Use of ND-42 related plasmid in preparation of fruit fly mitochondrial protein expression kit

Abstract

The application discloses an application of an ND-42 related plasmid in preparation of a Drosophila mitochondrial protein expression kit, and the kit is mainly designed for a preparation method of mitochondrial related protein expression in S2 cells for the ND-42 protein. The kit constructs a pUAS-attB-3xHA-ND-42CDS full-length plasmid, a pUAS-attB-3xHA-ND-42-DNK-dom plasmid and a pUAS-attB-3xFlag-Nap1CDS full-length plasmid. The kit mainly uses a UAS-Gal4 system to realize the expression of the ND-42 protein, the DNK domain of the ND-42 protein and the Nap1 protein. The results of immunoprecipitation and Western blotting show that the Nap1 protein can strongly interact with the ND-42 protein. Further experimental observation shows that the Nap1 protein can also strongly physically combine with the DNK domain of the ND-42 protein. The kit can provide an important research method for the function and mechanism exploration of the mitochondrial related protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of ND-42 related plasmid in preparing mitochondrial protein expression kit in Drosophila S2 cells, belonging to the field of basic medicine. BACKGROUND

[0002] Mitochondria, as functional organelles in somatic and germ cells, not only serve as an important source of energy and reactive oxygen species, but also play a key role in signal transduction, cell proliferation and differentiation, and cell programmed death, thus participating in the occurrence of a variety of diseases. During spermatogenesis, mitochondria undergo great changes in morphology, including dynamic changes in shape and localization. In the post-meiotic spermatid, the ring-shaped mitochondrial sheath is organized around the axoneme. Subsequently, the spermatid begins to elongate, and the mitochondria unfold and fuse to form two large mitochondrial derivatives. In Drosophila, mitochondrial dynamics mainly includes the processes of mitochondrial fusion and fission, and is essential for the regulation of spermatogenesis. Fuzzy onions (fzo) is identified as a mitochondrial fusion gene, which can encode a transmembrane GTPase protein. Deletion of fzo gene can lead to mitochondrial fusion defects, eventually mediating the occurrence of male sterility. Porin is another important gene affecting mitochondrial fusion and fission, and its disruption can also impair the individualization process of spermatids and lead to male sterility. Through the study of Drosophila Pink1 gene, many scholars have found the targeting binding sequence and serine / threonine kinase domain of mitochondria, and its regulatory mode is similar to that of human Pink1 gene, and it is also related to the pathogenesis of Parkinson's disease. In the testis of Pink1 mutant, the nebenkern structure in spermatids presents a vacuole-like structure, and only one mitochondrial derivative is derived in the subsequent differentiation stage, which is similar to the phenotype of Parkin mutant testis, indicating that Pink1 and Parkin may regulate the development process of spermatids by reducing mitochondrial fission or increasing mitochondrial fusion.

[0003] ND-42 is also known as nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 42kDa subunit, which encodes the ortholog of human NDUFA10 and belongs to a subunit of mitochondrial electron transport chain complex I. In some biological events, ND-42 is generally considered to be an inhibitor of Pink1 and can interact with various proteins. Studies have shown that ND-42 is mainly located in mitochondria and can regulate mitochondrial function and related metabolic processes. Importantly, ND-42 is essential for the homeostasis of oxidative stress in various tissues, which involves multiple systems such as reproduction and nervous system. However, the function and mechanism of ND-42 in different tissues have not been fully elucidated. The kit is mainly designed for the preparation of mitochondrial related protein expression in S2 cells, which helps to further study the function and regulation mechanism of mitochondrial related proteins. SUMMARY

[0004] The purpose of the present application is to provide a kit for ectopic expression of ND-42 related plasmid in Drosophila S2 cells, which is used for the preparation of Drosophila mitochondrial related protein expression. ND-42 protein is a key mitochondrial protein identified in previous studies. By expressing related plasmid in Drosophila S2 cells through UAS-Gal4 system, ND-42 protein and DNK domain of ND-42 protein can be prepared, which helps to further study the function and regulation mechanism of mitochondrial related proteins. The present application solves the above technical problems by adopting the following technical solutions:

[0005] The application of ND-42 related plasmid in the preparation of Drosophila S2 cell mitochondrial protein expression kit, the kit is mainly designed for the preparation of mitochondrial related protein expression in S2 cells.

[0006] Further, the kit constructs pUAS-attB-3xHA-ND-42CDS full-length plasmid, pUAS-attB-3xHA-ND-42-DNK-dom plasmid and pUAS-attB-3xFlag-Nap1 CDS full-length plasmid.

[0007] Further, the UAS-Gal4 system is used to realize the expression of ND-42 protein, DNK domain of ND-42 protein and Nap1 protein.

[0008] Further, the kit uses immunoprecipitation and Western blot analysis to find that Nap1 protein can have strong physical interaction with ND-42 protein and DNK domain.

[0009] Advantages

[0010] The present application realizes the expression of ND-42 protein, DNK domain of ND-42 protein and Nap1 protein by using UAS-Gal4 system. The results of immunoprecipitation and Western blotting show that Nap1 protein can have strong interaction with ND-42 protein. Further observation shows that Nap1 protein can also have strong physical combination with DNK domain of ND-42. The kit is helpful for the in-depth study of the function and regulation mechanism of mitochondria-related proteins. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Figure A is the pUAS-attB-3xHA-ND-42CDS full-length plasmid map; Figure B is the pUAS-attB-3xHA-ND-42-DNK-dom plasmid map; Figure C is the pUAS-attB-3xFlag-Nap1 CDS full-length plasmid map; and Figure D is the action mode diagram of ND-42 protein and Nap1 protein.

[0012] Figure 2 Figure A is the pUAS-attB-3xHA-ND-42CDS full-length plasmid map; Figure B is the pUAS-attB-3xHA-ND-42-DNK-dom plasmid map; Figure C is the pUAS-attB-3xFlag-Nap1 CDS full-length plasmid map; and Figure D is the action mode diagram of ND-42 protein and Nap1 protein.

[0013] Figure 3 Figure A is the pUAS-attB-3xHA-ND-42CDS full-length plasmid map; Figure B is the pUAS-attB-3xHA-ND-42-DNK-dom plasmid map; Figure C is the pUAS-attB-3xFlag-Nap1 CDS full-length plasmid map; and Figure D is the action mode diagram of ND-42 protein and Nap1 protein. DETAILED DESCRIPTION

[0014] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be explained as a limitation of the present application.

[0015] The present application is used to solve the above technical problems by using the following technical solutions:

[0016] Example 1 Preparation and expression of ND-42 related plasmid

[0017] The kit mainly realizes the expression of ND-42 protein, DNK domain of ND-42 protein and Nap1 protein by using UAS-Gal4 system. The kit constructs pUAS-attB-3xHA-ND-42CDS full-length plasmid, pUAS-attB-3xHA-ND-42-DNK-dom plasmid and pUAS-attB-3xFlag-Nap1 CDS full-length plasmid.

[0018] (1) UAS-Gal4 system

[0019] The kit mainly drives the expression of pUAS-attB related plasmid by using ub-Gal4 (a widely expressed Gal4) through Drosophila UAS-Gal4 system to realize the specific expression of related proteins. The pUAS-attB plasmid designed in the kit can express mitochondrial related fusion proteins containing tagged proteins. Driving the expression of these tagged fusion proteins can be used for subsequent co-immunoprecipitation, immunofluorescence, Western blotting and other experiments.

[0020] (2) Plasmid construction

[0021] Construction of pUAS-attB-3xHA-ND-42 CDS full-length plasmid and pUAS-attB-3xHA-ND-42-DNK-dom plasmid: ND-42 CDS and ND-42 (DNK_dom) CDS sequences were amplified by PCR, and the CDS sequences were connected to the pUAS-attB-3xHA vector by using Not I and Xba I restriction endonuclease sites. Construction of pUAS-attB-3xFlag-Nap1 CDS full-length plasmid: 3xFlag-Nap1 CDS sequence was synthesized in vitro, and the CDS sequence was connected to the pUAS-attB vector by using EcoR I and Kpn I restriction endonuclease sites.

[0022] Example 2 Interaction of Nap1 protein with ND-42 DNK domain

[0023] HA-ND-42 protein, HA-ND-42 protein DNK domain and Flag-Nap1 protein were overexpressed in Drosophila S2 cells. The results of co-immunoprecipitation and Western blotting showed that ND-42 protein could have strong physical binding with Nap1 protein. By further observing the interaction between ND-42 protein DNK domain and Flag-Nap1 protein, it was found that there was also physical binding between them. The above results suggest that Nap1 protein can play a role by binding to the DNK domain of ND-42 protein.

[0024] (1) Drosophila S2 cell culture and plasmid transfection

[0025] 1) Cell recovery: The frozen Drosophila S2 cells were quickly removed from liquid nitrogen, then placed in a 42°C constant temperature water bath, and the frozen tube was gently shaken to accelerate cell melting. Try to melt all the cells within one minute. After melting, the outer wall of the frozen tube was disinfected with 75% alcohol and wiped clean with an alcohol cotton ball. Then, the melted cells were quickly transferred to a culture dish containing 5 mL of preheated 10% fetal bovine serum culture solution. First, the cells were incubated in a 28°C incubator for 30 min, then centrifuged at 300g for 5 min, the supernatant was removed to remove DMSO, and the cells were resuspended with 2 mL of fresh culture solution and transferred to a culture dish for further culture at 28°C.

[0026] 2) Cell passage: In order to obtain a stable cell strain or to expand a large number of cells in a short time, and to maintain the continuity of the cell species. When the cell growth density reaches 6-20 x 10 6 / mL, the cells need to be passaged at a ratio of 1:1-1:2. 1 mL of cell solution was removed from the culture dish and transferred to another clean sterile culture dish, and 1 mL of preheated 10% fetal bovine serum culture solution was added to each dish. The cells after passage continue to be incubated at 28°C, and then can be frozen or used for experiments.

[0027] 3) Cell freezing: During the experiment, the state is good and the excess cells can be frozen. After collecting 2-3 mL of cell solution, transfer it to a clean sterile EP tube, centrifuge at 300g for 5 min, remove the supernatant, add 1 mL of freezing solution, resuspend the cell pellet, mix well by blowing, and then transfer to the labeled freezing tube. Immediately place it in the programmed cooling box and store it at -80°C, and after 48 hours, transfer it to liquid nitrogen for long-term storage.

[0028] 4) Plasmid transfection: HA-ND-42 protein, HA-ND-42-DNK_dom truncated protein and Flag-Nap1 protein are expressed under the drive of ub-Gal4. The high-efficiency transfection reagent (Qiagen) can transfect plasmid (0.4 μg per well) into S2 cells. Use EC buffer to dilute the plasmid to 100 μL, and add 6.4 μL enhancer, vortex and incubate at room temperature for 5 min. Then, form a lipid complex by adding 20 μL Effectene Transfection Reagent, and incubate for 10 min. Subsequently, 600 μL of opti-MEM reagent is added to the lipid complex, mixed with S2 cells, and transfection is performed.

[0029] (2) Immunoprecipitation and Western Blot

[0030] RIPA lysis buffer was added to S2 cells to extract total protein, and the concentration was determined by BCA method. The protein suspension was incubated with anti-HA magnetic beads or anti-Flag magnetic beads at 4°C for 2h, and the magnetic beads were washed 3 times at 4°C (5min / time, centrifuged at 500g for 5min each time), and then eluted. After adding loading buffer to the eluate, heat at 95°C for 5min.

[0031] Take 20μg total protein for 10% SDS-PAGE gel electrophoresis, transfer to PVDF membrane, block with 5% skim milk at room temperature for 1h, add primary antibody and incubate at 4°C overnight, wash 3 times with TBST (5min / time), add secondary antibody and incubate at room temperature for 1h, wash the membrane and observe the immunoreaction protein band in the fluorescence image analyzer. Primary antibody information: HA antibody (Cell Signaling Technology, dilution ratio 1:1000), Flag antibody (Beyotime, dilution ratio 1:1000). Secondary antibody information: Alexa 680(Abcam, dilution ratio 1:3000), 800CW(Abcam, dilution ratio 1:3000).

Claims

1. A method of preparing for expression of Drosophila mitochondrial proteins in Drosophila S2 cells, characterized by, Comprising the following steps: Step 1: Constructing plasmid combination: Constructing pUAS-attB-3xHA-ND-42 CDS full-length plasmid, amplifying ND-42 CDS sequence by PCR, and connecting to pUAS-attB-3xHA vector by using Not I and Xba I restriction endonuclease sites; Constructing pUAS-attB-3xHA-ND-42-DNK-dom plasmid, amplifying DNK domain sequence of ND-42 by PCR, and connecting to pUAS-attB-3xHA vector by using Not I and Xba I restriction endonuclease sites; Constructing pUAS-attB-3xFlag-Nap1 CDS full-length plasmid, synthesizing 3xFlag-Nap1 CDS sequence in vitro, and connecting to pUAS-attB vector by using EcoR I and Kpn I restriction endonuclease sites; Step 2: Configuring expression system: Integrating the plasmid combination of step 1 with UAS-Gal4 system to express ND-42 protein, ND-42 protein DNK domain and Nap1 protein in Drosophila S2 cells.

2. The method of claim 1, wherein, The UAS-Gal4 system in step 2 drives plasmid expression by ub-Gal4 promoter.

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