Human derived monoclonal antibody for identifying activated integrin alpha 4 beta 7
A monoclonal antibody and integrin technology, applied in the direction of antibodies, anti-receptors/cell surface antigens/cell surface determinant immunoglobulins, anti-inflammatory agents, etc., can solve the problem of non-specific antibody recognition
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Embodiment 1
[0072] Embodiment 1, integrin α 4 beta 7 Construction of wild-type and mutant soluble protein expression vectors and protein expression and purification
[0073] The following description is based on α 4 The amino acid sequence of the subunit (human) is shown in SEQ ID NO: 33 (999aa). The following description is based on β 7 The amino acid sequence of the subunit (human) is shown in SEQ ID NO: 9 (779aa).
[0074] It has been confirmed that an N-glycosylation site is introduced between the I and Hybrid domains of the integrin β subunit, which can activate the integrin molecule. Based on this, construct the full-length integrin α 4 beta 7 Wild-type and mutant soluble protein expression vectors with this mutation, α 4 The subunit has a splice body in the body, so in order to obtain the full-length α 4 Subunit, the inventors mutated the splice site R558A (ie the amino acid at position 558 was mutated from R to A). Soluble integrins do not have transmembrane and intracell...
Embodiment 2
[0077] Example 2. Using phage antibody library technology to screen and express antibodies that specifically recognize activated integrins
[0078] (1) Using purified wild-type non-activated and mutant-activated α 4 beta 7 Integrin protein, by panning (panning), screening scFv antibody library Tomlinson I+J (purchased from Geneservice), after three rounds of screening and removal of wild-type non-activated α 4 beta 7 Integrin protein combined with phage, and finally can be able to express specific binding activated α 4 beta 7 Integrin scFv phage. The specific screening process is to first label the soluble integrin protein with biotin, and incubate it with the human single-chain antibody phage library. The coated magnetic beads are mixed with it and incubated, and all the biotin-labeled integrins are immobilized on the magnetic beads, and the phages that bind the integrin are also immobilized on the magnetic beads, and finally the magnetic beads are separated by a magneti...
Embodiment 3、J19
[0101] Example 3, J19 specifically recognizes activated integrin α 4 beta 7
[0102] After the full-length J19 was purified, in order to further verify its recognition specificity, the inventors used flow cytometry detection method.
[0103] figure 2 The specific process of A experiment: Stable expression of integrin α 4 beta 7 K562 cells were treated with 1mM Ca 2+ +1mM Mg 2+ and 2mM Mn 2+ Wash twice with HBS buffer, then add J19 (10μg / ml), add the same amount of hIgG (purchased from Pierce) to the control, and incubate at 4°C for 0.5 hours; 2+ +1mM Mg 2+ and 2mM Mn 2+ Wash twice with HBS buffer respectively, then add Alexa Fluor 488-labeled goat anti-human IgG (1:500) (purchased from Invitrogen), and incubate at 4°C for 0.5 hours; 2+ +1mM Mg 2+ and 2mM Mn 2+ Wash twice with HBS buffer, and detect with a flow cytometer (purchased from BD).
[0104] figure 2 Experimental procedure of B: basic operation and figure 2 A is the same, except that before adding the...
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