T-20 fusion protein, and its preparing method and use
A fusion protein, T-20 technology, applied in the direction of peptide/protein components, chemical instruments and methods, hybrid peptides, etc., to achieve the effect of high recovery rate, strong operability and simple method
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Embodiment 1
[0068] Example 1: The expression vector is the engineering bacteria construction of pPICZαC
[0069] In this example, the EasySelectTM Pichia expression system from Invitrogen was used to express the T-20 fusion protein. Using pPICZαC as an expression vector, the T-20 gene and Fc gene were cloned behind the α factor signal peptide gene.
[0070] 1.1 Gene cloning and construction of recombinant plasmid 1 ( figure 1 )
[0071] Design and synthesize T-201, T-202, T-203, T-204, Fc1, Fc2 (SEQ ID NO: 5, 6, 7, 8, 9, 10), and T-201, T-202, T -203, T-204 are primers, PCR amplifies the T-20 gene sequence, and Kpn I and Xba I sites are introduced into the 5' end and 3' end respectively. Using Fc1 and Fc2 as primers and the first-strand cDNA synthesized from the total RNA extracted from peripheral blood lymphocytes as a template, the Fc gene was amplified by PCR, and Cla I and Eco RI were introduced into the 5' end and 3' end respectively. site. The PCR conditions were: 94°C for 1 mi...
Embodiment 2
[0079] Embodiment 2: the construction of the engineering bacteria whose expression vector is pPIC9
[0080] In this example, the Pichia pastoris expression system of Invitrogen Company was used to express the T-20 fusion protein.
[0081] 2.1 Construction of recombinant plasmid 2 ( Figure 4 )
[0082] Design and synthesize primers T-205, T-206, Fc3, Fc4 (SEQ ID NO: 11, 12, 13, 14), introduce XhoI restriction site and yeast α signal peptide cleavage site (KEX2 enzyme recognition site), Fc4 primer was introduced into Sna BI, and T-205 primer and T-206 were introduced into Sna BI and Not I sites, respectively. Using pGEM-T-Fc as a template and Fc3 and Fc4 as primers, the Fc gene was amplified by PCR, and the gene fragment was recovered by electrophoresis, digested with XhoI and SnaBI and recovered. Simultaneously, the pPIC9 plasmid was digested with XhoI and SnaBI, recovered by electrophoresis, ligated with the Fc gene fragment treated with XhoI and SnaBI, and then transforme...
Embodiment 3
[0087] Example 3: Construction of engineering bacteria whose expression vector is pMETαA
[0088] In this example, the Pichia methonolica expression system of Invitrogen Company was used to express the T-20 fusion protein.
[0089] 3.1 Construction of recombinant plasmid 3
[0090] Recombinant plasmid 2 in Example 2 was double digested with Xho I and Not I, the T-20 and Fc fusion protein gene fragments were recovered, ligated with the pMETαA plasmid treated with Xho I and Not I digestion, transformed into Escherichia coli DH5α, and coated On the LB plate containing the ampicillin of 100ug / ml, screening transformant obtains recombinant plasmid 3 ( Figure 5 ).
[0091] 3.2 Transformation of recombinant plasmid 3 into host strain PMAD11
[0092] A large amount of recombinant plasmid 3 was extracted, linearized with Apa I, precipitated with absolute ethanol, washed with 70% ethanol, dried in the air, and dissolved in an appropriate amount of TE solution (concentration was abou...
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