Recombinant targeted fusion protein for treating acquired immunodeficiency syndrome
A fusion protein and recombinant technology, applied in the direction of peptide/protein components, recombinant DNA technology, animal/human protein, etc., to achieve the effect of reducing toxicity and reducing immunogenicity
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Embodiment 1
[0079] Example 1. Acquisition of gene CD4V1TNFαm1 encoding recombinant targeting fusion protein
[0080] 1.PCR primers and their sequences
[0081] 1.1 TNFαm1 primers synthesized by Boya Company:
[0082] Forward primer 5'-aaa gtaagaggtccaagat-3'25bp containing KpnI site
[0083] Reverse primer 5'-aaa ttatcacagggcaatg-3'25bp containing BamHI site
[0084] 1.2 The sequencing primers of pCW111 synthesized by Boya Company:
[0085] Forward primer 5'-AAAAAGGGCATCAAATTAAACC-3'
[0086] Reverse primer 5'-TCGGCGATATAGGCGCCAGC-3'
[0087] The PCR reaction conditions are: denaturation at 94°C for 3 minutes and cycle: denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds. After 25 cycles, extend at 72°C for 10 minutes. The amplicon used was High Fidelity Pfu. The target fragment in the PCR product was recovered by 1.0% agarose gel to obtain a DNA fragment of about 514bp.
[0088] 2. Acquisition of mutant TNFαm1
[0089] TNFα...
Embodiment 2
[0092] Example 2. Construction of fusion gene CD4V1-TNFαm1 temperature-controlled expression recombinant pCW-CD4V1TNFαm1
[0093] The pCW111 vector was treated with BamHI and NdeI double enzyme digestion, and the large fragment was recovered; the recombinant pGEM-CD4V1TNFαm1 was treated with the same scheme, and the small fragment (797bp) containing the fusion gene CD4V1TNFαm1 was recovered, and the two were ligated to obtain the recombinant pCW -CD4V1TNFαm1, thereby placing the gene encoding the CD4V1 TNFαm1 fusion protein in the temperature-regulated promoter P R P L under control. For the results of enzyme digestion and identification of recombinant pCW-CD4 V1TNFαm1, see image 3 , see the sequencing results Figure 7 ,8.
Embodiment 3
[0094] Example 3. Acquisition of the gene encoding TNFα mutant TNFαm2
[0095] 3.1 TNFαm2 primers synthesized by Boya Company:
[0096] Forward primer 5'-aaa cgt aaacgcaagcct-3' contains PstI / KpnI site
[0097] point
[0098] Reverse primer 5'-aaa ttatcacagggcaatg-3'25bp containing BamHI enzyme
[0099] cutting site
[0100] 3.2 Acquisition of mutant TNFαm2
[0101] Using the "3.1" PCR primers to amplify the plasmid pBS-TNFαm1, the result is to obtain the deletion mutation of the first 7 amino acids of the N-terminal of the prototype TNFα, and mutate Pro8, Ser9, Asp 10 into arg8, Lys9, Arg10, and obtain An attenuated potent mutant of TNFαm2. At the same time, a BamHI site ( GGA TCC ) and 2 stop codons ( UAAUGA ). After the obtained PCR product was treated by the BamHI / PstI double enzyme digestion scheme, after the glass milk was recovered, it was connected with the plasmid pBluescript SK treated by the same scheme to obtain the recombinant plasmid pBS-TNFαm2. ...
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