A recombinant clonorchis sinensis secreted phospholipase a2 protein and its in vitro soluble expression and purification preparation method

A technology for secreted phospholipase and Clonorchis sinensis, which is applied in the fields of biochemical equipment and methods, chemical instruments and methods, recombinant DNA technology, etc.

Inactive Publication Date: 2019-03-01
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there is no technology in the prior art that can realize the soluble expression of Clonorchis sinensis secreted phospholipase A2 protein

Method used

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  • A recombinant clonorchis sinensis secreted phospholipase a2 protein and its in vitro soluble expression and purification preparation method
  • A recombinant clonorchis sinensis secreted phospholipase a2 protein and its in vitro soluble expression and purification preparation method
  • A recombinant clonorchis sinensis secreted phospholipase a2 protein and its in vitro soluble expression and purification preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning and expression of embodiment 1 recombinant CssPLA2 protein (i.e. MBP-CssPLA2 fusion protein)

[0044] 1.1 Using the cloning plasmid of the CssPLA2 gene (coded as Cs4e05) in the Clonorchis sinensis adult cDNA library as a template (Template), apply the designed specific primers to amplify the target gene by PCR:

[0045] CssPLA2 upstream primer (P1): 5'-CTAG TCTAGA AAACCACGGTCAATTTCA-3' (SEQ ID NO.2) underlined is the XbaI restriction site);

[0046] CssPLA2 downstream primer (P2): 5'-GGG AAGCTT GCTCATACAGTAATGTACG-3' (SEQ ID NO.3) is underlined as the HindIII restriction site).

[0047] by Ex Taq TM Enzyme PCR amplification, the reaction conditions are as follows:

[0048] Pre-denatured at 95°C for 10 minutes, and the reaction cycle parameters were 95°C for 30s, 57°C for 30s, 72°C for 1min, a total of 30 cycles; 72°C for 10min.

[0049] The PCR reaction system is as follows:

[0050] 10×Ex Taq Buffer (Mg 2+ plus)

5.0μl

dNTPs (10mmol...

Embodiment 2 Embodiment 1

[0126] The enzymatic activity assay of embodiment 2 embodiment 1 gained recombinant CssPLA2 protein

[0127] Secreted PLA2 (sPLA2) enzyme activity assay kit was used to measure the enzymatic activity of the recombinant CssPLA2 protein obtained in Example 1, honeybee secreted PLA2 was used as a positive control, and MBP tagged protein (maltose binding protein) was used as a negative control.

[0128] Principle: Phospholipase A2 (Phospholipase A2), referred to as PLA2, can hydrolyze the second acyl bond (sn-2) of glycerophospholipids to generate lysophospholipids and fatty acids.

[0129] Steps:

[0130] (1) Negative control group: add 10ul DTNB, 5ul reaction buffer and 10ul MBP control protein to the microwell of the microtiter plate;

[0131] (2) Positive control group: add 10ul DTNB, 5ul reaction buffer and 10ul honeybee PLA2 protein to the wells of the microtiter plate;

[0132] (3) Experimental group: Add 10ul DTNB, 5ul reaction buffer and 10ul MBP-CssPLA2 fusion protein ...

Embodiment 3

[0137] Embodiment 3 removes the endotoxin of recombinant CssPLA2 protein

[0138] 3.1 According to Detoxi-Gel TM Endotoxin Removing Gel kit instructions, the operation is as follows:

[0139] (1) Wait for the resin to settle naturally at room temperature for about 30 minutes to recover its activity;

[0140] (2) Regenerate Detoxi-Gel resin: clean the resin with 1% sodium deoxycholate of 5 times the volume of the resin, then add 3 to 5 times the volume of water for injection to remove the cleaning agent;

[0141] (3) Equilibrium resin: Add 3 to 5 times the volume of water for injection into the column.

[0142] (4) Sample addition: put the recombinant CssPLA2 protein solution obtained in Example 1 into a dialysis bag, dialyze at 4°C in PBS buffer (pH 7.4), change the dialysate every 4 hours or so, and change the solution for 3-3 hours in total. 4 times; put the dialysis bag into sucrose, use the water absorption of sucrose to concentrate the protein; add 1ml concentrated re...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a recombinant CssPLA2 protein and a soluble expression in vitro and a purifying preparation method thereof. CssPLA2 protein obtained by existing expression technology is an inclusion body without enzymatic activity; and enzymatic activity thereof is weak even after renaturation of the inclusion body. The CssPLA2 protein comprises CssPLA2 protein and MBP tag protein. According to wide and deep research, soluble expression of CssPLA2 protein is achieved initially by optimization of special expression means; recombinant CssPLA2 protein with biological activity is prepared, and problems in the existing expression technology are solved effectively.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant clonorchis sinensis secreted phospholipase A2 protein and its in vitro soluble expression and purification preparation method. Background technique [0002] The existing expression technology for Clonorchis sinensis secreted phospholipase A2 protein (that is, CssPLA2 protein, GenBank accession number: ABL07371.1) can only express non-biologically active Clonorchis sinensis secreted phospholipids in the form of inclusion bodies Enzyme A2 protein, because the expressed Clonorchis sinensis secreted phospholipase A2 protein has no biological activity, it cannot be used for the functional research of this protein. Even after complex protein refolding techniques, the biological activity of the resulting protein is still quite low. [0003] For example: the existing literature F.Hu et al. / Molecular & Biochemical Parasitology 167 (2009) 127-134 uses the plasmid numb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N9/18C12N15/70
CPCC07K2319/24C12N9/18C12Y301/01004
Inventor 李学荣吴银娟余新炳
Owner SUN YAT SEN UNIV
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