Immune suppression fusion protein, its coded nucleic acid and application
A technology of fusion protein and toxin protein, which is applied in the field of immunology, drug design, and molecular biology, and can solve problems such as large toxic side effects, short immune tolerance period, and transplant failure
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Embodiment 1
[0095] Example 1: Cloning of B7-2 Extracellular Region cDNA
[0096] According to the published B7-2 and B7-1 gene sequences, primers were designed to amplify the cDNA encoding B7-2 extracellular region 225aa and B7-1 extracellular region 208aa, and were verified by GOLDKEY software. The primer sequence of B7-2 is: the forward primer PF7-2A is 5'-GCGGATCCGCTGCTCCTCTG-3' (SEQ ID NO: 5), and a BamHI site is introduced at the 5' end; the reverse primer PR7-2A is 5'-CCCGGGTTAAGGAATGTGGTCTGG -3' (SEQ ID NO: 6), a stop codon TAA and a SmaI site were introduced at the 3 end. The B7-1 primers are: the forward primer PF7-1A is 5'-GCGGATCC-3' (SEQ ID NO: 7), and a BamHI site is introduced at the 5' end; the reverse primer PR7-1A is 5'-CCCGGGTTA-3 '(SEQ ID NO: 8), a stop codon TAA and a SmaI site were introduced at the 3' end. Primers PF7-2A and PR7-2A, as well as PF7-1A and PR7-1A amplify the cDNAs encoding the 1-225aa and 1-208aa fragments of the extracellular region variable regions...
Embodiment 2
[0100] Example 2: Construction of a novel recombinant immunosuppressive toxin fusion protein B7-PE40KDEL expression vector
[0101] According to B7-2 225 , B7-1 208 and PE40 target gene sequence, designed to amplify B7-2 225 , B7-1 208 Forward and reverse primers PF7-2B and PR7-2B, PF7-1B and PR7-1B, and PF-PEA and PR-PEA of PE40. For fusion construction, make B7-2 225 , B7-1 208 and the PE40KDEL gene with a flexible linker (Gly 4 Ser) 4On this basis, the reverse primer PR-PEB was designed. The difference between PR-PEB and PR-PEA is that the 5' REDLK can be replaced by KDEL, and in primers PF7-2B and PF7 EcoRI and MunI restriction sites were introduced into -1B, a repeat sequence of primers PR7-2B and PR7-1B was included in the primer PF-PEA, and a SmaI restriction site and connecting peptide were introduced into the primer PR-PEA [(Gly 4 Ser) 4 ] coding sequence. See Table 1 for details.
[0102] Table 1. Construction of primer sequences containing the fusion gene...
Embodiment 3
[0127] Embodiment 3: Expression of recombinant toxin protein fusion gene B7-PE40KDEL
[0128] The plasmids pGEM-T-B7-2-PE40KDEL and pGEM-T-B7-1-PE40KDEL constructed in Example 2 were respectively used as templates, and PF7-2B or PF7-1B and PR-PEB were used as primers to amplify the target fragment. The 100ul volume PCR reaction system is: 100ngDNA template, 10× reaction buffer, 10pmol of upstream and downstream primers, 10mmol / l dNTP 10ul, 25mmol / l MgSO4 6ul, dimethyl sulfoxide 10ul, Taq DNA polymerase 2U, double distilled Make up the volume with water to 100ul. The PCR reaction conditions were: 98°C for 3min, cycle parameters: 94°C for 1min, 55°C for 1min, 72°C for 1.5min, after 25 cycles, extend at 72°C for 10min. The length fragments of B7-1-PE40KDEL DNA and B7-2-PE40KDEL amplified by PCR reaction were 1842bp and 1791bp, respectively. After the PCR reaction products were recovered, they were inserted into the pGEM-T vector respectively, and the about 1.8kb fragment was ex...
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