A fusion protein comprising PDGFRβ-specific affinity body and TNFα and its use
A fusion protein and recombinant vector technology, applied in DNA/RNA fragments, medical preparations containing active ingredients, fusion polypeptides, etc., can solve problems such as weak capacity and vascular leakage, and achieve small molecular weight, effective tumor treatment, inhibition of The effect of tumor growth
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Embodiment 1
[0059] Example 1 includes PDGFRβ-specific Affibody Z PDGFRβ Molecular Design and Cloning Construction of Fusion Protein Z-TNFα with TNFα
[0060] 1. Molecular design of Z-TNFα
[0061] Affibody Z PDGFRβ Consists of 58 amino acids (Table 1). TNFα is the extracellular segment of murine TNFα (a fragment consisting of 77-233 amino acids) (see Table 1). Priority design will Z PDGFRβ Linked to the N-terminus of TNFα to construct the fusion protein Z-TNFα.
[0062] 2. Construction of Z-TNFα expression vector
[0063] According to Z PDGFRβ The amino acid sequence (SEQ ID NO: 1) was designed, and its initial coding gene (SEQ ID NO: 2) was designed, and then optimized by nucleic acid analysis software. For the coding sequence of TNFα gene, refer to the sequence (SEQ ID NO: 3, SEQ ID NO: 4) provided by gene bank (NM_013693.3). According to the molecular design, the Z-TNFα coding gene (SEQ ID NO: 5, SEQ ID NO: 6) was constructed by software, and then artificially synthesized by a ...
Embodiment 2
[0064] Example 2 Expression and separation and purification of Z-TNFα
[0065] Transfer the pQE30-Z-TNFα plasmid into Escherichia coli M15 strain, pick the monoclonal strain and insert it into the double-resistant (ampicillin 100 μg / ml, kanamycin 30 μg / ml) LB liquid medium, shake culture at 37°C , when the bacterial concentration A 600 When the temperature reaches about 0.8, add 0.05 mM isopropyl-β-D-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside, IPTG), and induce culture at 26° C. for 14-16 hours. Centrifuge (7000g, 10min) to collect the bacteria, resuspend with Lysis buffer (50mM phosphate buffer, pH8.0; 300mM sodium chloride; 20mM imidazole; 10mM β-mercaptoethanol), add phenylmethanesulfonyl fluoride (Phenylmethanesulfonyl fluoride, PMSF) to a final concentration of 1 mM, and sonicate the bacteria in an ice bath (power 300 W, work for 10 s, interval 30 s, 40 min in total). After breaking the bacteria, centrifuge (4°C, 25000g, 10min), repeat 4 times, and col...
Embodiment 3
[0066] Example 3 Binding of Z-TNFα to pericytes
[0067] Firstly, after incubating pericytes with PDGFRβ-specific antibody, the expression of PDGFRβ receptor on the surface of pericytes was detected by flow cytometry. The result is as Figure 4 As shown in A, PDGFRβ is highly expressed on the surface of pericytes. In order to further analyze whether Z-TNFα can combine with pericytes, we use FAM dyes to label Z-TNFα and TNFα, then incubate them with pericytes for 1 h, and then analyze by flow cytometry. Figure 4 The result of B shows that Z-TNFα can bind to pericytes. Moreover, this binding can be weakened by a PDGFRβ-specific antibody (a-PDGFRβ) incubated with cells in advance, indicating that Z-TNFα binds to cells through PDGFRβ on the cell surface. However, TNFα is largely not bound to pericytes, suggesting that fusion Z PDGFRβ Endowed TNFα with the ability to bind to PDGFRβhighly expressed pericytes.
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