[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.
