CN116622778A - Fluorescence labeling baculovirus expression vector for co-immunoprecipitation and construction method thereof - Google Patents

Fluorescence labeling baculovirus expression vector for co-immunoprecipitation and construction method thereof Download PDF

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CN116622778A
CN116622778A CN202310522262.3A CN202310522262A CN116622778A CN 116622778 A CN116622778 A CN 116622778A CN 202310522262 A CN202310522262 A CN 202310522262A CN 116622778 A CN116622778 A CN 116622778A
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梁昌镛
马媛媛
赵淑玲
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Yangzhou University
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Abstract

The invention discloses a fluorescence labeling baculovirus expression vector for co-immunoprecipitation and a construction method thereof. The fluorescence labeling baculovirus expression vector is characterized in that after inserting a promoter SV40-IE2 into a pFastBac-Dual vector and connecting an eGFP sequence to the promoter SV40-IE2, an interaction protein A sequence fused with an HA tag and an interaction protein B sequence fused with a Flag tag are respectively connected to a promoter of the vectorp10A promoterpolhAnd then, the method is carried out. The expression vector can check the virus infection efficiency through fluorescent observation, and can express two proteins in the same cell, so that the successfully transfected recombinant virus can be better observed, and the analysis in insect cells is facilitatedInteractions between proteins.

Description

Fluorescence labeling baculovirus expression vector for co-immunoprecipitation and construction method thereof
Technical Field
The invention belongs to the technical field of construction of virus expression vectors, and relates to a fluorescence labeling baculovirus expression vector for co-immunoprecipitation and a construction method thereof.
Background
Co-immunoprecipitation (Co-IP) is a common method for in vivo analysis of protein interactions based on specific binding between antigen and antibody, and its principle is that two proteins to be verified for interactions are expressed in the same cell, then precipitation and immunoblotting detection are performed using a tag antibody or a protein autoantibody, and finally the nucleotide sequence thereof can be deduced by sequence analysis. Since immunoprecipitation is natural physiological binding and is not affected by human, the result has high reliability, and the advantage is that the interacted proteins exist in nature and are carried out in natural state, thus avoiding the human influence, and the interacted protein complex in natural state can be separated (Feng Xiaoqin, xu Feng, wang, etc. the weak interaction of the proteins is verified by optimizing the co-immunoprecipitation technology [ J ]. The Proc of Chinese academy of sciences research, 2013,30 (01): 18-23). However, this method requires a certain amount of antibody against the target protein, and requires a certain concentration of the protein, the binding of the two proteins may not be direct, and there may be a third party bridging the middle (Li, li Yi, gao Jianmei. Development of co-immunoprecipitation techniques [ J ]. J. Enmongolian medicine, 2008 (04): 452-454.), so that only highly expressed proteins are apparent in practical operation, and this method is not suitable for all cases.
The baculovirus-insect cell expression system has the function of high-level recombinant protein expression, and the insect cell has similar mode and capacity of translating and post-translating modification of recombinant protein as that of mammal cell, and the expressed recombinant protein has bioactivity similar to that of natural protein. Currently, co-transfection methods are generally used for the analysis of protein interactions in Sf9 cells using baculovirus expression systems, i.e., co-transfection of Sf9 cells with Bacmid carrying the protein a gene and baculovirus shuttle vector (Bacmid) carrying the protein B gene. However, this method has many disadvantages such as generally low co-transfection efficiency (i.e., the expression of two proteins in the same cell is small) and the inability to observe the infection efficiency. The target protein is fluorescently marked by using Green Fluorescent Protein (GFP), so that the visualization of transfection effect can be realized. However, since GFP is large, it is often fused with a target protein to observe the expression distribution or subcellular localization of the target protein in cells and tissues and organs, and the like, it is rarely used as a Co-IP tag protein (Wang Mingjiang, wu Jinxia, zhang Yugong, etc.. Recent progress in protein interaction experimental techniques [ J ]. Genetics, 2013,35 (11): 1274-1282.). If eGFP is fused to the rear end of the target gene, fluorescence is observed, but the fused eGFP is large, which affects the protein structure and thus interaction.
Disclosure of Invention
The invention provides a fluorescence labeling baculovirus expression vector for co-immunoprecipitation and a construction method thereof. According to the invention, a new promoter is inserted into the vector to independently express the eGFP green fluorescent gene, and the constructed baculovirus expression vector not only contains the independently expressed eGFP gene and has fluorescent markers, but also can simultaneously express two different proteins A and B without mutual influence. The invention can observe virus infection efficiency through fluorescence, and can express two proteins in the same cell, but does not affect the two inserted proteins, so that the successfully transfected recombinant virus can be observed better, and the interaction between the proteins can be analyzed in insect cells conveniently.
The technical scheme of the invention is as follows:
the fluorescence labeling baculovirus expression vector for co-immunoprecipitation is a recombinant pFastBac-Dual vector inserted with a promoter SV40-IE2 shown in SEQ ID No.1, an eGFP sequence shown in SEQ ID No.2, an interaction protein A sequence fused with an HA tag and an interaction protein B sequence fused with a Flag tag, wherein the eGFP is connected with the inserted promoter SV40-IE2, the interaction protein A sequence fused with the HA tag is connected with the promoter p10 of the self-contained promoter pFastBac-Dual vector, the interaction protein B sequence fused with the Flag tag is connected with the self-contained promoter polh of the pFastBac-Dual vector, and the sequence of the pFastBac-Dual vector is shown in SEQ ID No. 3.
The interaction protein A and the interaction protein B are two different proteins of interaction to be analyzed, in a specific embodiment, the interaction protein A is RSV Pc4 protein, the nucleotide sequence of the protein is shown as SEQ ID No.4, the interaction protein B is Laodelphax striatellus action protein, and the nucleotide sequence of the protein is shown as SEQ ID No. 5.
The invention relates to a construction method of a fluorescence labeling baculovirus expression vector, which comprises the following steps:
inserting the promoter SV40-IE2 shown in SEQ ID No.1 and the eGFP sequence shown in SEQ ID No.2 into the pFastBac-Dual vector shown in SEQ ID No.3, constructing a green fluorescent protein expression vector pBacDual-IE2-eGFP under the initiation of the IE2 promoter, transforming the pBacDual-IE2-eGFP plasmid into DH10Bac cells, obtaining DH10Bac cells containing recombinant Bacmid-pBacDual-IE2-eGFP through blue-white spot screening and PCR identification, respectively inserting the fluorescent marker baculovirus expression vectors pBacDual-eGFP-A-B containing eGFP, interacting protein A and interacting protein B under the polh and p10 promoters of pBacDual-IE2-eGFP after fusion of HA tag and interacting protein B.
In the specific embodiment of the invention, the fluorescence labeling baculovirus expression vector is pBacDual-eGFP-Pc4-AT, and the specific construction method is as follows:
inserting the promoter SV40-IE2 shown in SEQ ID No.1 and the eGFP sequence shown in SEQ ID No.2 into the pFastBac-Dual vector shown in SEQ ID No.3, constructing a green fluorescent protein expression vector pBacDual-IE2-eGFP under the initiation of the IE2 promoter, transforming the pBacDual-IE2-eGFP plasmid into DH10Bac cells, obtaining DH10Bac cells containing recombinant Bacmid-pBacDual-IE2-eGFP through blue-white spot screening and PCR identification, respectively inserting RSV Pc4 shown in SEQ ID No.4 into the HA tag and the Alodelpha chinensis Actin shown in SEQ ID No.5 into the polh and p10 promoters of pBacDual-IE2-eGFP, and constructing a fluorescent marker baculovirus expression vector pBacDual-eGFP-Pc4-AT containing eGFP, interacting protein A and interacting protein B.
Compared with the prior art, the invention has the following advantages:
the invention constructs a fluorescence-marked Co-IP carrier on the basis of a pFastBac-Dual carrier, and performs an immune coprecipitation experiment. The pFastBac-Dual vector features two promoters on a single vector for simultaneous expression of two proteins in insect cells. In order to verify interaction between RSV Pc4 and Laodelphax striatellus Actin, in order to better judge the transfection condition of the generated recombinant virus, the invention constructs a fluorescence-labeled baculovirus expression vector for Co-IP based on a pFastBac-Dual vector with two promoters, and the novel promoter SV40-IE2 is inserted into the vector to independently express eGFP green fluorescence genes, so that the virus infection efficiency can be checked through fluorescence observation on the premise of not influencing the expression of the two inserted interaction proteins, and the two proteins can be expressed in the same cell, so that the successfully transfected recombinant virus can be better observed, and the interaction between the proteins can be conveniently analyzed in insect cells.
Drawings
FIG. 1 is a diagram showing the double-restriction identification electrophoresis after each target fragment was ligated into T vector. Wherein M: DNA Marker (DL-5000); 1: xbaI/PstI double cleavage T-SV40-IE2;2: pstI/HidIII double cleavage of T-eGFP.
FIG. 2 is a diagram of the cleavage map of the recombinant vector pBacDual-IE2. Wherein M: DNAMaroker (DL-10000); 1: xbaI/PstI double cleavage pBacDual-IE2.
FIG. 3 is a diagram of the cleavage map of the recombinant vector pBacDual-IE2-eGFP. Wherein M: DNAMaroker (DL-5000); 1: xbaI/HidIII double cleavage pBacDual-IE2-eGFP.
FIG. 4 is a microscopic view of recombinant virus vAc pBacDual-IE2-eGFP Transfected Sf9 cells.
FIG. 5 shows a double digestion identification electrophoresis pattern of HA-Pc4, flag-AT after ligation into T vector. Wherein M: DNAMaroker (DL-5000); 1: xhoI/SphI double-enzyme cutting T-HA-Pc4;2: bamHI/NotI double cleaves T-Flag-AT.
FIG. 6 is a diagram of the cleavage of the recombinant vector pFastBacDual-HA-Pc4-IE2-eGFP. Wherein M: DNA Marker (DL-10000); 1: xhoI/SphI double enzyme digestion pFastBacDual-HA-Pc4-IE2-eGFP.
FIG. 7 is a diagram of the cleavage of the recombinant vector pFastBacDual-HA-Pc4-Flag-AT-IE2-eGFP. Wherein M: DNA Marker (DL-10000); 1: bamHI/NotI double enzyme cuts pFastBacDual-HA-Pc4-Flag-AT-IE2-eGFP.
FIG. 8 is a microscopic view of recombinant virus vAc HA-Pc4-Flag-AT Transfected Sf9 cells.
FIG. 9 is a graph showing the interaction between Co-IP detection Pc4 and action. Wherein, HA represents the detection of intracellular proteins by immunoblotting with HA antibody; IB Flag represents detection of intracellular proteins by immunoblotting with Flag antibodies; HAIB: flag represents immunoprecipitation with HA antibody and immunoblotting detection with Flag antibody.
Detailed Description
The invention is further described below with reference to the detailed description and the accompanying drawings.
Example 1
Constructing a recombinant expression vector Bacmid-pBacDual-IE2-eGFP:
primers were designed, SV40-IE2 (SEQ ID No. 1) and eGFP (SEQ ID No. 2) were amplified by PCR, and cloned into pMD19-T vectors, respectively, to construct recombinant plasmids T-SV40-IE2 and T-eGFP. After sequencing verification, the extracted plasmids are subjected to double digestion by XbaI/PstI and PstI/HidIII respectively (shown in FIG. 1), SV40-IE2 is firstly connected to a pFastBac-Dual vector by using T4 DNA ligase to construct a recombinant plasmid pBacdual-IE2 (shown in FIG. 2), eGFP is then connected to the pBacdual-IE2 vector, and after double digestion verification, the recombinant vector pBacdual-IE2-eGFP is successfully constructed (shown in FIG. 3). After double enzyme digestion verification, extracting plasmids to transform DH10Bac competent strains, and successfully obtaining a recombinant baculovirus expression vector Bacmid-pBacDual-IE2-eGFP through blue and white spot screening and PCR verification.
The designed primers are as follows:
SV40-IE2-F: as shown in SEQ ID No.6,
SV40-IE2-R: as shown in SEQ ID No.7,
eGFP-F: as shown in SEQ ID No.8,
eGFP-R: SEQ ID No. 9.
SV40-IE2-F, SV40-IE2-R is an upstream and downstream amplification primer of the SV40-IE2 gene, and the introduced enzyme digestion sites are XbaI and PstI respectively; eGFP-F, eGFP-R is respectively an upstream and a downstream amplification primer of eGFP gene, and the introduced enzyme cutting sites are respectively PstI and HidIII. The SV40 pA and OpIE2 gene fragments were amplified using the commercial plasmid pIZ/V5-His as template and the primers SV40-IE2-F, SV-IE 2-R. The commercial plasmid pEGFP-N1 is used as a template, and the primer eGFPPH-F, eGFPPH-R is used for amplifying the eGFP gene fragment.
Example 2
Preparation of recombinant Virus vAc pBacDual-IE2-eGFP ：
The DH10Bac competent strain is transformed by the recombinant vector pBacDual-IE2-eGFP, and is successfully verified by blue and white spot screening and PCR to obtain the recombinant baculovirus expression vector Bacmid-pBacDThe ul-IE 2-eGFP. The recombinant baculovirus expression vector Bacmid-pBacDual-IE2-eGFP is transfected into Sf9 insect cells by DOTAP eukaryotic cell transfection reagent, and after 5-6 days of transfection, fluorescence is observed under an inverted fluorescence microscope, and the result is shown in FIG. 4, a large amount of green fluorescent protein expression is observed, which indicates that the recombinant virus vAc pBacDual-IE2-eGFP The construction was successful.
Example 3
Construction of recombinant pBacDual-HA-Pc4-Flag-AT-IE2-eGFP vector:
PCR amplification of the Pc4 and action gene fragments was performed using primers HA-Pc4-F (SEQ ID No. 10)/HA-Pc 4-R (SEQ ID No. 11), flag-AT-F (SEQ ID No. 12)/Flag-AT-R (SEQ ID No. 13). The recombinant plasmids T-HA-Pc4 and T-Flag-AT are respectively cloned into a pMD19-T vector, and after identification is correct, pc4 and action genes are respectively cut off from the pMD19-T vector by using XhoI/SphI and BamHI/NotI (as shown in figure 5), and the same digested pBacDual-IE2-eGFP vector is connected (as shown in figure 6). And (3) performing enzyme digestion identification (shown in figure 7), and successfully constructing a recombinant pBacDual-HA-Pc4-Flag-AT-IE2-eGFP vector. The constructed pBacDual-HA-Pc4-Flag-Actin-IE2-eGFP converts DH10Bac competent cells. And extracting plasmid DNA from the white single colony for PCR identification, and extracting recombinant Bacmid DNA by using an OMEGABAC/PAC DNA purification kit after the identification is correct to obtain Bacmid-HA-Pc4-Flag-AT.
Example 4
Preparation of recombinant Virus vAc HA-Pc4-Flag-AT ：
The DH10Bac competent cells are transformed by pBacDual-HA-Pc4-Flag-Actin-IE2-eGFP, and the recombinant baculovirus expression vector Bacmid-pBacDual-HA-Pc4-Flag-Actin-IE2-eGFP is successfully obtained through blue and white spot screening and PCR verification. The recombinant baculovirus expression vector Bacmid-pBacDual-HA-Pc4-Flag-Actin-IE2-eGFP was used to transfect Sf9 insect cells with DOTAP eukaryotic cell transfection reagent, after 5-6 days of transfection, the fluorescent conditions were observed under an inverted fluorescent microscope (as in FIG. 8), and the expression of a large amount of green fluorescent protein was observed, indicating recombinant virus vAc HA-Pc4-Flag-AT The construction was successful.
Example 5
Co-immunoprecipitation (Co-IP):
recombinant virus vAc HA-Pc4-Flag-AT And (3) infecting Sf9 cells, and expressing the protein to be tested in the cells. The RSV Pc4 protein was immunoprecipitated by HA tag antibody by lysing the cells, and the Laodelphax striatellus Actin was detected by Flag tag antibody to determine whether the RSV Pc4 protein and AT protein were expressed successfully (see FIG. 9). In FIG. 9, three columns from left to right are pBacDual-HA-Pc4-Flag-AT-IE2-eGFP, pBacDual-HA-Pc4-IE2-eGFP and pBacDual-Flag-AT-IE2-eGFP recombinant vectors, respectively. HA and IB Flag results show that the labeled RSV Pc4 protein and AT protein are successfully expressed; HA IB: flag is the HA-tagged RSV Pc4 protein precipitated by the HA-tagged antibody, then the Flag-tagged AT protein is detected by the Flag-tagged antibody, and the experimental result shows that the RSV Pc4 HAs an interaction relation with actin.
In summary, the recombinant vector constructed by the invention not only contains the eGFP gene which is expressed independently, but also can express the A protein and the B protein simultaneously, and the two proteins have no mutual influence. The virus infection efficiency can be checked through fluorescent observation, and two proteins can be expressed in the same cell, so that the interaction between Pc4 and Laodelphax striatellus action is successfully verified.

Claims (4)

1. A fluorescence-labeled baculovirus expression vector for co-immunoprecipitation, characterized in that it is a recombinant pFastBac-Dual vector having inserted therein a promoter SV40-IE2 shown in SEQ ID No.1, an eGFP sequence shown in SEQ ID No.2, an interacting protein A sequence fused with an HA tag and an interacting protein B sequence fused with a Flag tag, wherein the eGFP is ligated to the inserted promoter SV40-IE2 followed by ligation of the interacting protein A sequence fused with an HA tag to the promoter self-contained in the pFastBac-Dual vectorp10After that, the Flag-tag-fused interacting protein B sequence was ligated to the promoter self-contained in the pFastBac-Dual vectorpolh The sequence of the pFastBac-Dual vector is shown in SEQ ID No. 3.
2. The fluorescence-labeled baculovirus expression vector of claim 1, wherein the interacting protein A is RSV Pc4 protein, the nucleotide sequence of which is shown as SEQ ID No.4, and the interacting protein B is Laodelphax striatellus Actin protein, the nucleotide sequence of which is shown as SEQ ID No. 5.
3. The method for constructing a fluorescence-labeled baculovirus expression vector as defined in claim 1, which is characterized by comprising the following steps:
inserting the promoter SV40-IE2 shown in SEQ ID No.1 and the eGFP sequence shown in SEQ ID No.2 into the pFastBac-Dual vector shown in SEQ ID No.3, constructing a green fluorescent protein expression vector pBacDual-IE2-eGFP under the initiation of the IE2 promoter, transforming the pBacDual-IE2-eGFP plasmid into DH10Bac cells, obtaining DH10Bac cells containing recombinant Bacmid-pBacDual-IE2-eGFP through blue-white spot screening and PCR identification, respectively inserting pBacDual-IE2-eGFP after fusing HA tag and Flag tag with interactive protein ApolhAnd a p10 promoter, a fluorescence labeling baculovirus expression vector pBacDual-eGFP-A-B containing eGFP, an interaction protein A and an interaction protein B is constructed.
4. The method for constructing a fluorescence-labeled baculovirus expression vector as defined in claim 2, which is characterized by comprising the following steps:
inserting the promoter SV40-IE2 shown in SEQ ID No.1 and the eGFP sequence shown in SEQ ID No.2 into the pFastBac-Dual vector shown in SEQ ID No.3, constructing a green fluorescent protein expression vector pBacDual-IE2-eGFP under the initiation of the IE2 promoter, transforming the pBacDual-IE2-eGFP plasmid into DH10Bac cells, obtaining DH10Bac cells containing recombinant Bacmid-pBacDual-IE2-eGFP through blue-white spot screening and PCR identification, respectively inserting RSV Pc4 shown in SEQ ID No.4 into HA tag and Laodelphax striatus Actin shown in SEQ ID No.5 into pBacDual-IE2-eGFP after fusion of Flag tagpolhAnd under the p10 promoter, constructing a fluorescence labeling baculovirus expression vector pBacDual-eGFP-Pc4-AT containing eGFP, interaction protein A and interaction protein B.
CN202310522262.3A 2023-05-10 2023-05-10 Fluorescence labeling baculovirus expression vector for co-immunoprecipitation and construction method thereof Pending CN116622778A (en)

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