CN106519002A - GRP78 truncated gene and application thereof - Google Patents

Abstract

The invention discloses a GRP78 truncation body and its application. The amino acid sequence of GRP78 truncated body is shown in SEQ ID NO.1. The invention utilizes bioengineering technology to genetically recombine a DNA sequence corresponding to a GRP78 truncated polypeptide into a p3*flag-cmv-13-14 eukaryotic expression vector. After enzyme digestion and sequence analysis proved that the recombination was successful, the eukaryotic expression recombinant polypeptide was transfected into HepG2 cells, and immunoblotting proved the protein expression of the polypeptide, and the recombination of the polypeptide was realized. Then, the improvement of IR by the polypeptide was studied. Cytological experiments showed that the polypeptide has an important function of improving IR, and it will be widely used in the preparation of drugs for improving IR.

Description

GRP78 truncated body and its application

technical field

The invention relates to the technical field of bioengineering, in particular to a GRP78 truncated body and its application.

Background technique

Type II diabetes is a common disease with a high incidence in my country and the world. In my country, type II diabetes has become the fastest growing disease. The latest data show that there are more than 60 million diabetic patients in my country, accounting for one-fifth of the global diabetic patients. It is estimated that the total number of diabetic patients in my country will be close to 100 million by 2025.

Type II diabetes is a metabolic disease with multiple etiologies. In addition to internal genetic factors, it also has a great relationship with people's living environment, lifestyle and behavior. In particular, some metabolic disorders caused by excessive obesity can easily lead to Type II diabetes. Type II diabetes, also known as non-insulin-dependent diabetes, refers to the resistance of muscle and fat tissue to insulin. As compensation, the pancreatic beta cells secrete more insulin, but they still cannot maintain blood sugar within the normal range. In addition, excessive insulin secretion damages the function of pancreatic β cells, resulting in insufficient insulin secretion. Insulin resistance refers to a pathophysiological state in which the responsiveness of the body's target tissue to insulin is lower than normal. It is often accompanied by clinical diseases such as obesity and the early stage of type II diabetes. It is an important feature of these diseases and is also the cause of these diseases. important factors that occur. But the molecular mechanisms that cause insulin resistance and the resulting obesity, type II diabetes and other related diseases are not very clear. This makes its prevention and treatment difficult.

Glucose Regulated Protein 78 (Glucose Regulated Protein, GRP78), which is located in the lumen of the endoplasmic reticulum, is an endoplasmic reticulum molecular chaperone related to the cell defense system. GRP78 is a hallmark protein of endoplasmic reticulum stress. Under normal conditions, three transmembrane proteins on the ER membrane, double-stranded RNA-dependent protein kinase-like ER kinase, activated transcription factor 6, and inositol-required kinase, bind to GRP78 and are in an inactive state; when not When folded or misfolded proteins accumulate in the lumen of the endoplasmic reticulum, GRP78 is dissociated and activated, enters the cytoplasm, its expression and stability are significantly increased, and it mediates different protein processing procedures through different pathways, participating in Endoplasmic reticulum stress process.

Studies have shown that compared with male GRP78 ^+/+ mice, male GRP78 ^+/- mice can increase their energy consumption levels, thereby alleviating high-fat induced diet (High-fat Induced Diet, HFD)-induced obesity, and It is resistant to diet-induced hyperglycemia, hyperinsulinemia, and hepatic steatosis, thereby alleviating insulin resistance and type II diabetes.

Contents of the invention

Purpose of the invention: Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a GRP78 truncation body, which has the important function of improving insulin resistance (Insulin Resistance, IR). Another object of the present invention is to provide the application of GRP78 truncation body.

Technical solution: In order to realize the above-mentioned purpose of the invention, the technical solution adopted in the present invention is:

The amino acid sequence of GRP78 truncated body is shown in SEQ ID NO.1.

The DNA sequence of the gene encoding the GRP78 truncation is shown in SEQ ID NO.2.

A vector containing the DNA sequence of the gene encoding the GRP78 truncation.

In the vector, the DNA sequence of the GRP78 truncated body is connected into the p3*flag-cmv-13-14 eukaryotic expression vector to construct a recombinant expression plasmid.

Application of the GRP78 truncated body in the preparation of medicines for improving IR.

Beneficial effects: Compared with the prior art, the present invention uses bioengineering technology to genetically recombine a DNA sequence corresponding to a GRP78 truncated polypeptide into the p3*flag-cmv-13-14 eukaryotic expression vector. After enzyme digestion and sequence analysis proved that the recombination was successful, the eukaryotic expression recombinant polypeptide was transfected into HepG2 cells, and immunoblotting proved the protein expression of the polypeptide, and the recombination of the polypeptide was realized. Then, the improvement of IR by the polypeptide was studied. Cytological experiments showed that the polypeptide has an important function of improving IR, and it will be widely used in the preparation of drugs for improving IR.

Description of drawings

Figure 1 is a diagram of the Western blot protein expression results of the DNA sequence corresponding to the GRP78 truncated polypeptide, with a size of 40KD;

Figure 2 is a graph showing the protein expression levels of insulin resistance indicators p-AKT and p-GSK3β detected by Western blot analysis;

Fig. 3 is a graph showing protein expression levels of endoplasmic reticulum stress-related pathway molecules detected by Western blot analysis.

detailed description

The present invention will be further described below in conjunction with specific embodiments. The experimental method that does not indicate specific condition in the embodiment, usually according to conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide (the fourth edition, M.R. Green, J. Sambrook book, Science Press, 2013), Or follow the conditions recommended by the manufacturer.

Example 1

1) GRP78 truncated polypeptide recombination

Extract human mRNA, reverse transcribe it into cDNA, use cDNA as template, apply PCR technology (primer sequence: 5'-AAGCTT ATG GAG GTA GAA AAG GCC AAA CG-3', 5'-CTC GAG CAA CTC ATC TTT TTC TGC TGT -3', reaction conditions: 95°C, 5min→95°C, 30s→55°C, 20s→72°C, 1min (total 30 cycles)→72°C, 1min/kb) successfully amplified GRP78 truncated body (SEQ ID NO.1) corresponding to the 1092bp mRNA sequence (SEQ ID NO.2). The amplified fragment was ligated with the p3*flag-cmv-13-14 vector, and the ligated product was transferred into competent Escherichia coli DH5α, and clones were selected on the Amp+ agar plate, and the recombinant plasmid was extracted by alkaline lysis method, and then ECOR1 was digested and identified and sequenced, and the verification results were correct.

2) Construction of polypeptide analog eukaryotic expression vector and its expression detection

After the p3*flag-cmv-13-14 plasmid containing the GRP78 truncated polypeptide nucleic acid fragment was digested by ECOR1, the fragment was obtained using the recovery kit, and the plasmid p3*flag-cmv-13-14 was treated with the same enzyme , and then ligate the recovered polypeptide nucleic acid fragment and the digested vector p3*flag-cmv-13-14 at 16° C. overnight under the action of T4 DNA ligase. Recombinants were identified by enzyme digestion. The correctly connected 364aa polypeptide eukaryotic expression vector was transfected into HepG2 cells, and samples were collected 48 hours later, lysed with RIPA cell lysate, and the results of immunoblotting were shown in Figure 1, which proved the expression of the polypeptide.

Example 2 Detection of Recombinant Peptide Improvement of Insulin Resistance Function

1) Analysis of the expression changes of AKT, p-AKT, GSK3β, p-GSK3β in IR HepG2 cells

①Cell model: HepG2 cells were first incubated in DMEM medium containing 0.25mm/L PA without serum for 24 hours; then incubated in culture medium containing 100nmol/L insulin for 15 minutes, and then the protein was extracted.

②Discard the culture medium for the cells, wash with PBS in an ice bath, and then add protein lysate to the culture dish (according to a 6 cm culture dish with 80-90% confluence, add 1 mL of lysate) to scrape the cells, extract the protein, and use Western blot was used to detect the protein expression levels of insulin signaling pathway-related molecules (AKT, p-AKT, GSK3β, p-GSK3β). The results are shown in Figure 2. After transfection of 364aa polypeptide in liver cancer cells HepG2, insulin resistance markers: The expression levels of p-AKT and p-GSK3β increased.

2) Analysis of the expression changes of GRP78 and CHOP in HepG2 cells of IR

① Cell model construction is the same as above.

②Discard the culture medium for the cells, wash with PBS in an ice bath, and then add protein lysate to the culture dish (according to a 6cm culture dish with 80-90% fusion degree, add 1ml of lysate) to scrape the cells, extract the protein, and use The protein expression level of endoplasmic reticulum stress-related pathways (GRP78, CHOP) was detected by western blot experiment. The results are shown in Figure 3. After transfection of 364aa polypeptide in liver cancer cell HepG2, the expression level of CHOP downstream of endoplasmic reticulum stress decline.

Claims (5)

1. GRP78 truncated body, the amino acid sequence of which is shown in SEQ ID NO.1. 2. The gene encoding the GRP78 truncated body according to claim 1, its DNA sequence is as shown in SEQ ID NO.2. 3. A carrier containing the DNA sequence of the gene encoding the GRP78 truncation body of claim 2. 4. The vector according to claim 3, characterized in that: the DNA sequence of the GRP78 truncated body is connected into the p3*flag-cmv-13-14 eukaryotic expression vector to construct a recombinant expression plasmid. 5. The application of the GRP78 truncated body according to claim 1 in the preparation of medicines for improving IR.