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Method for biopanning affinity ligand of bromelin

A bromelain and affinity technology, which is applied in biochemical equipment and methods, microbial determination/inspection, and measurement devices, can solve the problems of cumbersome operation procedures, high separation costs, and long purification time, etc., and achieve rich biological resources, The preparation method is simple and the effect of low cost

Inactive Publication Date: 2011-07-13
DONGHUA UNIV
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  • Application Information

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Problems solved by technology

With the rapid development and expansion of biological technology, the demand for high-purity biomacromolecules is also increasing in the market, and the backward traditional chromatography technology cannot fully keep up with the market demand: high separation cost, long purification time, Scientists in the field of chromatography have been plagued by problems such as cumbersome operating procedures

Method used

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  • Method for biopanning affinity ligand of bromelin
  • Method for biopanning affinity ligand of bromelin
  • Method for biopanning affinity ligand of bromelin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The cultivation and growth curve determination of phage host bacteria ER2738, the specific steps are as follows:

[0031] figure 1 Shown: 2.5 hours after being inoculated into the liquid medium, Escherichia coli passed the lag phase and entered the logarithmic phase. After about 3-3.5 hours of culture, ODA 600 When reaching 0.5, we can see from the figure that the E. coli ER2738 colony begins to enter the logarithmic phase, and the OD value rises extremely fast. The culture time of 3.5 hours is consistent with the time that most researchers experience. It shows that it is most suitable to pick a single colony for liquid culture or generally 3.5 hours after dilution for phage infection.

Embodiment 2

[0033] The amplification of M13 filamentous phage, the specific steps are as follows:

[0034] (1) Pick the ER2738 strain and culture it in LB-Tet culture medium, shake it at 200 rpm at 37°C overnight, and use LB culture medium (without antibiotic Tet) to shake the 1:100 diluted culture medium to ODA on the next day 600 = 0.5 to be used.

[0035] (2) The phage eluate was quantitatively added to the LB bacterial liquid, and infected at 37° C., 85 rpm for 1 hour. Centrifuge at 5000rpm for 15min at 4°C. The supernatant was removed, and the bacteria were resuspended in 200ml LB culture medium (note: no antibiotics were added to the LB culture medium here to increase the infectivity and activity of bacteriophage) for 12 hours.

[0036](3) The next day, take the bacterial solution and centrifuge at 5000rpm for 15min at 4°C, take the supernatant, transfer it to a fresh centrifuge tube, add 1 / 5 (V / V) PEG / NaCl, vortex and mix well, and place on ice for 1h. Centrifuge at 12000rpm for...

Embodiment 3

[0039] To optimize the conditions of the biopanning method, the specific steps are as follows:

[0040] (1) Target molecule concentration selection optimization:

[0041] Four parallel elutriation processes were designed to be carried out at the same time, with BSA as the target molecule solution, and the concentrations of the incubated microtiter plates were 100 μg / ml, 80 μg / ml, 50 μg / ml, 20 μg / ml / . Randomly pick 10 phages screened out by each panning, and perform ELISA detection on them, such as figure 2 As shown: Overall, it can be found that the OD value screened by the target molecule concentration with the lowest concentration of 20 μg / ml is generally high, which indicates that the affinity of the ligands screened by the microplate plate coated with the low concentration target molecule solution is relatively low. say higher.

[0042] Therefore, it is designed to reduce the concentration of bromelain solution step by step during the four rounds of elutriation, from 10...

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Abstract

The invention relates to a method for biopanning the affinity ligand of bromelin. The method comprises the following steps: (1) biopanning; (2) performing directed amplification and enrichment on bacteriophage; (3) testing the titer; (4) performing monoclonal amplification on bacteriophage; (5) performing an enzyme-linked immuno sorbent assay (ELISA); and (6) performing DNA sequencing, wherein the sequence is Ile-XXX-Ser-Pro-XXX-XXX-XXX (LXSPXXX). The preparation method is simple and has low cost, the biological resources of Escherichia coli and bacteriophage are rich; the selected heptapeptide ligand which has affinity to bromelin has higher affinity to bromelin, and can be used to discover the concensus sequence and provide rich experimental data for the interaction of protein in the proteomics; and the heptapeptide ligand can also be used in the separation and purification researches of pineapple protein.

Description

technical field [0001] The invention belongs to the field of affinity ligand screening methods, in particular to a method for affinity screening bromelain affinity ligands. Background technique [0002] At present, affinity chromatography technology has become more and more mature, and it has been more used in production practice, but at the same time, it has also encountered corresponding bottleneck problems. With the rapid development and expansion of biological technology, the demand for high-purity biomacromolecules is also increasing in the market, and the backward traditional chromatography technology cannot fully keep up with the market demand: high separation cost, long purification time, Scientists in the field of chromatography have been plagued by problems such as cumbersome operating procedures. [0003] How to find a suitable ligand that can individually adsorb a biomacromolecule such as a certain protein or enzyme is a problem that needs to be solved urgently ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37G01N33/573
Inventor 朱利民陈天翔聂华丽汪雯
Owner DONGHUA UNIV
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