Apostichopus japonicus F type lectin AjFL-1 as well as preparation method and application

A technology of lectin and japonicus imitation, which is applied in the field of molecular biology and can solve the problems of no preparation method of japonicus japonicus f-type lectin

Inactive Publication Date: 2018-06-22
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there are no related reports about the imitation of A. japonicus F-type lectin, its preparation method and its application in the preparation of drugs for inhibiting Pichia pastoris

Method used

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  • Apostichopus japonicus F type lectin AjFL-1 as well as preparation method and application
  • Apostichopus japonicus F type lectin AjFL-1 as well as preparation method and application
  • Apostichopus japonicus F type lectin AjFL-1 as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0038] Apostichopus japonicus F-type lectin of the present invention Aj The binding activity detection of FL-1 and different PAMPs (LPS, PGN, Mannan and Fucose), the specific steps are as follows:

[0039] (1) Coating solution (50 mM Na 2 CO 3 -NaHCO 3 buffer, pH 9.8) to dilute the different PAMPs to 100 μgmL -1 , add 100 μL per well into a 96-well microtiter plate, and coat overnight at 4 °C;

[0040] (2) Wash 3 times with TBST, 5 min each time;

[0041] (3) Add 200 μL of 3% BSA solution (dissolved in TBST) to each well, and block in a 37 °C incubator for 1 h;

[0042](4) Wash 3 times with TBST, 5 min each time;

[0043] (5) Apostichopus japonicus F-type lectin of the present invention Aj After FL-1 was serially diluted to two-fold concentration, 100 μL per well (three parallels were set for each gradient, TBST was used as a blank control, and the tagged protein Trx was used as a negative control), and incubated at 18 °C for 3 h;

[0044] (6) Wash 3 times with TBST, 5...

experiment example 2

[0053] Apostichopus japonicus F-type lectin of the present invention Aj FL-1 and different bacteria ( P. pastoris , V. splendidus, E. coli, M. luteus ) agglutination activity assay. Escherichia coli ( Escherichia coli Top10, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), Vibrio splendidus ( Vibrio splendidus , purchased from Beijing Microorganism Culture Collection Center), Pichia pastoris ( Pichia pastoris GS115, purchased from Invitrogen), Micrococcus luteus ( Micrococcus luteus , purchased from Beijing Microorganism Culture Collection Center). Specific steps are as follows:

[0054] (1) FITC staining: the cells were collected by centrifugation at 5000 rpm for 5 min. After the medium was discarded, TBS buffer was added, and the cells were washed three times. Add FITC (final concentration 0.1mg ml -1 ), stained in the dark for 30 min. Cells were collected by centrifugation at 5000 rpm for 5 min. After discarding the supernatant, add TBS ...

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Abstract

The invention discloses apostichopus japonicus F type lectin AjFL-1. The amino acid sequence is shown by SEQ ID NO.1. The preparation method comprises the following steps: performing PCR amplificationon a coding region fragment of apostichopus japonicus F type lectin AjFL-1 by using specific primers P1 and P2, wherein the DNA sequence of the primer P1 is shown by SEQ ID NO.2, and the DNA sequenceof the primer P2 is shown by SEQ ID NO.3; performing Nde1 and Xho1 digestion on the PCR amplification product and a pET-22b(+) carrier; then connecting through T4 ligase, transforming, sequencing andidentifying the recon; transferring the recon into an escherichia coli Transetta (DE3) expression strain, and performing induction culture; then performing purification and renaturation to obtain recombinant protein with an amino acid sequence in the sequence table SEQ ID No.1. The apostichopus japonicus F type lectin AjFL-1 can be used for preparing a pichia pastoris inhibiting drug.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to an F-type lectin imitating Apostichopus japonicus Aj FL-1, preparation method and application. Background technique [0002] Imitation sea cucumber ( Apostichopus japonicus ) has high nutritional value and contains a variety of effective anticancer active ingredients. It is one of the important economic marine aquaculture species in the Yellow and Bohai Sea regions in northern my country. However, due to the deteriorating marine environment, the high concentration of pathogenic bacteria has caused serious disease problems in sea cucumber farming, which has restricted the healthy development of sea cucumber farming. In the process of sea cucumber disease occurrence, the key first step in the body's immune response is the recognition between pathogens and host immune factors. Because sea cucumbers do not have acquired immune systems and lack specific immune recognition...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/70A61K38/17A61P31/10
CPCA61K38/00C07K14/43504C12N15/70
Inventor 宋林生王英薛壮衣启麟宋小瑞
Owner DALIAN OCEAN UNIV
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