTGAGAAAGTGCTTCATTTAGTCCGTGACCACATGGTGCTGCATGAATATGTGAACGCGGCGGGCATCACG 18. TXN2-eGFP 1-10 Amino acid sequence (as shown in SEQ ID NO: 18): MVRSGIAAEAPLQMAQRLLLRRFLASVVSRKPSHSRWAPLASRALQTPQLSLSNLTGTPSQARSLYTTQVCTTTFNIQDGPDFQDRVVNSETPVVVDFHAQWCGPCKILGPRLEKVVAKQHGKVVMAKVDIDDHTDLAIEYEVSAVPTVLAIKNGDVVDKFVGIKDEDQLEAFLKKLIGLEMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK 19. PDCoV-eGFP 11 Amino acid sequence (as shown in SEQ ID NO: 19): MSDAEEWQIIVFIAIIWALGVILQGGYATRNRVIYVIKLILLWLLQPFTLVVTIWTAVDRSSKKDAVFIVSIIFAVLTFISWAKYWYDSIRLLMKTRSAWALSPESRLLAGIMDPMGTWRCIPIDHMAPILTPVVKHGKLKLHGQELANGISVRNPPQDMVIVSPSDTFHYTFKKPVESNNDPEFAVLIYQGDRASNAGLHTITTSKAGDARLYKYMRDHMVLHEYVNAAGIT 20. PEDV-eGFP 11 Amino acid sequence (as shown in SEQ ID NO: 20): MSNGSIPVDEVIQHLRNWNFTWNIILTILLVVLQYGHYKYSAFLYGVKMAILWILWPLVLALSLFDAWASFQVNWVFFAFSILMACITLMLWIMYFVNSIRLWRRTHSWWSFNPETDALLT TSVMGRQVCIPVLGAPTGVTLTLLSGTLLVEGYKVATGVQVSQLPNFVTVAKATTTIVYGRVGRSVNASSGTGWAFYVRSKHGDYSAVSNPSSVLTDSEKVLHLVRDHMVLHEYVNAAGIT The raw materials and reagents used in the construction and application of the mitochondrial fluorescent labeling tool provided by this invention are all commercially available.

[0035] The following examples further illustrate the construction of the porcine TXN2 mitochondrial fluorescent labeling tool and the fission-type fluorescent interaction detection system of the present invention: Example 1. Construction of eukaryotic recombinant expression vector (1) Mitochondrial tag gene amplification and vector recombination Reference sequences of four porcine mitochondrial localization genes, Fis1, COX8, TOM20, and TXN2, were obtained from the NCBI database. Their gene sequences are shown in SEQ ID NO:1-4; their amino acid sequences are shown in SEQ ID NO:5-8; and a schematic diagram of the eukaryotic expression vector construction is shown below. Figure 1 As shown.

[0036] The primer sequences are as follows: Fis1-F (as shown in SEQ ID NO:21): 5'-ACTAGTCCAGTGTGGTGGAATTCTATGGAGGCCGTGCTGAACGAGT-3' Fis1-R (as shown in SEQ ID NO:22): 5'-CTCGCCCTTGCTCACCATCTCGAGGGATTTGGACTTGGATACAGCAAG-3' COX8-F (as shown in SEQ ID NO:23): 5'-ACTAGTCCAGTGTGGTGGAATTCTATGTCCGCGCTGACTCCGCTGCTG-3' COX8-R (as shown in SEQ ID NO:24): 5'-CTCGCCCTTGCTCACCATCTCGAGCTCCCGCTTCTTGTAGCTGTCCAG-3' TOM20-F (as shown in SEQ ID NO:25): 5'-ACTAGTCCAGTGTGGTGGAATTCTATGGTGGGCCGGAACAGCGCCATTGC-3' TOM20-R (as shown in SEQ ID NO:26): 5'-CTCGCCCTTGCTCACCATCTCGAGCTCCACATCATCTTCAGCCAAGCT-3' TXN2-F (as shown in SEQ ID NO:27): 5'-ACTAGTCCAGTGTGGTGGAATTCTATGGTTCGAAGTGGTATCGCGG-3' TXN2-R (as shown in SEQ ID NO:28): 5'-CTCGCCCTTGCTCACCATCTCGAGGCCAATCAGCTTCTTCAGAAAGGC-3' The PCR reaction system is as follows: 2×Phanta Flash Master Mix (Dye Plus) 25 μl; corresponding upstream primer 2 μl; corresponding downstream primer 2 μl; corresponding cDNA template 2 μl; ddH2O 19 μl; The reaction conditions were: 98℃ pre-denaturation for 30s, 35 cycles (98℃ denaturation for 10s, 60℃ annealing for 5s, 72℃ extension for 15s), and 72℃ final extension for 5min; the obtained PCR products were detected by gel electrophoresis with 1% agarose, and the target fragments were extracted and purified.

[0037] The vector pcDNA4.0-eGFP was linearized using a double enzyme digestion method. Its gene sequence is shown in SEQ ID NO:9; and its amino acid sequence is shown in SEQ ID NO:10. The double enzyme digestion reaction system was as follows: pcDNA4.0-eGFP 1 μg; EcoRI 1 μl; XhoRI 1 μl; 10× digestion buffer 2 μl; ddH2O to a final volume of 20 μl; incubated at 37℃ for 30 min; the digestion products were verified by electrophoresis and then recovered by gel extraction.

[0038] Four tag genes were ligated into the linearized pcDNA4.0-eGFP vector using homologous recombination technology. The ligation reaction system was as follows: 50 ng of linearized vector; 30 ng of target gene; 5 μl of homologous recombination enzyme; ddH2O to bring the total to 10 μl; and the reaction was carried out in a water bath at 50°C for 15 min.

[0039] The ligation product was transformed into DH5α competent cells and plated on LB agar plates containing 100 μg / ml ampicillin, and cultured overnight. Single colonies were picked for colony PCR verification. Primers were CMV-F and BGH-R. The reaction system was: Rapid TaqMaster Mix 12.5 μl; CMV-F 1 μl; BGH-R 1 μl; bacterial culture 2 μl; ddH2O 8.5 μl. The reaction conditions were: 95℃ pre-denaturation for 30 min, 35 cycles (95℃ denaturation for 15 s, 55℃ annealing for 5 s, 72℃ extension for 2 min), and 72℃ final extension for 5 min. Positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO:11-14, and the gel imaging results are shown in [image missing]. Figure 2 As shown. Plasmids were extracted from the correctly sequenced strains after amplification culture for later use.

[0040] (2) Construction of Split-eGFP series eukaryotic expression vectors

[0041] Using the Split-eGFP dual-fragment system and pcDNA4.0 as a unified vector backbone, two types of recombinant plasmids were constructed: ① Template preparation: The TXN2 gene fragment was amplified using the validated pcDNA4.0-TXN2-eGFP plasmid as a template; eGFP was amplified using the commercially available Split-eGFP standard plasmid as a template. 1-10 Large fragments and eGFP 11 Small fragments; using cDNA from laboratory-preserved PDCoV CHN-HN-1601 strain (GenBank accession no. MG832584) and PEDV CV777 strain (GenBank accession no. AF353511) as templates, the M protein encoding gene was amplified respectively.

[0042] ② Preparation of fusion genes by overlap PCR: This invention uses overlap PCR technology to construct TXN2-eGFP1-10, PDCoV M-eGFP11, and PEDV M-eGFP, respectively. 11 Three sets of fusion genes; for cases where the fragments to be fused lack naturally complementary overlapping sequences, complementary overlapping regions and corresponding restriction enzyme sites are first introduced at the ends of the fragments using specific primers, followed by two rounds of PCR reactions to complete fusion amplification and product purification. Among these, TXN2-eGFP was constructed. 1-10 When fusing genes, the TXN2 encoding gene is used as the insert fragment 1, eGFP. 1-10The coding sequence is insert 2. The forward amplification primer sequence for insert 1 is 5'-CTTGGTACCGAGCTCGGATCCATGGTTCGAAGTGGTATCGCG-3' (as shown in SEQ ID NO:29), which contains the BamHI restriction site GGATCC at its 5' end. The reverse amplification primer sequence for insert 1 is 5'-TCTTCTCCTTTGGACTCGAGGCCAATCAGCTTCTT-3' (as shown in SEQ ID NO:30). The forward amplification primer sequence for insert 2 is 5'-CTCGAGTCCAAAGGAGAAGAACTGTTTACCG-3' (as shown in SEQ ID NO:31). The reverse amplification primer sequence for insert 2 is 5'-AACGGGCCCTCTAGACTCGAGTTTTTCATTTGGATCTTTGCTCAG-3' (as shown in SEQ ID NO:32), which contains the XhoI restriction site CTCGAG at its 5' end. PDCoV M-eGFP was constructed. 11 When fusing genes, the PDCoV M encoding gene is used as the insert fragment 1, eGFP. 11 The coding sequence is insert 2. The forward amplification primer sequence for insert 1 is 5'-CTTGGTACCGAGCTCGGATCCATGTCTGACGCAGAAGAGTGGC-3' (as shown in SEQ ID NO:33), which contains the BamHI restriction site GGATCC at its 5' end. The reverse amplification primer sequence for insert 1 is 5'-CCATGTGGTCACGCATATACTTATACAGGCGAGCGTCACC-3' (as shown in SEQ ID NO:34). The forward amplification primer sequence for insert 2 is 5'-GTATATGCGTGACCACATGGTGC-3' (as shown in SEQ ID NO:35). The reverse amplification primer sequence for insert 2 is 5'-AACGGGCCCTCTAGACTCGAGCGTGATGCCCGCCGCGTT-3' (as shown in SEQ ID NO:36), which contains the XhoI restriction site CTCGAG at its 5' end. PEDV M-eGFP is constructed. 11 When fusing genes, the PEDV M encoding gene is used as the insert fragment 1, eGFP. 11The coding sequence is insert 2. The forward amplification primer sequence for insert 1 is 5'-CTTGGTACCGAGCTCGGATCCATGTCTAACGGTTCTATTCCCGTT-3' (as shown in SEQ ID NO:37), which contains the BamHI restriction site GGATCC at its 5' end. The reverse amplification primer sequence for insert 1 is 5'-CATGTGGTCACGCATGACTAAATGAAGCACTTTCTCACTATCTG-3' (as shown in SEQ ID NO:38). The forward amplification primer sequence for insert 2 is 5'-TAGTCATGCGTGACCACATGGTGC-3' (as shown in SEQ ID NO:39). The reverse amplification primer sequence for insert 2 is 5'-AACGGGCCCTCTAGACTCGAGCGTGATGCCCGCCGCGTT-3' (as shown in SEQ ID NO:40), which contains the XhoI restriction site CTCGAG at its 5' end. The specific construction process of the three sets of fusion genes is as follows: PCR amplification was performed using upstream and downstream fragment templates to obtain purified fragments to be fused with complementary overlapping regions at the ends. The two fragments to be fused from each group were mixed in a 1:1 molar ratio, and 2×Phanta Flash Master Mix was added for the first round of primerless fusion reaction. After 5 PCR cycles, the two fragments were allowed to pair complementaryly at the overlapping regions at the ends and complete polymerization extension, splicing them to form a double-stranded template of the full-length fusion gene. Then, the corresponding upstream and downstream primers of the fusion gene were added to the reaction system for the second round of PCR amplification. 30 cycles were set to enrich the full-length fusion gene product. After amplification, the product was separated by agarose gel electrophoresis, and the target band corresponding to the expected molecular weight was cut out. After gel recovery and purification, the three sets of purified full-length fusion genes were obtained.

[0043] ③ Homologous recombination cloning: After double digestion and linearization of the pcDNA4.0 vector, homologous recombination ligation was performed with three sets of fusion genes. The cloning was then transformed into DH5α, followed by initial screening by bacterial culture PCR and sequencing verification. The gene sequences are shown in SEQ ID NO:15-17, and the amino acid sequences are shown in SEQ ID NO:18-20. Endotoxin-free plasmids were extracted from the correctly sequenced strains and stored at -20℃ in the dark for later use. A schematic diagram of the Split-eGFP system is shown below. Figure 3 As shown.

[0044] Example 2. Performance Validation of the Mitochondrial Labeling Tool

[0045] (1) Protein expression identification: LLC-PK1 cells in logarithmic growth phase were washed twice with PBS, digested with 1× 0.25% Trypsin-EDTA (Gibco, product number 3224459), digestion was stopped with 10% DMEM, centrifuged and resuspended, and then seeded into 6-well plates and cultured for 12 h. After cell adhesion, four mitochondrial tag plasmids were transfected, and an empty vector control was set up. The transfection complex was prepared by first diluting 1 μg of plasmid with 100 μl of 0% DMEM and 3 μl of transfection reagent (Shanghai Nolai Biotechnology Co., Ltd., product number CT801) with 100 μl of 0% DMEM, incubating for 5 min, mixing, and continuing incubation for 30 min. The complex was then added to the cell wells and cultured at 37℃ with 5% CO2 for 24 h. Discard the culture medium, add RIPA cell lysis buffer (Beyotime Biotechnology Co., Ltd., product number P0013D), lyse on ice for 30 min, centrifuge at 12000 rpm for 10 min at 4℃, collect the supernatant, and detect the expression of the target protein by Western blotting. The results are as follows. Figure 4 As shown.

[0046] (2) Laser confocal two-dimensional imaging detection: LLC-PK1 cells were seeded in 24-well plates containing climbing slides and cultured for 12 h. Afterward, plasmids were transfected using the method described above. 24 h after transfection, the cells were fixed overnight at 4°C with pre-cooled 4% paraformaldehyde fixative (Beyotime Biotechnology Co., Ltd., P0099). After washing with PBS, permeabilization buffer Triton X-100 (Beyotime Biotechnology Co., Ltd., P0096) was added for 10 min. After washing with PBS, DAPI (Beyotime Biotechnology Co., Ltd., C1006) was added and incubated for 10 min. After washing three times with PBS, the slides were mounted, and the imaging effect was observed using an inverted laser confocal microscope. The results are as follows: Figure 5 As shown.

[0047] (3) Comparison of mitochondrial three-dimensional reconstruction imaging: Two labeling plasmids, Fis1-eGFP and TXN2-eGFP, which showed better two-dimensional imaging effects, were selected and transfected into LLC-PK1 cells. Fixation and staining were performed according to the above method. Continuous tomographic images were acquired along the Z-axis of the cells using an inverted laser confocal microscope. After acquiring a series of tomographic images, three-dimensional volume reconstruction was performed using the accompanying imaging software to compare the restoration effect and precision of the mitochondrial three-dimensional network structure by the two labeling tools. The results are as follows: Figure 6 As shown, the 3D images of mitochondria labeled with Fis1-eGFP have blurred edges and are difficult to show the delicate mitochondrial branches; while the 3D outlines of mitochondria labeled with TXN2-eGFP are clear and the network structure is coherent and complete. It can clearly show the spatial distribution, branching morphology and connection details of mitochondria. The 3D reconstruction imaging quality is significantly better than that of Fis1-eGFP, which can meet the needs of 3D observation and quantitative analysis of fine morphology of mitochondria in pig cells.

[0048] (4) Establishment of a mitochondrial-endoplasmic reticulum interaction detection system

[0049] LLC-PK1 cells were seeded in 24-well plates containing slides and cultured for 12 h. Afterward, they were co-transfected with TXN2-eGFP plasmid and the endoplasmic reticulum marker plasmid PDI-mCherry. The cells were cultured at 37°C with 5% CO2 for 24 h, then fixed overnight at 4°C with pre-cooled 4% paraformaldehyde fixative (Beyotime Biotechnology Co., Ltd., P0099). After washing with PBS, permeabilization was performed for 10 min with Triton X-100 permeabilization buffer (Beyotime Biotechnology Co., Ltd., P0096). After further washing with PBS, DAPI (Beyotime Biotechnology Co., Ltd., C1006) was added and incubated for 10 min. The cells were then washed three times with PBS and mounted. Green and red fluorescence signals were acquired using a dual-channel laser confocal microscope to observe the spatial distribution and contact sites of mitochondria and endoplasmic reticulum, establishing a visual detection system for porcine mitochondrial-endoplasmic reticulum interaction. Results are as follows: Figure 7 As shown, the mitochondrial fluorescence signal of TXN2-eGFP and the endoplasmic reticulum fluorescence signal of PDI-mCherry are clear, and their co-localization regions can be accurately identified. This can intuitively present the spatial contact and interaction regions between mitochondria and endoplasmic reticulum, confirming that this labeling tool can support qualitative observation and semi-quantitative analysis of organelle interactions, and expanding its application scope in cell biology research.

[0050] (5) Sequence difference analysis and protein half-life detection

[0051] The full-length sequences of the TXN2 gene from four species—pig, human, chicken, and mouse—were obtained from NCBI. Multiple sequence alignment and homology analysis were performed using MEGA software. The results are as follows: Figure 8 As shown, the TXN2 gene sequences from different species exhibit significant differences and low overall homology, suggesting that universal mitochondrial marker molecules are difficult to achieve precise mitochondrial localization and functional tracing across species. Therefore, the development of species-specific mitochondrial fluorescent labeling tools has clear application necessity and practical value.

[0052] LLC-PK1 cells were seeded in 6-well plates and transfected with TXN2-eGFP plasmid and other tag plasmids. After 24 h, cells were treated with 50 μg / ml actinomycin. Cell samples were collected at 0, 3, 6, 9, 12, and 18 h. The expression levels of eGFP fusion proteins with different mitochondrial locations were detected by Western blot. β-actin was used as an internal control for gray-scale quantification. The gray-scale ratio of the target protein to the internal control at each time point was normalized to the 0 h group to obtain the relative protein level. The results are as follows: Figure 9As shown, the protein levels of COX8-eGFP, Fis-eGFP, and TOM20-eGFP proteins all decreased significantly at 12 and 18 hours after CHX treatment. COX8-eGFP showed the fastest degradation rate, with its relative protein level at 12 h being only 18% of that at 0 h, and further decreasing to 12% at 18 h. TOM20-eGFP degradation accelerated significantly after 12 h, with its relative protein level at 18 h being less than 10% of that at 0 h. Fis-eGFP exhibited a time-dependent, gradual degradation, with relative protein levels at 12 h and 18 h being 55% and 38% of those at 0 h, respectively. After correction with β-actin as an internal reference, the protein abundance of all three proteins was significantly lower at 12 h and 18 h compared to 0 h. In stark contrast, the protein level of TXN2-eGFP remained stable within the 0–18 h range after CHX treatment, with the relative protein level at each time point maintained above 93% of that at 0 h after internal reference normalization, showing no significant degradation. The above results confirm that among all mitochondrial localization proteins involved in this experiment, TXN2 has the longest protein half-life and excellent intracellular stability. Compared with traditional easily degradable mitochondrial marker molecules, TXN2 can achieve long-term, high-intensity stable mitochondrial tracking, and is especially suitable for experimental scenarios such as long-term live-cell imaging, dynamic mitochondrial tracking, and long-term drug treatment, with significant application advantages.

[0053] (6) Cell safety testing

[0054] LLC-PK1 cells were seeded in 96-well plates and cultured for 12 h. Afterward, they were transfected with different doses of TXN2-eGFP plasmid, with dose and time gradients set up, and each group had 3 replicates. After the set time, 10 μl of CCK8 reagent was added to each well, and the cells were incubated for 1 h. The absorbance at 450 nm was measured using a microplate reader to evaluate cell viability and vector safety. The results are shown below. Figure 10 As shown in the left figure, the time gradient detection results indicate that at 24 h and 48 h post-transfection, there was no statistically significant difference in cell viability between the pcDNA3.1-TXN2-eGFP transfected group and the empty vector control group at the same time points. P >0.05, ns). As shown in the dose gradient detection results (right figure), under the two transfection doses of 1 μg and 4 μg, the cell viability of the pcDNA3.1-TXN2-eGFP transfection group was not significantly different from that of the control group with the same dose of empty vector. P >0.05, ns). In summary, under the experimental conditions, TXN2 protein overexpression did not interfere with normal cell proliferation activity, the recombinant expression plasmid showed no significant cytotoxicity, and possessed good cell safety, providing a safe basis for subsequent cellular-level mechanism research and applications.

[0055] Example 3. Construction and Validation of Split-eGFP Splitting Fluorescent Interaction Detection System

[0056] LLC-PK1 cells were seeded in 24-well plates containing climbing smears and cultured for 12 h. Afterward, they were co-transfected with plasmids. Experimental groups were as follows: Blank control group: TXN2-eGFP. 1-10 +free eGFP 11 Plasmid; Experimental group: TXN2-eGFP 1-10 + PEDV M-eGFP 11 Fusion plasmid, TXN2-eGFP 1-10 + PDCoV M-eGFP 11 Fusion plasmid. After incubation at 37℃ and 5% CO2 for 24 h, fixation, staining, and mounting were performed as described above. Fluorescence reconstruction signals were detected using an inverted laser confocal microscope. Results are as follows: Figure 11 As shown, obvious mitochondrial-localized green fluorescence was observed in the experimental group, while no specific fluorescence signal was observed in the control group, confirming that the system can effectively detect the interaction between viral proteins and mitochondria and can capture the interaction events between the virus and host organelles.

[0057] This invention is the first to apply porcine TXN2 to the Split-eGFP system, constructing a virus-mitochondrial interaction visualization detection tool specific to porcine cells, thus avoiding the species adaptation defects of heterologous tools. It is also broadly compatible with multiple porcine coronaviruses, including PDCoV and PEDV, enabling in situ, dynamic, and specific detection of mitochondrial localization and interaction characteristics of viral proteins. Compared to traditional immunoprecipitation and conventional co-localization imaging techniques, this system offers higher sensitivity and specificity, capturing transient and weak virus-host interactions. It forms a complete visualization detection technology system for the pathogenic mechanisms of porcine coronaviruses, possessing extremely high research application and technology transfer value.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a porcine TXN2 mitochondrial fluorescent labeling plasmid, characterized in that, Includes the following steps: Step A, Amplification TXN2 Porcine mitochondrial tag genes; Step B: Linearize the pcDNA4.0-eGFP vector by double digestion with EcoRI and XhoI to obtain a linearized vector; Step C: Recombining the... TXN2 The porcine mitochondrial tag gene was ligated into the linearized vector, transformed into DH5α competent cells, and verified by bacterial PCR and sequencing. The positive plasmid pcDNA4.0-TXN2-eGFP was extracted. The TXN2 The porcine mitochondrial tag gene has the nucleotide sequence shown in SEQ ID No.

4.

2. The construction method as described in claim 1, characterized in that, The TXN2 The protein encoded by the porcine mitochondrial tag gene has the amino acid sequence shown in SEQ ID No.

8.

3. The construction method as described in claim 1, characterized in that, The linearized vector has a nucleotide sequence as shown in SEQ ID No.

9.

4. The construction method as described in claim 1, characterized in that, The linearized vector has an amino acid sequence as shown in SEQ ID No.

10.

5. The porcine TXN2 mitochondrial fluorescent labeling plasmid obtained by the construction method according to any one of claims 1 to 4.

6. A method for constructing a Split-eGFP splitting fluorescence interaction detection system, characterized in that, Based on the porcine TXN2 mitochondrial fluorescent labeling plasmid obtained by the construction method according to any one of claims 1 to 4, the following steps are included: Amplification was performed using the porcine TXN2 mitochondrial fluorescent labeling plasmid as a template. TXN2 Gene fragments; Using the Split-eGFP dual-fragment system with pcDNA4.0 as a unified backbone, TXN2-eGFP was constructed separately via overlap PCR. 1-10 PDCoV M-eGFP 11 PEDV M-eGFP 11 Fusion recombinant plasmid; The TXN2-eGFP 1-10 It has the nucleotide sequence shown in SEQ ID No. 15; The TXN2-eGFP 1-10 It has the amino acid sequence shown in SEQ ID No. 18; The PDCoV M-eGFP 11 It has the nucleotide sequence shown in SEQ ID No. 16; The PDCoV M-eGFP 11 It has the amino acid sequence shown in SEQ ID No. 18; The PEDV M-eGFP 11 It has the nucleotide sequence shown in SEQ ID No. 15; The PEDV M-eGFP 11 It has the amino acid sequence shown in SEQ ID No.

18.

7. The Split-eGFP splitting fluorescence interaction detection system obtained by the construction method as described in claim 6.

8. The application of the porcine TXN2 mitochondrial fluorescent labeling plasmid as described in claim 5 and / or the Split-eGFP splitting fluorescent interaction detection system as described in claim 7 in any of the following: (I) Observation of mitochondrial morphology in pig cells; (II) Detection of porcine organelle interactions; (III) Research on the pathogenic mechanism of porcine coronavirus; in, The detection of porcine organelle interactions includes visual observation and / or quantitative analysis of mitochondrial-endoplasmic reticulum spatial interactions; The study on the pathogenesis mechanism of porcine coronavirus includes in situ visualization detection of virus-mitochondrial interactions.

9. A method for detecting mitochondrial-endoplasmic reticulum spatial interactions, characterized in that, The porcine TXN2 mitochondrial fluorescent labeling plasmid as described in claim 5 was co-transfected with the endoplasmic reticulum labeling molecule PDI-mCherry to establish a dual fluorescent labeling detection system, thereby enabling visual observation and quantitative analysis of the spatial interaction between mitochondria and endoplasmic reticulum.

10. A method for in-situ visualization detection of virus-mitochondrial interactions, characterized in that, Mitochondrial-anchored TXN2-eGFP in the Split-eGFP splitting fluorescence interaction detection system as described in claim 7 1-10 eGFP, a plasmid fused with viral proteins 11 Plasmids were co-transfected into porcine cells, and fluorescence signals were detected using laser confocal microscopy after culture. When the viral protein interacted spatially with the mitochondria and the distance reached a threshold, eGFP was activated. 1-10 With eGFP 11 The fragment spontaneously reconstructs into a complete fluorescent protein and restores green fluorescence, enabling in-situ visualization detection of virus-mitochondrial interactions.

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