CN116656706A - Recombinant plasmid of acid-sensitive fusion protein and construction and application thereof - Google Patents
Recombinant plasmid of acid-sensitive fusion protein and construction and application thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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Abstract
The invention provides an acid-sensitive fusion protein recombinant plasmid, a construction method and application thereof, and relates to the technical field of biological medicine. The invention utilizes PCR amplification technology, overlap extension PCR, transformation and connection technology to fuse pHLIP with C1C2 structural domain of exosome membrane protein Lactadherein to obtain acid-sensitive fusion peptide gene sequence pHLIP-C1C2-1 and acid-sensitive fusion peptide gene sequence pHLIP-C1C2-2 added with glycosylation motif (GNSTM) site with protective effect; recombinant plasmids pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro (respectively abbreviated as CT1 and CT 2) are constructed by using the two acid-sensitive fusion proteins, and the plasmids can be used for cell transfection and lay a foundation for obtaining modified receptor cell tracing. Meanwhile, the invention also obtains HEK293T monoclonal cell strain HEK293TC1/C2 capable of stably expressing the acid-sensitive peptide fusion protein, and creates conditions for preparation of targeted exosomes and targeting of exosomes.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to an acid-sensitive fusion protein recombinant plasmid and construction and application thereof.
Background
Malignant tumor is one of important factors threatening human health, is not easy to diagnose in early stage and has low cure rate, and effective clinical diagnosis and treatment are important for improving survival rate of cancer patients. The targeted tumor specific biomarker is beneficial to early and accurate diagnosis of tumors and effective delivery of drugs, and has small toxic and side effects on normal cells. However, targeted therapies have certain limitations in clinical applications due to low genetic, phenotypic heterogeneity and receptor specificity of tumor cells. Several studies have found that different types of monoclonal antibodies, ligand proteins, localized peptides or nucleic acids can be used as tumor-specific biomarkers. A "universal" biomarker that can be adapted for use with a variety of tumors has not yet been identified. The tumor microenvironment is in an acidic state due to faster metabolism of tumor cells, increased glycolysis, strong carbonic anhydrase activity and the like, and is a common feature of almost all tumors. By utilizing the characteristic, the tumor and normal tissue cells can be distinguished, and the cell surface acidity can be used as a general biomarker characteristic of the tumor.
The low pH intercalating peptide (pH low insertion peptide, pHLIP) is a water-soluble molecule that interacts with the lipid membrane in a pH dependent manner, derived from the bacterial rhodopsin. pHLIP has three forms, and under normal physiological pH (pH 7.4), pHLIP maintains balance between water-soluble state (I) and membrane surface adsorption state (II); when the pH value is lowered, aspartic acid or acidic amino acid residues in pHLIP are protonated, so that the hydrophobicity of pHLIP is enhanced, and pHLIP spontaneously folds into an alpha-helix transmembrane structure (III). The carboxyl (C) end of the peptide can be quickly inserted into cytoplasm of target cells, and the amino (N) end of the peptide locates pHLIP on plasma membrane lipid bilayer outside cell membrane. Thus, pHLIP has the ability to target acidic tissues and dual delivery, and various exogenous molecules can be delivered to the cell surface and within the cell by coupling to the N-or C-terminus of pHLIP. While many molecules in cells delivered by pHLIP can be roughly divided into: pharmaceutical agents, cell permeable molecules and molecules that are polar cell impermeable.
pHLIP has been demonstrated to target tumor acidic microenvironment, and its targeting delivery system has strong specificity advantage compared with traditional targeting nanocarriers and transmembrane peptides, and has the following advantages in application: pHLIP is insensitive to the heterogeneous expression of receptors or antigens in tumor cells; pHLIP has specific markers that actively target the acidic tumor microenvironment; pHLIP not only can accurately target primary tumor focus and metastasis focus, but also can develop more application aspects by utilizing the characteristic that pHLIP is inserted into the surface of tumor cell membrane. The pHLIP technology can deliver different types of functional molecules into cells for treatment and diagnosis, has better application prospect in clinic, but still has the defect of needing to be optimized at present, wherein the improvement of the synthesis efficiency of pHLIP and exogenous molecules can obviously improve the clinical transformation capability of pHLIP and exogenous molecules. Although pHLIP has been demonstrated to have specific targeting effect on acidic tumor microenvironment, there is still a need to further improve the targeting efficiency, and in clinical application, it is required to screen patients for inflammatory diseases, thereby improving the high specificity of pHLIP.
The exosomes are membrane vesicles with the diameter of about 10-100nm released outside cells after fusion of intracellular multivesicular bodies and cell membranes, and comprise a plurality of intracellular substances such as RNA, protein, DNA fragments and the like. The exosomes can shuttle among cells, are favorable for the exchange of intercellular substances and information, and can be loaded with chemotherapeutic drugs and siRNA for targeted treatment. Exosomes have natural advantages as drug carriers: (1) The nanometer size is provided, so that the capture of the reticuloendothelial system can be easily escaped in the body; (2) The phospholipid bilayer structure has a phospholipid bilayer structure, is similar to the cell membrane in composition, has stronger affinity to the cell membrane, and is convenient for entering cells; (3) Belongs to endogenous vesicles, can not cause immune response when entering the body as a carrier, and can not be identified as non-self substances by the immune system in the body to be phagocytized; (4) It is derived from cells, contains a plurality of transmembrane proteins on the surface, can be modified by gene, and adding ligand short peptide capable of targeting target cells at the tail end of a proper protein, so as to realize in vivo targeted treatment.
Disclosure of Invention
The invention aims to provide an acid-sensitive fusion protein recombinant plasmid, a construction method and application thereof, and the invention successfully constructs two recombinant plasmids of pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro, and obtains a monoclonal cell strain HEK293TCT1/CT2 for stably expressing the acid-sensitive peptide fusion protein HEK293T, thereby providing a foundation for preparation of targeted exosomes and targeting of exosomes.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
in one aspect, the invention provides an acid-sensitive fusion protein, which is formed by fusing pHILP and a C1C2 domain of exosome membrane protein Lactadherein, wherein the gene sequence of the fusion protein is shown as SEQ ID NO.1, and the fusion protein is named pHILP-C1C2-1.
Further, the fusion protein is formed by connecting a Lactadherein signal peptide-WT-pHLIP-Flag and C1C2-HA into a pHILP-C1C2-1 fusion protein gene sequence by using an overlap extension PCR technology, and adding XbaI and NotI double enzyme cutting sites at two ends of the fusion protein; wherein the sequence of the Lactadherein signal peptide-WT-pHLIP-Flag is shown as SEQ ID NO. 2; the sequence of the C1C2-HA is shown as SEQ ID NO. 3.
Furthermore, the fusion protein also comprises a glycosylation motif with a protective effect, the gene sequence of the fusion protein is shown as SEQ ID NO.4, and the fusion protein is named pHILP-C1C2-2.
In another aspect, the present invention provides an acid-sensitive fusion protein recombinant plasmid, which is prepared according to the following steps:
(1) Amplification of the C1C2-HA Gene sequence of interest
Amplifying the C1C2 structural domain gene sequence of the Lactadherein gene by utilizing a PCR amplification technology, wherein the sequence is shown as SEQ ID NO.9; and adding an HA tag sequence at the sequence end by utilizing a PCR technology, wherein the obtained C1C2-HA sequence is shown as SEQ ID NO. 3;
(2) pHLIP-Flag gene sequence amplification
Amplifying a Lactadherein signal peptide-WT-pHLIP-Flag tag sequence by using a PCR amplification technology, wherein the obtained Lactadherein signal peptide-WT-pHLIP-Flag sequence is shown as SEQ ID NO. 2;
(3) Construction of pHLIP-C1C2-1 and pHLIP-C1C2-2 fusion protein genes
Connecting a Lactadherein signal peptide-WT-pHLIP-Flag acid-sensitive peptide sequence obtained by PCR amplification and a C1C2-HA gene sequence into fusion proteins pHLIP-C1C2-1 and pHLIP-C1C2-2 by using an overlap extension PCR technology;
(4) Construction of recombinant plasmid of acid-sensitive fusion peptide
Cloning the acid-sensitive fusion peptide gene to a multiple cloning site on a pCDH-CMV-MCS-EF1-GFP-Puro lentiviral vector to obtain a recombinant plasmid pCDH-pHLIP-C1C2-1-Puro and a recombinant plasmid pCDH-PHLIP-C1C2-2-Puro, which are respectively abbreviated as CT1 and CT2;
(5) Identification of recombinant plasmid of acid-sensitive fusion peptide
The constructed recombinant plasmids CT1 and CT2 are detected and identified by agarose gel electrophoresis and are identified by gene sequencing, and the recombinant plasmids of the acid-sensitive fusion peptide are successfully constructed by identification and are named pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro respectively.
Further, recombinant plasmids CT1 and CT2 are packaged into lentivirus to transfect HEK293T cells, and lentivirus liquid is collected and concentrated to infect HEK293T cells; the combination of puromycin and fold dilution is then used to screen monoclonal cells to obtain HEK293T monoclonal cell line HEK293TCT1/CT2 which stably expresses the acid sensitive peptide fusion protein.
Advantageous effects
The invention provides an acid-sensitive fusion protein recombinant plasmid, a construction method and application thereof. The invention utilizes PCR amplification technology, overlap extension PCR, transformation and connection technology to fuse pHLIP with C1C2 of exosome membrane protein Lactadherein to obtain acid-sensitive fusion peptide gene sequence pHLIP-C1C2-1 and acid-sensitive fusion peptide gene sequence pHLIP-C1C2-2 added with glycosylation motif (GNSTM) site with protection function; recombinant plasmids pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro (respectively abbreviated as CT1 and CT 2) are constructed by using the two acid-sensitive fusion peptide proteins, and the plasmids can be used for cell transfection and co-expression with lentiviral packaging plasmids to pack carrying genes into lentiviral particles, so that foundation is laid for stably inserting exogenous genes into cell genome and obtaining the trace of a modified receptor cell. Meanwhile, the invention also obtains HEK293T monoclonal cell strain HEK293TCT1/LCT2 capable of stably expressing the acid-sensitive peptide fusion protein, and creates conditions for preparation of targeted exosomes and targeting of exosomes.
Drawings
FIG. 1 shows that the C1C2 target gene is amplified by a PCR amplification experiment; wherein M is DL5000; C1C2-HA;2:C1C2 genes of interest.
FIG. 2 shows that the target gene of Lactadherein signal peptide-WT-pHLIP-Flag is amplified by PCR amplification experiment, wherein M is DL5000;1, 2:Lactadherein signal peptide-WT-pHLIP-Flag target gene.
FIG. 3 shows the amplification of the acid-sensitive fusion peptide pHLIP-C1C2-1 and pHLIP-C1C2-2 in a PCR overlap extension PCR experiment; DL5000;1 pHLIP-C1C2-1 target gene; 2 pHLIP-C1C2-2 target gene.
FIG. 4 shows the patterns of recombinant plasmids pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro.
FIG. 5 shows agarose gel electrophoresis of recombinant plasmids pCDH-pHLIP-C1C2-1-Puro (left) and pCDH-pHLIP-C1C2-2-Puro (right).
FIG. 6 shows the sequencing results of pCDH-pHLIP-C1C2-1-Puro (A) and pCDH-pHLIP-C1C2-2-Puro (B).
FIG. 7 is a graphical representation of the information of the packaging plasmids psPAX2 and pMD2. G.
FIG. 8 is a fluorescent image of recombinant plasmids pCDH-pHLIP-C1C2-1-Puro (A) and pCDH-pHLIP-C1C2-2-Puro (B) transfected HEK293T cells.
FIG. 9 shows the results of stable expression of pHLIP-C1C2-1, pHLIP-C1C2-2 monoclonal cells; FIG. 9A is a fluorescent, white light state of HEK293T cells of the acid sensitive fusion protein pHLIP-C1C2-1; FIG. 9B shows the cell state of HEK293T cells, fluorescent, white light, of the acid sensitive fusion protein pHLIP-C1C 2-2.
FIG. 10 shows the results of infection of HEK293T cells with the lentiviral filtrate concentrate; FIG. 10A shows the cell state of fluorescent, white light of HEK293T cells infected with the recombinant plasmid pCDH-pHLIP-C1C2-1-Puro lentiviral fluid; FIG. 10B shows the fluorescent, white light cell status of HEK293T cells infected with the recombinant plasmid pCDH-pHLIP-C1C2-2-Puro lentiviral fluid.
FIG. 11 shows the differential expression levels of C1C2 and pHLIP in stably transfected cell lines.
FIG. 12 shows the expression of the tag protein Flag and HA in stably transfected cell lines.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Test 1
Construction of recombinant plasmid of pHILP-C1C2-1/12 fusion protein
1. Polymerase Chain Reaction (PCR) amplification to obtain C1C2 target gene
Obtaining a gene fragment of a C1C2 domain of the Lactadherein protein by utilizing a PCR amplification technology, and designing a C1C2 sequence Primer by using Primer premier5.0 software; the primer sequences are shown below:
the PCR amplification reaction system and conditions of the C1C2F 5'-AAATGTGTCGAGCCACTGG-3'SEQ ID NO.5C1C2R:5'-ACAGCCCAGCAGCTCCAG-3' SEQ ID NO.6 are shown as follows,
after completion of the reaction, the reaction mixture was subjected to 1% agarose gel electrophoresis (FIG. 1), and the gel was recovered.
2. PCR amplification to obtain C1C2-HA gene
The HA tag was added to the tail end of the C1C2 sequence by PCR, and the primer sequence was as follows:
C1C2F：5′-AAATGTGTCGAGCCACTGG-3′SEQ ID NO.7C1C2-HA：
the 5'-GCGGCCGCTTAAGCGTAATCTGGCACATCGTATGGGTATCCGCTTCCACAGCC CAGCAGCTCCAG-3' SEQ ID NO.8PCR amplification reaction system and the conditions are shown below,
after completion of the reaction, the reaction mixture was subjected to 1% agarose gel electrophoresis, and the gel was recovered.
The result of 1% agarose gel electrophoresis detection shows that the band is positioned near 1000bp of the DNA marker, the position of the band is the same as the 1004bp of the theoretical band of the C1C2-HA target gene, the C1C2-HA target gene is successful, the result is shown in figure 1, lane 1 is C1C2-HA, and lane 3 is C1C 2. The sequence of the C1C2 target gene is shown as SEQ ID NO.9; the sequence of the C1C2-HA gene is shown as SEQ ID NO. 3.
3. PCR amplification of the target gene of Lactadherein Signal peptide-WT-pHLIP-Flag
The target gene fragment of the Lactadherein signal peptide-WT-pHLIP-Flag is obtained by utilizing a PCR amplification technology, and the primer sequences are as follows:
C-pHLIP F：5′-ATGCCGCGCCCCCGCCTGC-3′SEQ ID NO.10C-pHLIP R：
5′-TTTATCGTCATCGTCCTTATAATCAGCACATTCCTGGTTCGGGA-3′
the PCR amplification reaction system and conditions are shown below,
after completion of the reaction, the reaction mixture was subjected to 1% agarose gel electrophoresis, and the gel was recovered.
The result of 1% agarose gel electrophoresis detection shows that the band is positioned between 240bp of DNAmarker, and the position of the band is identical to that of a theoretical value 237bp, and the target gene of the Lactadherein signal peptide-WT-pHLIP-Flag is successful, and the result is shown in figure 2.
4. Construction of pHLIP-C1C2-1 fusion proteins
The synthesized pHLIP acid-sensitive peptide sequence and the C1C2 gene sequence are connected into fusion protein pHLIP-C1C2-1 by using an overlap extension PCR technology, and the fusion protein pHLIP-C1C2-1 gene sequence is shown as SEQ ID NO. 1.
The specific operation is as follows:
(1) The recovered product of the Lactadherein signal peptide-WT-pHLIP-Flag gel in the step 3 is used as a template, an XbaI cleavage site is added in the front section of the pHLIP sequence, and the PCR amplification reaction system and conditions are as follows.
(2) Taking the C1C2-HA gel recovery product in the step 2 as a template, adding NotI enzyme cutting sites at the rear section of the sequence, and carrying out PCR amplification reaction under the following conditions.
(3) And (3) performing overlap extension PCR reaction by taking the products of the steps (1) and (2) as templates, wherein a PCR amplification reaction system and conditions are shown as follows.
(4) After completion of the reaction, the reaction mixture was subjected to 1% agarose gel electrophoresis, and the gel was recovered.
(5) The 1% agarose gel electrophoresis detection shows that the band is about DNAmarker 1200, and the band is consistent with the theoretical value, and the pHLIP-C1C2-1 fusion protein is successfully constructed, and the result is shown in FIG. 3.
5. Construction of pHLIP-C1C2-2 fusion proteins
The PCR overlapping extension technology is utilized to take the fusion protein pHLIP-C2C2-1 sequence as a template, a glycosylation protection site (the glycosylation protection site has the sequence of GGAAATTCAACAATG, SEQ ID NO. 12) is added in front of the pHLIP sequence, and then the fusion protein pHLIP-C1C2-2 is connected with the C1C2 gene sequence to form the fusion protein pHLIP-C1C2-2, and the gene sequence of the fusion protein pHLIP-C1C2-2 is shown as SEQ ID NO. 4.
The PCR amplification reaction system and conditions are shown below.
The 1% agarose gel electrophoresis detection shows that the band is about 1200bp of the DNA marker, and the band accords with the theoretical value, and the pHLIP-C1C2-2 fusion protein is successfully constructed, and the result is shown in figure 3.
6. Construction of recombinant plasmid of acid-sensitive fusion peptide
(1) Double enzyme digestion is carried out on pHLIP-C1C2-1/2 fusion protein by using QuickCut restriction enzyme, after enzyme digestion is finished, agarose gel electrophoresis is carried out on the reaction product, whether the reaction product is consistent with a target gene fragment or not is identified, if so, gel recovery is carried out to purify DNA, and enzyme digestion fragment connection is carried out.
(2) The fusion gene of interest (pHLIP-C1C 2-1/2) recovered by agarose gel electrophoresis was subjected to ligation experiments with a plasmid vector (pCDH-GFP+Puro) by using T4 DNA ligase to construct recombinant plasmids pCDH-pHLIP-C1C2-1-GFP+Puro1 and pCDH-pHLIP-C1C2-2-GFP+Puro2 (abbreviated as CT1 and CT2, respectively), the plasmid maps were shown in FIG. 4.
The agarose gel electrophoresis identifies that the recombinant plasmid bands of CT1 and CT2 are positioned between DNAmarker 9000bp and 10000bp, and are consistent with the theoretical value, and the result is shown in figure 5; the constructed recombinant plasmids CT1 and CT2 are identified by gene sequencing, the gene sequences are consistent with expectations, and the results are shown in figure 6; the above results demonstrate that recombinant plasmids CT1 and CT2 were successfully constructed.
7. Conclusion(s)
(1) The invention utilizes PCR amplification technology, overlap extension PCR, transformation and connection technology to fuse pHLIP with C1C2 structural domain of exosome membrane protein Lactadherein to obtain acid-sensitive fusion peptide gene sequence pHLIP-C1C2-1;
(2) In order to prevent the pHLIP peptide from being degraded, a glycosylation motif (GNSTM) site with a protective effect is added on the basis of the fusion protein pHLIP-C1C2-1 sequence to obtain an acid-sensitive fusion peptide gene sequence pHLIP-C1C2-2;
(3) The acid-sensitive fusion protein genes are respectively introduced into pCDH-CMV-MCS-EF1-GFP-Puro lentiviral vectors to construct recombinant plasmids pCDH-PHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro (respectively abbreviated as CT1 and CT 2), and are verified by double enzyme digestion experiments and gene sequencing ratio, the results are completely consistent with theoretical values and gene sequences, and the recombinant plasmid of the acid-sensitive fusion peptide has been successfully constructed, can be used for cell transfection, and lays a foundation for obtaining the trace of the modified receptor cells.
Test 2
Establishment and identification of pHLIP-C1C2-1/2 fusion protein stable transgenic cell line
1. HEK293T cells transfected by CT1/CT2 recombinant plasmid
The lentivirus is packaged by using a liposome TM2000Reagent transfection method, plasmids used in a lentivirus system comprise acid-sensitive fusion peptide recombinant plasmids CT1 and CT2 containing target genes, and packaging plasmids psPAX2 and pMD2.G (shown in figure 7), wherein the psPAX2 can express lentivirus shells, the pMD2.G is a membrane protein plasmid of the lentivirus, the two plasmids together form a packaging system of two plasmids, and the plasmids are transferred into HEK293T cells together to package the lentivirus. Fluorescence was visible under an inverted fluorescence microscope after transfection, indicating that plasmid transfection works as shown in FIG. 8.
2. Infection of HEK293T cells with CT1/CT2 lentivirus
Collecting the slow virus liquid for 48h and 72 h respectively after the recombinant plasmid is transfected, concentrating the slow virus liquid, infecting HEK293T, and screening HEK293T cells expressing fluorescent protein by using puromycin. The monoclonal fluorescent cell strain is screened by a limiting dilution method, a monoclonal HEK293T cell strain which stably expresses pHLIP-C1C2-1/2 fusion protein is screened, fluorescence is visible under an inverted fluorescence microscope after infection, and the result is shown in figure 9.
3. Acquisition of C1/C2 monoclonal cells
Screening monoclonal fluorescent cell lines by using a limiting dilution method, screening monoclonal HEK293T cell lines which stably express pHLIP-C1C2-1/2 fusion protein, and carrying out visible fluorescence under an inverted fluorescent microscope to show that the infected cells are successful, wherein the fusion gene is introduced into a cell genome, and the result is shown in figure 10.
4. Verification of pHLIP and C1C2 expression in monoclonal cells
The total RNA in each cell was extracted by Trizol method and cDNA was obtained by reverse transcription, and the expression levels of pHLIP and C1C2 in the monoclonal cells and human kidney epithelial cells were detected by real-time quantitative PCR technique, and the expression levels of C1C2 in the monoclonal cell lines were calculated to be increased by about 33.75-fold, 36.45-fold, 32.75-fold, 29.54-fold, 23.54-fold and 25.62-fold, respectively, as compared with the human kidney epithelial cell 293T, and the results were statistically significant (P < 0.05), as shown in FIG. 11A. The expression level of pHLIP in the monoclonal cell line was increased by about 341.23-fold, 341.06-fold, 422.17-fold, 274.93-fold, 274.10-fold and 261.51-fold, respectively, as compared with that of human kidney epithelial cell 293T, and the results were statistically significant (P < 0.05), and the results are shown in FIG. 11B. In summary, pHLIP and C1C2 expression fold in monoclonal cells were both significantly increased compared to human kidney epithelial 293T cells.
5. Flag and HA protein expression verification in monoclonal cells
And selecting a cell strain with higher acid-sensitive fusion peptide expression quantity according to the RT-qPCR verification result, detecting the expression conditions of tag proteins Flag and HA in the stable transfer cell line by using a Western Blot method, verifying that the tag proteins are expressed in both the stable transfer cell line CT1 and CT2 by comparing with a normal cell HEK293T, and the result is shown in figure 12.
6. Conclusion(s)
(1) Packaging recombinant plasmids CT1 and CT2 into lentivirus to transfect HEK293T cells, and collecting lentivirus liquid for concentration to infect HEK293T cells; the combination of puromycin and fold dilution is then used to screen monoclonal cells to obtain HEK293T monoclonal cell line HEK293TCT1/CT2 which stably expresses the acid sensitive peptide fusion protein.
(2) The expression condition of the acid-sensitive fusion protein gene in the stable transgenic cell line is detected by using RT-qPCR and Western Blot method, and compared with the normal cell HEK293T, the expression condition of the acid-sensitive fusion protein gene in the stable transgenic cell line HEK293TCT1/CT2 is carried out, thereby obtaining the HEK293T monoclonal cell strain for stably expressing the acid-sensitive fusion protein, and creating conditions for the preparation of targeted exosomes and the targeting of exosomes.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. The acid-sensitive fusion protein is characterized by comprising a low-pH insertion peptide and a C1C2 domain of exosome membrane protein Lactadherein, wherein the gene sequence of the fusion protein is shown as SEQ ID NO.1, and the fusion protein is named pHILP-C1C2-1.
2. An acid sensitive fusion protein according to claim 1, wherein the fusion protein utilizes overlap extension PCR technique to ligate the Lactadherin signal peptide-WT-pHLIP-Flag sequence and C1C2-HA into a pHILP-C1C2-1 fusion protein gene sequence, and to add XbaI and NotI double cleavage sites at both ends thereof; wherein the sequence of the Lactadherein signal peptide-WT-pHLIP-Flag is shown as SEQ ID NO. 2; the sequence of the C1C2-HA is shown as SEQ ID NO. 3.
3. An acid sensitive fusion protein according to claim 1, wherein the fusion protein further comprises a protective glycosylation motif, the gene sequence of the fusion protein is shown in SEQ ID NO.4, and the fusion protein is named pHILP-C1C2-2.
4. An acid-sensitive fusion protein recombinant plasmid, which is characterized by comprising the following steps:
(1) Amplification of the C1C2-HA Gene sequence of interest
Amplifying a C1C2 structural domain gene sequence of the Lactadherein gene by using a PCR amplification technology, and adding an HA tag sequence at the sequence end by using a PCR technology, wherein the obtained C1C2-HA sequence is shown as SEQ ID NO. 3;
(2) Amplification of the Lactadherein Signal peptide-WT-pHLIP-Flag Gene sequence
Amplifying the Lactadherein signal peptide-pHLIP-Flag by using a PCR technology, wherein the sequence of the obtained Lactadherein signal peptide-WT-pHLIP-Flag is shown as SEQ ID NO. 2;
(3) Construction of pHLIP-C1C2-1 and pHLIP-C1C2-2 fusion protein genes
Connecting amplified Lactadherein signal peptide-pHLIP-Flag and C1C2-HA gene sequences into fusion proteins pHLIP-C1C2-1 and pHLIP-C1C2-2 by using an overlap extension PCR technology;
(4) Construction of recombinant plasmid of acid-sensitive fusion peptide
Cloning the acid-sensitive fusion peptide gene to a multiple cloning site on a pCDH-CMV-MCS-EF1-GFP-Puro lentiviral vector to obtain a recombinant plasmid pCDH-pHLIP-C1C2-1-Puro and a recombinant plasmid pCDH-pHLIP-C1C2-2-Puro, which are respectively abbreviated as CT1 and CT2;
(5) Identification of recombinant plasmid of acid-sensitive fusion peptide
The constructed recombinant plasmids CT1 and CT2 are subjected to double digestion test by restriction endonucleases XbaI and NotI, DNA sequencing is carried out, and the recombinant plasmids of the acid-sensitive fusion peptide are successfully constructed through identification, and are named as pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro respectively.
5. The use of a recombinant plasmid of an acid sensitive fusion protein according to claim 4, wherein the recombinant plasmids pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro are packaged as lentiviruses for transfecting HEK293T cells and the lentivirus fluid is collected for concentration to infect HEK293T cells; the combination of puromycin and fold dilution is then used to screen monoclonal cells to obtain HEK293T monoclonal cell line HEK293TCT1/CT2 which stably expresses the acid sensitive peptide fusion protein.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322233A (en) * | 1998-11-25 | 2001-11-14 | 宝洁公司 | Process for forming cleaning composition |
| US20020198144A1 (en) * | 1994-07-29 | 2002-12-26 | Dade International, Inc. | MHC complexes and uses thereof |
| CN1452617A (en) * | 2000-09-05 | 2003-10-29 | 甘瑟尔股份有限公司 | Acid-sensitive compounds, preparation and uses thereof |
| CN103555746A (en) * | 2013-10-10 | 2014-02-05 | 长春西诺生物科技有限公司 | Recombinant porcine circovirus type 2 virus-like particle, and preparation method and application thereof |
-
2023
- 2023-04-18 CN CN202310414773.3A patent/CN116656706A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020198144A1 (en) * | 1994-07-29 | 2002-12-26 | Dade International, Inc. | MHC complexes and uses thereof |
| CN1322233A (en) * | 1998-11-25 | 2001-11-14 | 宝洁公司 | Process for forming cleaning composition |
| CN1452617A (en) * | 2000-09-05 | 2003-10-29 | 甘瑟尔股份有限公司 | Acid-sensitive compounds, preparation and uses thereof |
| CN103555746A (en) * | 2013-10-10 | 2014-02-05 | 长春西诺生物科技有限公司 | Recombinant porcine circovirus type 2 virus-like particle, and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 周永安;张景萍;: "慢性髓性白血病bcr/abl融合基因的克隆与表达", 临床医药实践, no. 28, 25 October 2009 (2009-10-25) * |
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