CN-121975022-A - Preparation method and application of label-free sebastes schlegeli IGF2 recombinant protein

Abstract

The invention belongs to the technical field of genetic engineering, but not limited to, and particularly relates to a preparation method and application of a label-free Sebastes schlegeli IGF2 recombinant protein, wherein the IGF2 has important regulation and control effects on ovarian matrixes and embryos after fertilization by detecting IGF2 expression levels of ovaries in different development periods. The invention uses cDNA of Sebastes schlegeli as a template to carry out PCR amplification to obtain the Sebastes schlegeli IGF2 mature peptide, and the amino acid sequence of the Sebastes schlegeli is shown as SEQ ID NO. 2. The recombinant protein comprises the IGF2 mature peptide, a His-Sumo tag connected with the N end of the IGF2 mature peptide, a connecting peptide segment between the His tag and the Sumo tag, and the amino acid sequence is shown as SEQ ID NO. 1. The invention also provides application of the Sebastes schlegeli IGF2 mature peptide recombinant protein in promoting the proliferation of ovarian primary cells and the development of germ cells of Sebastes schlegeli.

Inventors

• QI XIN
• LI JIANSHUANG
• WANG XIAOJIE
• YANG JING
• WEN HAISHEN
• LI YUN

Assignees

• 中国海洋大学

Dates

Publication Date

20260505

Application Date

20260206

Claims (9)

1. 1. The Sebastes schlegeli IGF2 recombinant protein with the His-Sumo tag is characterized by comprising an N-terminal 6×histidine tag, a Sumo tag sequence and an IGF2 mature peptide amino acid sequence, wherein the amino acid sequence of the IGF2 recombinant protein with the His-Sumo tag is shown as SEQ ID NO. 1.
2. 2. The recombinant protein of Sebastes schlegeli with His-Sumo tag according to claim 1, wherein the amino acid sequence of the IGF2 mature peptide is shown in SEQ ID NO. 2.
3. 3. The recombinant protein of Selaginella schlegeli with His-Sumo tag according to claim 1, wherein the amino acid sequence of the Sumo tag is shown as SEQ ID NO. 3, the tertiary structure of which can be recognized by Sumo protease with high specificity and is cleaved between the C-terminal and the N-terminal of the fusion protein, thereby obtaining the recombinant protein without tag and residual amino acid at the N-terminal.
4. 4. The Sebastes schlegeli IGF2 recombinant protein with His-Sumo tag according to claim 1, wherein the encoding gene of the Sebastes schlegeli IGF2 recombinant protein with His-Sumo tag comprises a short nonpolar flexible connecting peptide segment between the His tag and the Sumo tag encoding N-terminal His tag, the Sumo tag and IGF2 mature peptide, and the DNA sequence of the encoding gene is shown as SEQ ID NO. 4.
5. 5. The Sebastes schlegeli IGF2 recombinant protein with the His-Sumo tag according to claim 1, wherein the amino acid sequence of the His tag added to the N end of the recombinant protein and the connecting peptide is HHHHHHGSSNNSGSGSGS, the 6 XHis tag is used for purifying IGF2 recombinant protein through nickel column affinity chromatography, the GGS connecting peptide is used as a short nonpolar and flexible connecting peptide, space and flexibility can be provided, and the His tag is prevented from being wrapped by Sumo tag and IGF2 mature peptide, so that space interference is caused, and the binding effect with a nickel column is affected.
6. 6. An expression vector for expressing the flat-banbastes schlegeli IGF2 recombinant protein with His-Sumo tag according to any one of claims 1-5, wherein the expression vector comprises an encoding gene of IGF2 recombinant protein and a skeleton plasmid, and the skeleton plasmid is obtained by modifying pET-32a (+).
7. 7. A recombinant engineering bacterium for expressing the flat-banseris schlegeli IGF2 recombinant protein with His-Sumo tag according to any one of claims 1 to 5, wherein the recombinant engineering bacterium comprises the expression vector, and the host bacterium of the recombinant engineering bacterium is selected from Rosetta-gami B (DE 3).
8. 8. A method for preparing the label-free flat-fish schlegeli IGF2 recombinant protein according to any one of claims 1-5, which is characterized by comprising the following steps: (1) Constructing the encoding gene of the Sebastes schlegeli IGF2 recombinant protein with the His-Sumo tag, and connecting the encoding gene to a skeleton plasmid to obtain an expression vector of the IGF2 recombinant protein; (2) Transforming the expression vector into host bacteria, and inducing to express IGF2 recombinant protein with His-Sumo label; (3) Separating by His tag affinity chromatography to obtain IGF2 recombinant protein with His-Sumo tag; (4) Adding SUMO protease for low-temperature overnight digestion, and cutting off His-Sumo tag; (5) Removing incompletely digested His-Sumo tag IGF2 recombinant protein, excised His-Sumo tag and Sumo protease through His tag affinity purification, binding the recombinant protein to a nickel column, removing the recombinant protein, and finally collecting IGF2 mature peptide recombinant protein without tag; (6) The purified protein solution was dialyzed into 1 XPBS, 1 XPBS+10% glycerol buffer, ultracentrifuged and concentrated, and then bovine serum albumin was added to a final concentration of 0.1%, and stored at-80 ℃.
9. 9. Use of the non-tagged flat-fish-based IGF2 recombinant protein according to any one of claims 1-7 in ovarian stromal cell proliferation and oocyte development.

Description

Preparation method and application of label-free sebastes schlegeli IGF2 recombinant protein Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method of a sebastes schlegeli no-label IGF2 mature peptide recombinant protein and application of the recombinant protein in the aspects of ovarian stromal cell proliferation, follicular development and the like. Background Sebastes schlegeli (Sebastes schlegelii) commonly known as black head fish, black Dan Lu, etc., belong to the genus Perciformes (Perciformes), the subgenera (Scorpaenoidei), pingke (Sebastidae), and the genus Pelargonium (Sebastes). The rice has the advantages of delicious meat quality, strong disease resistance and stress resistance and the like, and becomes an important object for the cultivation, proliferation and release of the northern net cages in China. The sebastes schlegeli is bred for the embryo, the gonad development of the male and female fishes is asynchronous, the sperm of the male fishes is mature in 11 to 12 months each year, then the female fishes are bred, and the sperm is stored in the crypt outside the follicle. After 3 to 4 months of the next year, the female fish egg nest is mature, in-situ fertilization is carried out, and then the follicular layer outside the fertilized egg is continuously proliferated, folded and loosened to form a connective tissue wrapped embryo rich in blood vessels. The energy required by embryo development in the mother is about 2 times of the energy of yolk, so that the embryo passes through the placenta-like structure formed by the filtering bleb layer, and oxygen and nutrient substances required by growth and development are obtained from the mother, so that the pregnancy process is successfully completed. The process is influenced by various in-vivo and in-vitro factors, and once dysplasia occurs, a large number of embryos can die and in the abdomen due to insufficient nutrition or hypoxia and the like, so that the yield of the Selaginella schlegeli is seriously reduced, and the healthy development of the culture industry is hindered. Among the various growth factors, insulin-like growth factors (Insulin like growth factors, IGFs) are thought to play a dominant role in embryonic development and fetal growth. Wherein, IGF2 overexpression results in overgrowth of placenta and fetus, and IGF2 gene deletion reduces the weight of placenta and fetus, igf2 deleted mice weigh 40% less than wild-type pups. In vitro experiments have found that when human umbilical vein endothelial cells are treated with IGF2, the cells proliferate rapidly and align and form a capillary-like network on the artificial basement membrane. Meanwhile, IGF2 stimulates follicular development and steroid hormone production by stimulating granulosa cells, thereby promoting meiosis and maturation of oocytes. Thus, IGF2 has an important regulatory role in the overall reproductive cycle of mammals. In addition, IGF2 promotes the final maturation of teleosts oocytes. However, it is not reported whether IGF2 affects proliferation and follicular development of ovaries stromal cells of teleosts. Like other vertebrates, sebastes schlegeli IGF2 is A classical secreted protein with A typical N-terminal signal peptide sequence that is evolutionarily conserved, and the mature peptide consists of A B-C-A-D domain, containing 3 disulfide structures. There are many reports of prokaryotic expression of fish IGF2 recombinant proteins. However, inclusion bodies are present in the IGF2 recombinant protein in these reports, and cumbersome renaturation procedures are required to restore the biological activity, whereas the denaturant urea can adversely affect the amino covalent modification of the recombinant protein. Secondly, CN 119286772A discloses a prokaryotic expression and purification method and application of the leopard gill-like perch IGF2 recombinant protein, but the obtained recombinant protein does not only contain mature peptide, and cannot exclude the influence of structures such as signal peptide and the like on the bioactivity of the recombinant protein. If the recombinant protein can induce escherichia coli to express soluble and active IGF2 mature peptide, the purity and biological activity of the protein can be greatly improved, and the production cost and time are saved, so that the recombinant protein has important practical significance. In view of the above analysis, the technical problem that needs to be solved in the prior art is: (1) The expression rate of the recombinant protein of the escherichia coli is too high, inactive inclusion bodies are easy to form, and the bioactivity needs to be recovered through complicated renaturation operation. (2) Urea, a denaturant, can alter inclusion body conformation but can adversely affect the amino covalent modification of recombinant proteins. (3) The recombinant protein expressed by taking the CDS sequence o