Recombinant expression and purification method of vitellogenin from bostrichthys sinensis
A technology of vitellogenin and Chinese snakehead, which is applied in the field of fusion protein expression and purification, can solve the problems of increasing separation and purification procedures, and the renaturation rate cannot reach 100%, and achieves low cost, high recovery rate, and simple separation and purification Effect
Active Publication Date: 2020-01-07
XIAMEN UNIV
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Problems solved by technology
Moreover, all the reported recombinant VTGs currently exist in inclusion bodies, and renaturation is required to obtain active recombinant proteins, and the renaturation rate cannot reach 100%, and the separation and purification procedures are increased.
The presence of recombinant protein in soluble form is more advantageous than inclusion body form, and there is no report on the expression of VTG recombinant expression in soluble form.
[0007] There is no report about the recombinant expression and purification method of vitellogenin in Chinese snakehead snakehead
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[0054] The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings, and the specific implementation methods are as follows:
[0055] The recombinant expression and purification of vitellogenin of Channa sinensis, equipped with the following experimental supplies: 1 μL recombinant PMAL-c5x vector, BL21 strain, 100 μg / mL ampicillin, 0.5 mM IPTG, PBS buffer, 1.0 MMTris-HCL, 300 mM NaCl, 0.5MEDTA, Amylmose affinity column, Factor Xa and Qubit TM Protein Assay Kit protein concentration determination kit.
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A recombinant expression and purification method of vitellogenin from bostrichthys sinensis belongs to the technical field of fusion protein expression and purification method. The method of the invention comprises the following steps: providing a vitellogenin gene based on bostrichthys sinensis, cloning part of the gene, recombining plasmids in vitro, transferring to escherichia coli for prokaryote expression, obtaining high-efficiency fusion expression at 16 DEG C of IPTG induction temperature, wherein the expressed target protein exists in a soluble state and accounts for more than 25% of total of bacterial soluble protein; applying a purification technology to obtain a fusion protein MNP-VTG, and separating a label MNP from the fusion protein to obtain a target protein VTG monomer through sucrose digestion. The method of the invention has high-efficiency expression, simple purification of VTG, more simple separation than inclusion bodies due to solubility and no need for renaturation. The obtained recombinant VTG provides stable and uniform raw material basis for subsequent applications. The recombinant protein can be used for biological activity function researches, and made into corresponding antibodies for detection research.
Description
technical field [0001] The invention relates to the technical field of fusion protein expression and purification methods, in particular to a method for expressing and purifying vitellogenin fusion proteins of the Chinese snakehead snakehead. Background technique [0002] Vitellogenin VTG is the precursor of vitellin in the egg development of oviparous animals. It is expressed by liver cells under the induction of estrogen, and the protein produced provides the hemolymph system to enter the ovary and enter the egg to mature into vitellin. Provides nutrients for egg development. As a reproductive nutritional protein, VTG is generally induced by hormones during yolk development (1. Bownes M. The roles of juvenile hormone, ecdysone and the ovary in the control of Drosophila vitellogenesis [J]. J Insect Physiol, 1989, 35: 409-413; 2.C.M.Campbell DRI.Hormonal control of vitellogenesis inhypophysectomized winter flounder(Pseudopleuronectes americanusWalbaum)[J].Gen Comp Endocrino...
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IPC IPC(8): C07K14/46C07K1/22C07K16/18C12N15/12C12N15/70A61P31/04A61P31/10G01N33/68
CPCA61P31/04A61P31/10C07K14/461C07K16/18C12N15/70G01N33/68G01N2333/4603
Inventor 周克夫孟坤龙敏
Owner XIAMEN UNIV