Yak DDX6 protein and application of yak DDX6 protein in influence on growth of embryo fibroblasts

Abstract

The invention belongs to the field of cell biology, and particularly relates to yak DDX6 protein and application of the yak DDX6 protein in influence on growth of embryo fibroblasts. According to the specific technical scheme, the coding gene sequence of the DDX6 protein is shown as SEQ ID NO: 1, and the amino acid sequence of the protein is shown as SEQ ID NO: 2. New DDX6 protein is synthesized from yaks, it is found for the first time that the activity of embryo fibroblasts can be improved or inhibited by using a specific amount of DDX6 protein, growth and proliferation of the fibroblasts can be promoted or inhibited, and great potential is achieved in research and application of the fibroblasts.

Description

Technical Field

[0001] The invention belongs to the field of cell biology, and specifically relates to yak DDX6 protein and its application in influencing the growth of embryonic fibroblasts. Background Art

[0002] Embryonic fibroblasts (EF cells) are a type of fibroblasts isolated from early embryos, which play an important supporting and regulatory role in the development process. EF cells are pluripotent and can differentiate into multiple cell types. If the growth and proliferation of EF cells can be promoted, it will be of great value to study their role.

[0003] DDX6 is an important RNA helicase and a member of the DEAD-box protein family. It is mainly distributed in the cytoplasm, with a small amount distributed in the nucleus and mitochondria. At present, research on DDX6 mainly focuses on disease prevention and treatment. Studies have shown that DDX6 can inhibit tumor cell proliferation and induce cell apoptosis, thereby playing an anti-cancer role; it can participate in the formation of drug resistance in certain tumors and is one of the new targets for tumor treatment; it can affect the branching and growth of neuronal axons; it can affect protein synthesis at the presynaptic end; it can regulate the recognition and response of cells to viral RNA, thereby affecting the body's resistance to viruses and immune response.

[0004] However, there are no studies on the role of DDX6 in stromal cell proliferation. Summary of the invention

[0005] The purpose of the invention is to extract yak DDX6 protein and use it in influencing the growth of embryonic fibroblasts.

[0006] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a DDX6 protein, the coding gene sequence of the protein is shown in SEQ ID NO:1.

[0007] Correspondingly, a DDX6 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0008] Accordingly, the DDX6 protein is used to improve fibroblast viability and / or promote fibroblast growth and / or promote fibroblast proliferation.

[0009] Preferably, the fibroblasts are from yak.

[0010] Preferably, the application method is: co-culturing the fibroblasts with DDX6.

[0011] Preferably, in the co-culture system, the DDX6 concentration is <5 μg / mL.

[0012] Accordingly, the DDX6 protein is used to reduce fibroblast viability and / or inhibit fibroblast growth and / or promote fibroblast proliferation.

[0013] Preferably, the fibroblasts are from yak.

[0014] Preferably, the application comprises the following steps: the application method is: co-culturing the fibroblasts with DDX6.

[0015] Preferably, in the co-culture system, the concentration of DDX6 is ≥5 μg / mL.

[0016] The present invention has the following beneficial effects: the present invention synthesizes a new DDX6 protein from yaks, and for the first time finds that the use of a specific amount of the DDX6 protein can increase or inhibit the activity of embryonic fibroblasts, promote or inhibit the growth and proliferation of fibroblasts, and has great potential in the research and application of fibroblasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the spatial conformation of DDX6 protein;

[0018] Figure 2 This is a comparison chart of the effects of yak DDX6 protein on the activity of yak embryonic fibroblasts. DETAILED DESCRIPTION

[0019] The present invention discovers for the first time that the DDX6 protein from yak can promote the activity of yak embryonic fibroblasts and promote cell proliferation.

[0020] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0021] Example 1: Acquisition of yak DDX6 protein

[0022] The DDX6 gene was amplified using yak cDNA template to obtain a target fragment of about 1653 bp. The sequence after sequencing is shown in SEQ ID NO: 1. The homology with 97% of wild yak DDX6 (predicted) genes is 99.75% after NCBI comparison. The yak DDX6 has the highest homology with wild yaks, followed by cattle. The molecular formula of yak DDX6 is C 2403 H 3894 N 672 O717 S 22 The coding region is 1452 bp in length, encoding 483 amino acids. The amino acid sequence is shown in SEQ ID NO: 2. The schematic diagram of the spatial conformation of DDX6 protein is shown in Figure 1 shown.

[0023] Expasy predicted that the molecular weight of DDX6 protein is 54376.74Da, positively charged, and has an isoelectric point of 8.85. DDX6 is composed of 20 amino acid residues, of which leucine (Leu, 10.6%) is the most abundant, followed by isoleucine (Ile, 8.1%). According to calculations, the proportion of hydrophobic amino acid residues in DDX6 is 36.65%, proving that it is a hydrophilic protein. According to predictions, DDX6 protein does not contain a signal peptide and a transmembrane domain, indicating that the protein is not a membrane protein or a secretory protein.

[0024] The CDS region fragment of the DDX6 gene obtained by PCR amplification was cloned into the pET-32a vector to construct the pET-32a-DDX6 recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells to obtain positive recombinant bacteria, and cultured until OD 600 The protein was 0.4 to 0.6. 0.5 mmol / L IPTG was added for induction for 6 h, and the protein was purified by ultrasonic fragmentation and nickel column affinity chromatography, and finally a yak DDX6 protein of about 57.2 kDa was obtained.

[0025] Example 2: Demonstration of the effect of yak DDX6 protein on yak embryonic fibroblasts

[0026] Primary yak embryonic fibroblasts isolated under sterile conditions were cultured in DEME culture medium supplemented with 0, 0.1, 1, 5, and 10 μg / mL of DDX6 protein at 37°C for 24 h and 72 h, and the effect of DDX6 protein on cell proliferation was detected using the CCK-8 method. Figure 1 As shown, the results showed that adding DDX6 at a final concentration of 1 μg / mL and culturing for 72 hours could significantly increase the viability of yak embryonic fibroblasts, and adding DDX6 at a concentration of 5 μg / mL and above would inhibit the viability of fibroblasts.

[0027] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A DDX6 protein, characterized in that: The coding gene sequence of the protein is shown in SEQ ID NO:

1.

2. A DDX6 protein, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. Use of the DDX6 protein according to claim 1 or 2 in improving fibroblast viability and / or promoting fibroblast growth and / or promoting fibroblast proliferation.

4. The application according to claim 3, characterized in that: The fibroblasts are derived from yaks.

5. The application according to claim 3, characterized in that: The application method is: co-culturing the fibroblasts and DDX6.

6. The application according to claim 5, characterized in that: In the co-culture system, the DDX6 concentration was <5 μg / mL.

7. Use of the DDX6 protein according to claim 1 or 2 in reducing fibroblast viability and / or inhibiting fibroblast growth and / or promoting fibroblast proliferation.

8. The application according to claim 7, characterized in that: The fibroblasts are derived from yaks.

9. The use according to claim 6, characterized in that: The application comprises the following steps: the application method is: co-culturing the fibroblasts with DDX6.

10. The use according to claim 5, characterized in that: In the co-culture system, the DDX6 concentration was ≥5 μg / mL.

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