Double-gene recombinant complex macrophage vaccine and preparation method and application thereof

A macrophage and complex technology, which is applied in the field of vaccines prepared by the same and its preparation, and dual-gene recombination complexes, can solve the problems of many influencing factors, unfavorable target gene expression and subsequent treatment, difficult evaluation and analysis of experimental results, and the like, Achieve the effect of inhibiting inflammatory response and brain edema, good development and application prospects, and improving neurological function scores

Pending Publication Date: 2019-06-28
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
0 Cites 1 Cited by

AI-Extracted Technical Summary

Problems solved by technology

[0005] There are two main methods of gene combination therapy: one is to simultaneously transfect target cells with a variety of recombinant expression vectors carrying different genes. The construction of recombinant expression vectors is cumbersome, time-consuming, and has many influencing factors. In addition, due to th...
View more

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a double-gene recombinant complex, a vaccine prepared from the same, and a preparation method and application ofthe complex and the vaccine. The recombinant complex is composed of a polypeptide complex and a double-gene expression cassette, and the portion from the upstream to the downstream of the double-geneexpression cassette is sequentially provided with a GFAP promoter, an IRAK2 RNAi gene, a terminator, an internal ribosome entry site IRES, an SMURF1 gene and a terminator. The polypeptide complex isformed by binding cell-penetrating peptide and a positive charge polypeptide. The double-gene recombinant complex macrophage vaccine can be used for transfecting hematoma surrounding tissue of cerebral hemorrhage in vitro, the activity of macrophages can be inhibited, inflammatory reaction and brain edema of cerebral hemorrhage are effectively inhibited, the neurological score is improved, and anideal strategy is provided for treatment of cerebral hemorrhage. The preparation method is simple and low in cost and has good development and application prospects in the field of cerebral hemorrhagetreatment.

Application Domain

Antibody medical ingredientsBlood disorder +5

Technology Topic

Gene expressionCell-penetrating peptide +8

Image

  • Double-gene recombinant complex macrophage vaccine and preparation method and application thereof
  • Double-gene recombinant complex macrophage vaccine and preparation method and application thereof
  • Double-gene recombinant complex macrophage vaccine and preparation method and application thereof

Examples

  • Experimental program(4)

Example Embodiment

[0043] Example 1 Preparation of double gene recombination complex
[0044] 1.1 Cloning of IRAK2RNAi
[0045] According to the Trizol kit instructions, extract total RNA from human liver tissue, reverse transcription to obtain total cDNA, and then use the total cDNA as a template to F1: 5’-aat ctgcag acttttcgcccgcacagagcgg-3' (SEQ ID No. 3, the underlined part is the Bgl1 restriction site) and R1: 5'-tac gaattc aagcgcgtgcacgaggaggtcacg-3' (SEQ ID No.4, the underlined part is the Apo1 restriction site) is the upstream and downstream primers, PCR amplifies IRAK2 full-length RNAi, the PCR reaction system is 50μl, and the cycle conditions are: 94℃ pre-denaturation 4 minutes ; Then denaturation at 94°C for 45 seconds, annealing at 58°C for 60 seconds, extension at 72°C for 1 minute, a total of 30 cycles; finally, extension at 72°C for 5 minutes. The PCR product was identified by agarose gel electrophoresis, gel-recovery kit was cut and purified, and then ligated with pT-Easy vector. The ligation product was transformed into E. coli DH5α competent cells. Positive clones were screened with LB plates containing ampicillin and extracted. The plasmid was identified by sequencing, and the positive clone plasmid was named pT-IRAK2RNAi.
[0046] 1.2 Cloning of SMURF1 full-length cDNA
[0047] According to the Trizol kit instructions, extract total RNA from human pancreas tissue, reverse transcription to obtain total cDNA, and then use the total cDNA as a template to F2: 5’-gatc ggatcc gtagacaggtacccc-3' (SEQ ID No. 5, the underlined part is the BamHI restriction site) and R2: 5'-acgc gtcgac caaatttagaataggttcc-3'(SEQ ID No.6, the underlined part is the NotI restriction site) is the upstream and downstream primers, PCR amplifies the full-length cDNA of SMURF1, the PCR reaction system is 50μl, and the cycle condition parameters are: 94℃ pre-denaturation 4 minutes ; Then denaturation at 94°C for 45 seconds, annealing at 58°C for 60 seconds, extension at 72°C for 1 minute, a total of 30 cycles; finally, extension at 72°C for 5 minutes. The PCR product was identified by agarose gel electrophoresis, gel-recovery kit was cut and purified, and then ligated with pT-Easy vector. The ligation product was transformed into E. coli DH5α competent cells. Positive clones were screened with LB plates containing ampicillin and extracted. The plasmid was identified by sequencing, and the positive clone plasmid was named pT-SMURF1.
[0048] The results of agarose gel electrophoresis showed that the PCR product showed a single specific band at about 1000 bp ( figure 1 ), consistent with the expected results; the sequencing results showed that the inserted gene sequence of the positive clone plasmid was consistent with the SMURF1 full-length cDNA sequence of GenBank (nm_001199847.1) (lane 1 is the DNA molecular weight standard, lane 2 is the PCR product).
[0049] 1.3 Construction of the recombinant vector pCMV-IRAK2RNAi-SMURF1
[0050] According to the restriction sites designed at both ends of the IRAK2 and SMURF1 full-length cDNA, first insert the IRAK2 full-length cDNA into the IRES upstream of the pCMV vector, and then insert the SMURF1 full-length cDNA into the IRES downstream of the pCMV vector. The specific method is as follows: firstly pT-IRAK2 vector is digested with Bgl1 and Apo1, and the product of the digestion is digested and purified by the gel recovery kit, and then connected to the pCMV vector that is also digested with Bgl1 and Apo1. Transform E. coli DH5α competent cells, screen positive clones with ampicillin-containing LB plates, extract plasmids, double-enzyme digestion with Bgl1 and Apo1, and name the positive clone plasmid pCMV-IRAK2; then use BamHI and NotI for pT-SMURF1 vector After double enzyme digestion, the product of double enzyme digestion was recovered and purified by gel recovery kit, and then ligated with the pCMV-IRAK2 vector that was also double digested with BamHI and NotI. The ligation product was transformed into E. coli DH5α competent cells with ampicillin The positive clones were screened on the LB plate, the plasmids were extracted, and identified by BamHI and NotI double enzyme digestion. The positive clone plasmid was named pCMV-IRAK2RNAi-IRES-SMURF1.
[0051] 1.4 Construction of recombinant vector pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1
[0052] Use pCMV-IRAK2RNAi-IRES-SMURF1 vector as template and F3: 5’-acgc gtcgac cacatcgcggccggagcacttt-3' (SEQ ID No. 7, the underlined part is NotI restriction site) and R3: 5'-agaat gcggccgc agaacaaatatttggttcc-3' (SEQ ID No.8, the underlined part is the NotI restriction site) is the upstream and downstream primers, PCR amplifies the IRAK2RNAi-IRES-SMURF1 fragment and replaces the restriction sites at both ends of the fragment with NotI restriction sites Point and NotI restriction site, PCR reaction system and cycle conditions parameters are the same as before. The PCR product was identified by agarose gel electrophoresis, gel-recovery kit was cut and purified, and then ligated with pT-Easy vector. The ligation product was transformed into E. coli DH5α competent cells. Positive clones were screened with LB plates containing ampicillin and extracted. The plasmid was identified by sequencing, and the positive clone plasmid was named pT-IRAK2RNAi-IRES-SMURF1.
[0053] The pT-IRAK2RNAi-IRES-SMURF1 vector was double digested with NotI and NotI, and the double digested product was recovered and purified by gel recovery kit, and then connected to the pShuttle-GFAP vector that was also digested with NotI and NotI. The product was transformed into E. coli DH5α competent cells, positive clones were screened on LB plates containing ampicillin, the plasmids were extracted, and not I and Not I were digested with enzymes. The positive clone plasmid was named pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1.
[0054] 1.5 Construction of the recombinant complex vector pAd-IRAK2RNAi-SMURF1
[0055] The pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1 vector was digested and linearized with PmeⅠ, and then electrotransformed into E. coli BJ5183 competent cells simultaneously with the pAdEasy-1 vector, and homologous recombination in the bacteria was carried out. The LB plate containing kanamycin was used Cultivate, pick smaller colonies, culture overnight with LB liquid medium containing kanamycin, extract plasmid, PacI restriction enzyme digestion, and name the positive plasmid pAd-IRAK2RNAi-SMURF1.
[0056] 1.6 Packaging of the recombinant complex vector pAd-IRAK2RNAi-SMURF1
[0057] Inoculate human embryonic kidney 293T cells into a T-25 culture flask at a cell density of 40%-60%. When the cells grow to 30%-50% confluence, discard the culture medium, and use Lipofectamine 2000 reagent to pAd-IRAK2RNAi-SMURF1 The vector was transfected into 293T cells, the cytopathological changes were observed under a microscope, and the cells were collected by centrifugation when the obvious cytopathic effect appeared. After the cell pellet was resuspended in PBS, the cells were lysed by repeated freezing and thawing four times, and the cell debris was removed by centrifugation to collect virus-containing The supernatant is the Ad-IRAK2RNAi-SMURF1 virus stock; the virus stock is reinfected with 293T cells at a ratio of 1:10, the supernatant containing the virus is collected, and the above operation is repeated 3 times to obtain the fourth-generation recombinant complex Ad -IRAK2RNAi-SMURF1.
[0058] 1.7 Preparation of double gene recombination complex
[0059] Under stirring conditions, add dropwise the aqueous solution of the peptide complex to the 0.9% Nacl solution in which Ad-IRAK2RNAi-SMURF1 is dissolved. After the addition, continue to stir for 10 minutes and then stand for another 10 minutes to obtain.

Example Embodiment

[0060] Example 2 Preparation of Macrophage Vaccine Transfected by Double Gene Recombination Complex
[0061] 2.1. Sorting of macrophages
[0062] Take C57BL/6 normal mice, remove their necks and put them to death, and routinely disinfect them with 75% alcohol. Inject 5 mL of serum-free RPMI 1640 medium into the abdominal cavity of mice, rub the abdomen repeatedly for 5 minutes, open the abdominal cavity after standing for 3 minutes, extract the abdominal cavity liquid and centrifuge, check the cell density, and plant it in a 6-well plate for 3 hours to remove non-adherent cells , Re-add culture medium and cytokines: IFN-γ10ng/ml, LPS 100ng/ml; M2 type macrophages: IL-4 10ng/ml) stimulate culture for 24h.
[0063] 2.2. Transfection of the double-gene recombination complex into macrophages
[0064] Macrophages were seeded on a 6-well plate at a cell concentration of 1×106 cells/ml, and the fourth-generation double-gene recombination complex was added at a multiplicity of infection (MOI) of 100. The cells were collected 48 hours after infection, washed with PBS and resuspended , The double-gene recombination complex is transfected into macrophages.
[0065] Western Blot detection: ultrasonically lyse the macrophages transfected with the double-gene recombination complex and the macrophages transfected with the empty virus, collect the total cell protein for SDS-PAGE, electrotransfer the PVDF membrane after electrophoresis, wash the membrane, and block. Add rabbit anti-human SMURF1 polyclonal antibody, incubate at 37°C for 1 hour, wash the membrane, add goat anti-rabbit IgG, incubate at 37°C for 1 hour, wash the membrane, and develop color. See the result figure 2 The double-gene recombination complex can express SMURF1 protein in macrophages (lane 1 is the total protein of Ad-IRAK2/SMURF1 virus transfected dendritic cells, and lane 2 is the total protein of empty virus transfected dendritic cells) .
[0066] 2.3. Detection of inflammatory factors in macrophages after red blood cell lysate treatment,
[0067] Add 10μl of red blood cell lysate to macrophages incubated with 0.1mg of the double-gene recombination complex or control macrophages, 37℃, 24h, and detect the red blood cell lysate stimulated the secretion of IL-8 and TNF-α by ELISA method ability. The research results are as image 3 As shown, the macrophages incubated with the double-gene recombination complex secreted less IL-8 and TNF-α than the control macrophages.

Example Embodiment

[0068] Example 3 Detection of cerebral edema in mice with cerebral hemorrhage
[0069] 25μl of autologous whole blood was injected into the basal ganglia of mice. After 10 minutes, 1mg of the double-gene recombination complex macrophage vaccine or control (PBS) was injected at the same site. Three days later, the mice were sacrificed, brain tissues were obtained, and the brain edema of the mice was detected by dry and wet weight method. The research results are as Figure 4 As shown, compared with the control (PBS), the double-gene recombination complex macrophage vaccine can significantly reduce cerebral edema in mice with cerebral hemorrhage.

PUM

no PUM

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.

Similar technology patents

gRNA subjected to wild type T cell TCR alpha chain knockout and method

InactiveCN107236741AHigh gene knockout efficiencyEasy to prepare
Owner:THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products