Application of nanobody I22 to detection of recombinant human interferon alpha 2b
A technology of recombinant human interferon and nano-antibody, which is applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems of unfavorable rapid detection, lack of research, and reduce production costs, so as to achieve rapid detection, accurate experimental results, The effect of reducing production costs
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Embodiment 1
[0046] Embodiment 1: Construction of expression system
[0047]The screened target protein anti-recombinant human interferon α2b nanobody I22 gene sequence was constructed into the vector pET28a, and pET28a-His-I22-His with His tags at the N-terminal and C-terminal was constructed. The enzyme cutting site of this strain vector is NcoI / XHoI, such as figure 1 .
Embodiment 2
[0048] Example 2: Induced Expression Purification
[0049] In the first step, the nanobody I22 expression vector pET28a-His-I 22 -His were transformed into BL21 host bacteria respectively.
[0050] In the second step, the bacterial culture medium was placed in LB / Kan liquid medium (containing 1 mM IPTG), and the expression was induced in a shaker at 16° C. at 220 rpm.
[0051] In the third step, after 13 hours, centrifuge at 4°C and 13,000 rpm for 28 minutes to collect the bacterial liquid precipitate. Suspend the precipitate with an equilibration buffer, sonicate, centrifuge at 13,000 rpm at 4°C for 26 minutes, and collect the supernatant.
[0052] In the fourth step, use the nickel column to purify the protein, use the original bacterial solution as a positive control, and obtain a single target band at about 15kDa, which is the target protein, such as figure 2 .
[0053] The fifth step is to test the purity of the purified target protein and analyze the quality of the ...
Embodiment 3
[0055] Example 3: Whole-column imaging isoelectric focusing capillary electrophoresis to measure the isoelectric point of nanobody I22 protein
[0056] The first step is to prepare the solution, prepare positive electrolyte (85% phosphoric acid solution) and negative electrolyte (50% NaOH solution), 1% methylcellulose solution, and 8mol / L urea solution.
[0057] In the second step, the sample determination solution was thoroughly mixed, taking 2 μL each of Marker pI=4.65 and pI=8.96, 140 μL of 1% methylcellulose (methylcellulose), 25 μL of 8M Urea solution, and a total volume of 169 μL.
[0058] The third step is the preparation of samples and standards. Concentrate the protein sample to about 4 mg / mL, take 5 μL, mix it with 169 μL of measurement solution, and make up to 200 μL with deionized water.
[0059] In the fourth step, the sample is degassed, and the sample solution is centrifuged for 3 minutes at a speed of 11,000 rpm, quantitatively transferred to an automatic sampl...
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