Insulin receptor protein expressing cell lines and uses thereof

Abstract

The present application belongs to the technical field of drug bioactivity detection, and particularly relates to a method for detecting the bioactivity of insulin by establishing a gene engineering cell strain of insulin receptor protein and detecting the change in the phosphorylation level of the insulin receptor after the cell strain is stimulated by insulin. Specifically, the cell strain expressing the INSR protein is CHO-K1-INSR-6B3, and after insulin binds with the insulin receptor, the insulin receptor is phosphorylated, and the activity of the insulin and the amount of the phosphorylated receptor are in a dose-effect relationship. The activity of the insulin can be quantitatively detected by detecting the phosphorylation level of the insulin receptor. The detection method is easy to standardize and has good repeatability, low cost, convenience and accuracy, and thus has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of drug bioactivity detection technology, specifically relating to the establishment of a genetically engineered cell line of insulin receptor protein, and a detection method for determining insulin bioactivity based on the change in the phosphorylation level of the insulin receptor after the cell line is stimulated by insulin. Background Technology

[0002] Insulin was first discovered in 1921 by Canadians F.G. Banting and C.H. Best. Its clinical use began in 1922, saving previously incurable diabetic patients. Until the early 1980s, the insulin used clinically by the Institute of Nephrology, Chinese Academy of Sciences, was almost entirely extracted from pig and bovine pancreas. Human insulin consists of two peptide chains, A and B. Chain A contains 21 amino acids of 11 types, and chain B contains 30 amino acids of 15 types, for a total of 51 amino acids of 16 types. Two disulfide bonds are formed between the sulfhydryl groups of the four cysteine ​​residues A7(Cys)-B7(Cys) and A20(Cys)-B19(Cys), linking the A and B chains. Additionally, a disulfide bond also exists between A6(Cys) and A11(Cys) in chain A.

[0003] In 1955, F. Sanger's group in the UK determined the complete amino acid sequence of bovine insulin, paving the way for human understanding of the chemical structure of protein molecules. In 1958, Chinese scientists Wang Yinglai, Zou Chenglu, and others began exploring the chemical synthesis of insulin. In September 1965, Chinese scholars completed the total synthesis of crystalline bovine insulin, becoming the world's first artificially synthesized protein. After 1982, the world's first artificially synthesized E. coli recombinant human insulin (Humulin) was launched, followed by yeast recombinant human insulin, which was widely used in the treatment of diabetes. Subsequently, glargine insulin, detemir insulin, aspart insulin, and insulin analogs were launched, ushering in a new era of insulin therapy for diabetes.

[0004] The human insulin receptor (IR) is a (αβ)2 homodimeric receptor tyrosine kinase that plays a crucial role in maintaining homeostasis and regulating lipid, protein, and carbohydrate metabolism within the glucose system. IR signaling also regulates neurotransmitter levels in the brain, and abnormal IR signaling is associated with type 2 diabetes, cancer, and Alzheimer's disease. The extracellular portion of the IR αβ monomer comprises two leucine-rich repeat domains (L1 and L2), a cysteine-rich region (CR), and three fibronectin type III domains. The insertion domain (ID) is located at FnIII-2 and contains a protease hydrolysis site for the αβ subunit. Intramolecular disulfide bonds link the α chain to the β chain, with (αβ)2 being a stable disulfide bond at two positions. The human insulin receptor precursor contains 1382 amino acids, of which positions 1-27 are the signal peptide, which is cleaved during processing; positions 28-758 are processed into the α subunit; positions 763-1382 are processed into the β subunit; positions 759-762 are the RKRR site, which is recognized and cleaved by the furin protease; positions 733-741 are the insulin binding site; positions 1361-1364 are the PIK3R1 binding site; and positions 1023-1298 are the cytoplasmic kinase sites.

[0005] The human insulin receptor precursor begins at the N-terminus with a leucine-rich repeat domain (L1, residues 1-157), a cysteine-rich region (CR, residues 158-310), a leucine-rich repeat domain (L2, residues 311-470), and three fibronectin type III domains: FnIII-1 (residues 471-595), FnIII-2 (residues 596-808), and FnIII-3 (residues 809-906). Additionally, an insertion domain (ID, residues 638-756) is located within the FnIII-2 domain, containing the α / β furin cleavage site. Hydrolysis of this protein results in the IDα and IDβ domains. The β-chain, FnIII-3 domain, is located within a transmembrane helix (TH) and intracellular juxtamembrane (JM) region, immediately following a tyrosine kinase (TK) catalytic domain responsible for subsequent intracellular signaling pathways.

[0006] The α subunit of the insulin receptor is entirely extracellular and contains the insulin-binding domain. The β subunit consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The intracellular domain contains receptor tyrosine kinases. After insulin binds to the receptor's binding site, it catalyzes the phosphorylation of the receptor itself, activating downstream receptor tyrosine kinases and initiating signal transduction.

[0007] With the development of modern science and technology, insulin has become more diverse, evolving from animal insulin to recombinant human insulin and finally to insulin analogs. Insulin analogs are third-generation insulins produced using genetic engineering, modifying the insulin structure or altering its physicochemical properties to better meet physiological needs. Currently, commonly used ultra-short-acting insulins such as aspart insulin and ultra-long-acting insulins such as glargine insulin and detemir insulin are all insulin analogs. In the current insulin research and development process, it is necessary to test insulin activity. Currently, the internationally accepted method for determining the biopotency of human insulin and its analogs or conjugates is the in vivo animal method, mainly including biopotency analysis methods based on changes in blood glucose concentration in mice and rabbits, such as the double-cross mouse method in the Chinese Pharmacopoeia and the rabbit hypoglycemic method in the United States Pharmacopeia. The target unit definition for insulin biopotency is: a blood glucose level of 2.5 mmol / L after injection of the minimum dose of human insulin in a rabbit is defined as 1 unit of potency. The biopotency of various human insulins and their preparations is expressed using this definition. During measurement, the potency is calculated by cross-comparison using insulin potency standards. In conventional preparations, each ml of injection is calculated as 1 unit. The bioactivity of insulin is indicated by units, not by mass. Since there is currently no effective and reliable in vitro cellular method for analyzing and detecting bioactivity in insulin raw materials and formulations, the mass of one known unit of bioactivity is often used to reflect bioactivity. For example, one unit of regular insulin is equivalent to 0.03846 mg, and one unit of detemir insulin is equivalent to 0.142 mg. Because insulin is a biological product, especially in formulations, factors such as physical and chemical changes can affect its stability and alter its bioactivity. Converting bioactivity solely by mass would result in significant errors. Therefore, a more accurate in vitro analytical method for determining the bioactivity or bioactivity of human insulin and its formulations is needed.

[0008] The methods for determining the in vitro potency of human insulin reported in domestic and international literature include the rat primary mature adipocyte method and the 3T3-L1 preadipocyte induced adipocyte method. The rat primary mature adipocyte method uses 3H-labeled glucose to detect the glucose utilization rate of mature adipocytes and measures the intracellular isotope to analyze the biopotency. However, because primary mature adipocytes are easily broken, they cannot be effectively counted and have poor reproducibility, this method has only been reported in Novo Nordisk's literature on the quality research of detemir insulin. The 3T3-L1 preadipocyte induced adipocyte method has been limited to insulin research papers due to its low induction rate, easy cell shedding during induction, and uneven distribution of successfully induced adipocytes between wells.

[0009] Chinese patent CN105092490A discloses a method for inducing primary preadipocytes to differentiate into adipocytes, using the glucose oxidase method to determine the amount of glucose consumed in the culture medium of adipocytes under the action of insulin, and calculating the corresponding insulin biopotency. This method requires in vitro induction of preadipocytes, which involves the dissection of rats, extraction of preadipocytes, and primary culture. The operation is cumbersome, and the method has low sensitivity and significant variability.

[0010] There is still a need to develop simpler, lower-cost detection methods. Summary of the Invention

[0011] In view of the shortcomings of the prior art, the purpose of this invention is to provide a genetically engineered cell line of insulin receptor protein and a method for measuring insulin activity using the cell line, which is simpler and less expensive.

[0012] This invention constructs a cell line that highly expresses the insulin receptor. After insulin binds to the insulin receptor, the insulin receptor is phosphorylated. The Insulin Receptor Phosphorylation Detection Kit (CISBIO) can quantitatively detect the amount of phosphorylated insulin receptors. There is a dose-response relationship between insulin activity and the amount of phosphorylated receptors. By detecting the phosphorylation level of the insulin receptor, the activity of insulin can be quantitatively detected.

[0013] This invention provides a genetically engineered cell line, CHO-K1-INSR-6B3, for in vitro determination of insulin bioactivity. The working principle of the CHO-K1-INSR-6B3 cell line is as follows: The CHO-K1-INSR-6B3 cell line stably expresses the INSR protein. When this receptor protein is acted upon by insulin, insulin binds to the insulin receptor, and the downstream β-receptor is phosphorylated at positions Tyr1150 / 1151. The phosphorylation level of the β-receptor is positively correlated with the bioactivity of insulin in the stimulant. The bioactivity of insulin in unknown samples is determined by comparing it with that of insulin standards.

[0014] To construct a CHO-K1 cell line that can stably express INSR, this invention achieves this through the following technical solution.

[0015] In a first aspect, this invention provides a CHO-K1-INSR-6B3 cell line with high expression of the insulin receptor. The CHO-K1-INSR-6B3 cell line is classified as Chinese hamster ovary cells and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 19200 and deposit date of December 11, 2019. The CHO-K1-INSR-6B3 cell line was obtained by transfecting CHO-K1 cells with the insulin receptor expression vector, abbreviated as PGS2-INSR.

[0016] In a second aspect, the present invention provides a nucleotide sequence for encoding a human INSR protein, as shown in SEQ ID NO:2.

[0017] A third aspect of the present invention provides an expression vector comprising the nucleotide sequence described in the second aspect.

[0018] This invention also provides a method for constructing a recombinant expression plasmid expressing human INSR protein in eukaryotic cells. The specific construction method is as follows: The recombinant plasmid PUC57-INSR containing the INSR gene and the eukaryotic expression plasmid were digested with restriction endonucleases Hind III and ECOR I. The target fragment was recovered, ligated using T4 ligase, and incubated overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells, screened using LB medium containing kanamycin, and single clones were selected. The clones were cultured, and a small amount of plasmid was extracted and preliminarily identified by double enzyme digestion. The correctly identified recombinant plasmids were extracted in large quantities and sent to Shanghai Sangon Biotech for sequencing identification.

[0019] In a fourth aspect, the present invention provides a method for constructing a eukaryotic cell line expressing INSR protein.

[0020] Specifically, CHO-K1 cells were seeded in 6-well plates one day before transfection, and the cell density was adjusted. The linearized PGS2-INSR recombinant plasmid was mixed with Freestyle™ MAX transfection reagent in OptiPRO™ SFM medium and then added to the CHO-K1 culture wells for gene transfection. CHO-K1 cell lines with high INSR expression were selected under pressure.

[0021] A fifth aspect of the invention provides the use of the cell line in the determination of the bioactivity of human insulin or analogues.

[0022] Preferably, the human insulin includes insulin aspart, insulin detemir, insulin lispro, insulin lisgluten, insulin glargine, insulin degludec, and mixed recombinant human insulin injection.

[0023] In a sixth aspect, the present invention provides a method for determining insulin bioactivity based on the phosphorylation level of the β receptor in the CHO-K1-INSR-6B3 cell line.

[0024] Using insulin stimulation of growing CHO-K1-INSR-6B3 cells, and employing a kit provided by CISbio, the changes in β-receptor phosphorylation levels in CHO-K1-INSR-6B3 cells stimulated with agonists were measured to observe whether the insulin receptor expressed on the CHO-K1-INSR-6B3 cell membrane possesses biological activity. If the insulin receptor expressed on the CHO-K1-INSR-6B3 cell membrane is biologically active, stimulation with insulin agonists will enhance the physiological metabolic activity of CHO-K1-INSR-6B3 cells, increasing energy consumption. After activation of the intracellular insulin receptor, the intracellular β-receptor phosphorylation level will significantly increase, while in CHO-K1-INSR-6B3 cells that do not express insulin receptors, the intracellular β-receptor phosphorylation level does not change significantly after stimulation with insulin agonists.

[0025] Specifically, human insulin samples were added to the culture medium of the cell line. Different insulin samples were used to stimulate growing CHO-K1-INSR-6B3 cells. Changes in β-receptor phosphorylation were measured within 0.5–3 hours. An EC50 value was calculated by plotting concentration on the x-axis and signal value on the y-axis. A standard curve was then constructed based on the β-receptor phosphorylation levels after stimulation of CHO-K1-INSR-6B3 cells with the standard, thereby determining the biological activity of insulin in unknown samples.

[0026] The beneficial effects of this invention are as follows: A detection method for determining insulin bioactivity in CHO-K1-INSR-6B3 cell line after exposure to external stimuli has been established. This method is easy to standardize, has good repeatability, and is low-cost, convenient, and accurate, thus showing good application prospects. Attached Figure Description

[0027] Figure 1 : PGS2-INSR vector structure map;

[0028] Figure 2 : Detemir insulin dose-response results;

[0029] Figure 3 Aspart insulin dose-response results;

[0030] Figure 4 : Dose-response results of glargine insulin. Detailed Implementation

[0031] Example 1: Synthesis and Construction of INSR Expression Vector

[0032] 1. Amplification and identification of the INSR gene

[0033] The human INSR protein sequence, as shown in SEQ ID NO: 1, is 1382 amino acids in length. Sequence optimization and full-length DNA synthesis were outsourced. HindIII restriction sites and a kozak sequence were added to the N-terminus of the nucleotide sequence encoding the human INSR protein, and TGA and ECORI terminators were added to the C-terminus. The optimized full-length nucleotide sequence is shown in SEQ ID NO: 2. After full-sequence gene synthesis, the DNA was ligated into the PUC-57 vector to construct the PUC57-INSR vector.

[0034] Both the PUC57-INSR plasmid and the PGS2 vector were digested with restriction endonucleases HindIII and ECORI, and the target fragment was recovered and ligated using T4 ligase overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells, and single clones were selected using solid LB medium containing kanamycin. Plasmids were cultured and extracted in small quantities, and preliminarily identified by double enzyme digestion. The correctly identified recombinant plasmid was extracted in large quantities and sent to an external unit for sequencing identification. The sequencing confirmed the plasmid was correct. The structure of the PGS2-INSR vector is shown below. Figure 1 As shown. The correctly sequenced plasmid PVUI was linearized, then precipitated with ethanol and stored at -20℃ for later use.

[0035] 2. Construction of INSR-overexpressing cell lines

[0036] CHO-K1 cells were transfected with the linearized plasmid PGS2-INSR using a liposome transfection method (Freestyle MAX, Invitrogen), and protein expression was detected by flow cytometry. Plasmid transfection was performed according to the Freestyle™ MAX transfection reagent instructions. The plasmid was administered at a concentration of 0.5 × 10⁻⁶ cells / day prior to transfection with the reagent. 6 Pass cells per ml, count cells on the day of transfection, and adjust cell density to 1×10⁹ / ml. 6 / ml, cultured at 37℃. Then, prepare the transfection solution by adding 50ug of linearized plasmid PGS2-INSR to OptiPRO™ SFM medium to a final volume of 1.5ml, and 50ul of Freestyle™ MAX transfection reagent to 1.45ml of OptiPRO™ SFM medium. After mixing well, slowly add the transfection reagent mixture to the DNA mixture, mix well, and let stand at room temperature for 10 minutes. Then, slowly add the transfection mixture dropwise to 30ml of CHO-K1 cells. Incubate at 37℃ and 130rpm with 8% CO2. 48h after transfection, pressurize with 50um MSX. After a certain pressure selection, cells that did not integrate successfully died, and cells that successfully integrated recovered to 90% viability. Four vials were cryopreserved; and a portion of the cells were transfected at 0.3×10 6 30 ml of the solution was inoculated per ml for flow cytometry detection of cell monoclonalization.

[0037] Cells in logarithmic growth phase were diluted to 2 cells / ml, and 200 μL of cells were seeded into each well of a 96-well plate. After 7 days, single clones were observed under a microscope and labeled. After cell growth, the culture was expanded and flow cytometry was performed to detect the expression of INSR on the cell surface. Untransfected CHO-K1 cells were used as negative cells, and transfected cells were used as test cells. After washing with PBS, PE-labeled INSR antibody (Sinochem) was added and incubated. After washing, flow cytometry was used to detect and screen CHO-K1 cells with high INSR expression: CHO-K1-INSR-6B3 cell line, CHO-K1-INSR-2A4 cell line, and CHO-K1-INSR-3B1 cell line.

[0038] Example 2: Analysis of INSR activity expression in CHO-K1-INSR-6B3, CHO-K1-INSR-2A4, and CHO-K1-INSR-3B1 cells.

[0039] Insulin and its analogues, upon binding to the insulin receptor, phosphorylate the Tyr1150 / 1151 position of the downstream β-receptor. CISbio's kits specifically detect phosphorylated β-receptors using a sandwich ELISA method. One antibody labels an Eu3+ cryptoid, and another labels d2. Insulin and its analogues phosphorylate upon receptor binding; higher activity corresponds to higher phosphorylation levels. After cell lysis, the phosphorylated β-receptor is released. The Eu3+ cryptoid-labeled antibody (donor) and the d2-labeled antibody (receptor) specifically bind to the phosphorylated β-receptor. When these two antibodies bind to the phosphorylated β-receptor, and the dyes are very close, a light source (laser or flash lamp) excites the donor to emit fluorescence resonance energy transfer (FRET) to the receptor, causing the receptor to emit fluorescence at a specific wavelength (665 nm). The signal intensity is proportional to the phosphorylation level of the insulin receptor.

[0040] CHO-K1-INSR-6B3, CHO-K1-INSR-2A4, and CHO-K1-INSR-3B1 cells were suspended in PBS at a concentration of 5 × 10⁶ cells / mL. 5 50 μL / well was added to a 96-well plate and the cells were starved for 2 hours. Pre-diluted insulin (10 μL / well) was added to the starved cells and incubated for 20 minutes. Immediately after stimulation, 20 μL / well of a lysis working solution (a 1:24 mixture of blocking reagent and 4X lysis buffer) was added and the cells were incubated at room temperature for 30 minutes at 150 rpm. After lysis, the cells were mixed thoroughly, and 16 μL was transferred to a 96-well shallow plate. Then, 4 μL of the pre-mixed antibody working solution was added and the cells were incubated at room temperature for 2 hours in the dark at 150 rpm. The plates were read using the TRF method with a BMG multi-plate reader. A standard curve was plotted, and the EC50 value was calculated. The results are shown in Table 1. The β-receptor phosphorylation level was significantly increased in CHO-K1-INSR-6B3, CHO-K1-INSR-2A4, and CHO-K1-INSR-3B1 cells, while no dose-response trend was observed in CHO cells not transfected with the INSR gene. Experimental results confirmed that the INSR proteins expressed by recombinant CHO-K1-INSR-6B3, CHO-K1-INSR-2A4, and CHO-K1-INSR-3B1 cells have similar physiological activities to natural INSR proteins and are specific.

[0041] Meanwhile, the CV (%) of the EC50 of recombinant human insulin in three cell lines—CHO-K1-INSR-6B3, CHO-K1-INSR-2A4, and CHO-K1-INSR-3B1—were 1.507%, 4.504%, and 8.453%, respectively, all less than 10.0%. Specifically, the CV (%) of the EC50 of recombinant human insulin in CHO-K1-INSR-6B3 was less than 2.0%; the 95% confidence intervals (X±SD) were 17.988±0.337 ng / ml, 12.210±0.683 ng / ml, and 19.210±2.016 ng / ml, respectively; and the SDs were 0.271, 0.550, and 1.624, respectively. CHO-K1-INSR-6B3 showed a small error range and good stability after five measurements. Furthermore, the average half-acting dose (EC50) of the recombinant human insulin potency standard was determined to be 17.697 ng / ml using existing technology, and the results obtained by CHO-K1-INSR-6B3 cells for the recombinant human insulin potency standard were basically consistent with these results. This demonstrates that the CHO-K1-INSR-6B3 cell line can be used to determine the bioactivity of insulin. This cell line is classified and named Chinese hamster ovary cells, with accession number CGMCC No: 19200.

[0042] Table 1. Half-response dose data from five replicate experiments of different cell lines.

[0043]

[0044] Example 3: Detection of insulin bioactivity

[0045] The detection was performed using the IR beta phosphor-Y1150 / 1151 kit. CHO-K1-INSR-6B3 cells were suspended in PBS at a density of 5 × 10⁶ cells / mL. 550 μL / well was added to a 96-well plate and the cells were starved for 2 hours. Different insulins (insulin aspart (300 units / 3 ml / vial), insulin glargine (300 units / 3 ml / vial), and insulin detemir (300 units / 3 ml / vial)) were diluted 2-fold with PBS and added to each well at 10 μL / well. The cells were incubated for 20 min. Immediately after stimulation, 20 μL / well of a lysis working solution (a 1:24 mixture of blocking reagent and 4× lysis buffer) was added and the cells were incubated at room temperature for 30 min at 150 rpm. After lysis, the cells were mixed thoroughly, and 16 μL was transferred to a 96-well shallow plate. Then, 4 μL of pre-mixed antibody working solution (both working antibodies and detection buffer were mixed at a 1:19 ratio; the diluted working antibodies were then mixed again at a 1:1 ratio before use) was added and the plates were incubated at room temperature in the dark for 2 hours at 150 rpm. The plates were read using a BMG multi-plate reader via TRF method. A standard curve was plotted, and the EC50 value was calculated. The results are shown below. Figure 2 , Figure 3 , Figure 4 As shown in Table 2, the results indicated a dose-response relationship between the β-receptor phosphorylation levels produced by different concentrations of insulin stimulating CHO-K1-INSR-6B3 cells. Using the same method, the β-receptor phosphorylation levels produced by CHO-K1-INSR-6B3 cells stimulated with insulin aspart, insulin glargine, and insulin detemir (samples with unknown biological activity) were measured, and the biological activity of insulin in the unknown samples was determined using a standard curve.

[0046] Table 2. Half-effect response dose data from 5 repeated trials

[0047]

[0048]

[0049] The results showed that the CV (%) of the EC50 of detemir insulin, aspart insulin, and glargine insulin were 2.223%, 1.143%, and 1.513%, respectively, all less than 5.0%. The 95% confidence intervals (X±SD) were 7.723±0.213 ng / ml, 25.951±0.368 ng / ml, and 27.982±0.526 ng / ml, respectively. sequence list <110> Lunan Pharmaceutical Group Co., Ltd. <120> Insulin receptor protein expression cell lines and their applications <130> 1 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1382 <212> PRT <213> Artificial Sequence <400> 1 Met Ala Thr Gly Gly Arg Arg Gly Ala Ala Ala Ala Pro Leu Leu Val 1 5 10 15 Ala Val Ala Ala Leu Leu Leu Gly Ala Ala Gly His Leu Tyr Pro Gly 20 25 30 Glu Val Cys Pro Gly Met Asp Ile Arg Asn Asn Leu Thr Arg Leu His 35 40 45 Glu Leu Glu Asn Cys Ser Val Ile Glu Gly His Leu Gln Ile Leu Leu 50 55 60 Met Phe Lys Thr Arg Pro Glu Asp Phe Arg Asp Leu Ser Phe Pro Lys 65 70 75 80 Leu Ile Met Ile Thr Asp Tyr Leu Leu Leu Phe Arg Val Tyr Gly Leu 85 90 95 Glu Ser Leu Lys Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Ser 100 105 110 Arg Leu Phe Phe Asn Tyr Ala Leu Val Ile Phe Glu Met Val His Leu 115 120 125 Lys Glu Leu Gly Leu Tyr Asn Leu Met Asn Ile Thr Arg Gly Ser Val 130 135 140 Arg Ile Glu Lys Asn Asn Glu Leu Cys Tyr Leu Ala Thr Ile Asp Trp 145 150 155 160 Ser Arg Ile Leu Asp Ser Val Glu Asp Asn Tyr Ile Val Leu Asn Lys 165 170 175 Asp Asp Asn Glu Glu Cys Gly Asp Ile Cys Pro Gly Thr Ala Lys Gly 180 185 190 Lys Thr Asn Cys Pro Ala Thr Val Ile Asn Gly Gln Phe Val Glu Arg 195 200 205 Cys Trp Thr His Ser His Cys Gln Lys Val Cys Pro Thr Ile Cys Lys 210 215 220 Ser His Gly Cys Thr Ala Glu Gly Leu Cys Cys His Ser Glu Cys Leu 225 230 235 240 Gly Asn Cys Ser Gln Pro Asp Asp Pro Thr Lys Cys Val Ala Cys Arg 245 250 255 Asn Phe Tyr Leu Asp Gly Arg Cys Val Glu Thr Cys Pro Pro Pro Tyr 260 265 270 Tyr His Phe Gln Asp Trp Arg Cys Val Asn Phe Ser Phe Cys Gln Asp 275 280 285 Leu His His Lys Cys Lys Asn Ser Arg Arg Gln Gly Cys His Gln Tyr 290 295 300 Val Ile His Asn Asn Lys Cys Ile Pro Glu Cys Pro Ser Gly Tyr Thr 305 310 315 320 Met Asn Ser Ser Asn Leu Leu Cys Thr Pro Cys Leu Gly Pro Cys Pro 325 330 335 Lys Val Cys His Leu Leu Glu Gly Glu Lys Thr Ile Asp Ser Val Thr 340 345 350 Ser Ala Gln Glu Leu Arg Gly Cys Thr Val Ile Asn Gly Ser Leu Ile 355 360 365 Ile Asn Ile Arg Gly Gly Asn Asn Leu Ala Ala Glu Leu Glu Ala Asn 370 375 380 Leu Gly Leu Ile Glu Glu Ile Ser Gly Tyr Leu Lys Ile Arg Arg Ser 385 390 395 400 Tyr Ala Leu Val Ser Leu Ser Phe Phe Arg Lys Leu Arg Leu Ile Arg 405 410 415 Gly Glu Thr Leu Glu Ile Gly Asn Tyr Ser Phe Tyr Ala Leu Asp Asn 420 425 430 Gln Asn Leu Arg Gln Leu Trp Asp Trp Ser Lys His Asn Leu Thr Ile 435 440 445 Thr Gln Gly Lys Leu Phe Phe His Tyr Asn Pro Lys Leu Cys Leu Ser 450 455 460 Glu Ile His Lys Met Glu Glu Val Ser Gly Thr Lys Gly Arg Gln Glu 465 470 475 480 Arg Asn Asp Ile Ala Leu Lys Thr Asn Gly Asp Gln Ala Ser Cys Glu 485 490 495 Asn Glu Leu Leu Lys Phe Ser Tyr Ile Arg Thr Ser Phe Asp Lys Ile 500 505 510 Leu Leu Arg Trp Glu Pro Tyr Trp Pro Pro Asp Phe Arg Asp Leu Leu 515 520 525 Gly Phe Met Leu Phe Tyr Lys Glu Ala Pro Tyr Gln Asn Val Thr Glu 530 535 540 Phe Asp Gly Gln Asp Ala Cys Gly Ser Asn Ser Trp Thr Val Val Asp 545 550 555 560 Ile Asp Pro Pro Leu Arg Ser Asn Asp Pro Lys Ser Gln Asn His Pro 565 570 575 Gly Trp Leu Met Arg Gly Leu Lys Pro Trp Thr Gln Tyr Ala Ile Phe 580 585 590 Val Lys Thr Leu Val Thr Phe Ser Asp Glu Arg Arg Thr Tyr Gly Ala 595 600 605 Lys Ser Asp Ile Ile Tyr Val Gln Thr Asp Ala Thr Asn Pro Ser Val 610 615 620 Pro Leu Asp Pro Ile Ser Val Ser Asn Ser Ser Ser Gln Ile Ile Leu 625 630 635 640 Lys Trp Lys Pro Pro Ser Asp Pro Asn Gly Asn Ile Thr His Tyr Leu 645 650 655 Val Phe Trp Glu Arg Gln Ala Glu Asp Ser Glu Leu Phe Glu Leu Asp 660 665 670 Tyr Cys Leu Lys Gly Leu Lys Leu Pro Ser Arg Thr Trp Ser Pro Pro 675 680 685 Phe Glu Ser Glu Asp Ser Gln Lys His Asn Gln Ser Glu Tyr Glu Asp 690 695 700 Ser Ala Gly Glu Cys Cys Ser Cys Pro Lys Thr Asp Ser Gln Ile Leu 705 710 715 720 Lys Glu Leu Glu Glu Ser Ser Phe Arg Lys Thr Phe Glu Asp Tyr Leu 725 730 735 His Asn Val Val Phe Val Pro Arg Lys Thr Ser Ser Gly Thr Gly Ala 740 745 750 Glu Asp Pro Arg Pro Ser Arg Lys Arg Arg Ser Leu Gly Asp Val Gly 755 760 765 Asn Val Thr Val Ala Val Pro Thr Val Ala Ala Phe Pro Asn Thr Ser 770 775 780 Ser Thr Ser Val Pro Thr Ser Pro Glu Glu His Arg Pro Phe Glu Lys 785 790 795 800 Val Val Asn Lys Glu Ser Leu Val Ile Ser Gly Leu Arg His Phe Thr 805 810 815 Gly Tyr Arg Ile Glu Leu Gln Ala Cys Asn Gln Asp Thr Pro Glu Glu 820 825 830 Arg Cys Ser Val Ala Ala Tyr Val Ser Ala Arg Thr Met Pro Glu Ala 835 840 845 Lys Ala Asp Asp Ile Val Gly Pro Val Thr His Glu Ile Phe Glu Asn 850 855 860 Asn Val Val His Leu Met Trp Gln Glu Pro Lys Glu Pro Asn Gly Leu 865 870 875 880 Ile Val Leu Tyr Glu Val Ser Tyr Arg Arg Tyr Gly Asp Glu Glu Leu 885 890 895 His Leu Cys Val Ser Arg Lys His Phe Ala Leu Glu Arg Gly Cys Arg 900 905 910 Leu Arg Gly Leu Ser Pro Gly Asn Tyr Ser Val Arg Ile Arg Ala Thr 915 920 925 Ser Leu Ala Gly Asn Gly Ser Trp Thr Glu Pro Thr Tyr Phe Tyr Val 930 935 940 Thr Asp Tyr Leu Asp Val Pro Ser Asn Ile Ala Lys Ile Ile Ile Gly 945 950 955 960 Pro Leu Ile Phe Val Phe Leu Phe Ser Val Val Ile Gly Ser Ile Tyr 965 970 975 Leu Phe Leu Arg Lys Arg Gln Pro Asp Gly Pro Leu Gly Pro Leu Tyr 980 985 990 Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Ser Asp Val Phe Pro Cys 995 1000 1005 Ser Val Tyr Val Pro Asp Glu Trp Glu Val Ser Arg Glu Lys Ile Thr 1010 1015 1020 Leu Leu Arg Glu Leu Gly Gln Gly Ser Phe Gly Met Val Tyr Glu Gly 1025 1030 1035 1040 Asn Ala Arg Asp Ile Ile Lys Gly Glu Ala Glu Thr Arg Val Ala Val 1045 1050 1055 Lys Thr Val Asn Glu Ser Ala Ser Leu Arg Glu Arg Ile Glu Phe Leu 1060 1065 1070 Asn Glu Ala Ser Val Met Lys Gly Phe Thr Cys His His Val Val Arg 1075 1080 1085 Leu Leu Gly Val Val Ser Lys Gly Gln Pro Thr Leu Val Val Met Glu 1090 1095 1100 Leu Met Ala His Gly Asp Leu Lys Ser Tyr Leu Arg Ser Leu Arg Pro 1105 1110 1115 1120 Glu Ala Glu Asn Asn Pro Gly Arg Pro Pro Pro Thr Leu Gln Glu Met 1125 1130 1135 Ile Gln Met Ala Ala Glu Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala 1140 1145 1150 Lys Lys Phe Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Val Ala 1155 1160 1165 His Asp Phe Thr Val Lys Ile Gly Asp Phe Gly Met Thr Arg Asp Ile 1170 1175 1180 Tyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Lys Gly Leu Leu Pro Val 1185 1190 1195 1200 Arg Trp Met Ala Pro Glu Ser Leu Lys Asp Gly Val Phe Thr Thr Ser 1205 1210 1215 Ser Asp Met Trp Ser Phe Gly Val Val Leu Trp Glu Ile Thr Ser Leu 1220 1225 1230 Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val Leu Lys Phe 1235 1240 1245 Val Met Asp Gly Gly Tyr Leu Asp Gln Pro Asp Asn Cys Pro Glu Arg 1250 1255 1260 Val Thr Asp Leu Met Arg Met Cys Trp Gln Phe Asn Pro Lys Met Arg 1265 1270 1275 1280 Pro Thr Phe Leu Glu Ile Val Asn Leu Leu Lys Asp Asp Leu His Pro 1285 1290 1295 Ser Phe Pro Glu Val Ser Phe Phe His Ser Glu Glu Asn Lys Ala Pro 1300 1305 1310 Glu Ser Glu Glu Leu Glu Met Glu Phe Glu Asp Met Glu Asn Val Pro 1315 1320 1325 Leu Asp Arg Ser Ser His Cys Gln Arg Glu Glu Ala Gly Gly Arg Asp 1330 1335 1340 Gly Gly Ser Ser Leu Gly Phe Lys Arg Ser Tyr Glu Glu His Ile Pro 1345 1350 1355 1360 Tyr Thr His Met Asn Gly Gly Lys Lys Asn Gly Arg Ile Leu Thr Leu 1365 1370 1375 Pro Arg Ser Asn Pro Ser 1380 <210> 2 <211> 4170 <212> DNA <213> Artificial Sequence <400> 2 aagcttgccg ccaccatggc tacaggaggc aggcggggag ctgctgctgc tccactgctg 60 gtggccgtgg ccgctctgct gctgggagct gctggacacc tgtaccctgg cgaggtgtgc 120 ccaggcatgg acatccggaa caatctgacc agactgcacg agctggagaa ctgcagcgtg 180 atcgagggcc atctgcagat cctgctgatg ttcaagacac gccctgagga ctttagggat 240 ctgtctttcc caaagctgat catgatcacc gactacctgc tgctgtttag agtgtatggc 300 ctggagagcc tgaaggatct gttccctaac ctgaccgtga tcaggggctc tcggctgttc 360 tttaattacg ctctggtcat ctttgagatg gtgcatctga aggagctggg cctgtataac 420 ctgatgaata tcacacgcgg ctctgtgagg atcgagaaga acaatgagct gtgctacctg 480 gccaccatcg actggtccag gatcctggac agcgtggagg ataactatat cgtgctgaac 540 aaggacgata atgaggagtg cggcgatatc tgtcccggca ccgctaaggg caagacaaac 600 tgtcctgcca ccgtgatcaa tggccagttc gtggagcggt gctggaccca ctcccattgc 660 cagaaggtgt gccctacaat ctgcaagagc cacggatgca ccgctgaggg actgtgctgt 720 cattctgagt gtctgggcaa ctgctcccag cccgacgatc ctacaaagtg cgtggcctgt 780 aggaacttct acctggacgg caggtgcgtg gagacctgcc cacctccata ctatcacttc caggactggc gctgcgtgaa cttctctttt tgccaggatc tgcaccataa gtgtaagaac tccagacgcc agggctgcca ccagtacgtg atccataaca ataagtgtat cccagagtgc ccctccggct atacaatgaa ctccagcaat ctgctgtgca ccccatgtct gggcccatgt cccaaggtgt gccatctgct ggagggcgag aagacaatcg attctgtgac ctccgctcag 1080 gagctgagag gctgtacagt gatcaacggc agcctgatca tcaatatccg cggcggcaac aatctggccg ctgagctgga ggctaacctg ggcctgatcg aggagatcag cggctacctg aagatcaggc ggtcttatgc cctggtgagc ctgtctttct ttagaaagct gcgcctgatc aggggcgaga ccctggagat cggcaattac tctttctatg ccctggacaa ccagaatctg aggcagctgt gggattggtc caagcacaac ctgaccatca cacagggcaa gctgttcttt 1380 cactacaatc ccaagctgtg cctgtccgag atccataaga tggaggaggt gagcggcaca 1440 aagggccggc aggagagaaa cgacatcgct ctgaagacca atggcgatca ggccagctgc 1500 gagaacgagc tgctgaagtt ttcttacatc agaacctcct tcgacaagat cctgctgcgc 1560 tgggagccat attggccccc tgactttcgg gatctgctgg gctttatgct gttctacaag 1620 gaggctccct atcagaacgt gacagagttc gacggccagg atgcctgcgg ctccaatagc 1680 tggaccgtgg tggacatcga tccacccctg aggagcaacg accccaagtc tcagaatcac 1740 cctggatggc tgatgagggg cctgaagcct tggacacagt acgccatctt tgtgaagacc 1800 ctggtgacat tctctgatga gagacgcacc tacggcgcta agtccgacat catctatgtg 1860 cagaccgatg ccacaaaccc ttctgtgcca ctggacccca tctccgtgtc caactcctcc 1920 agccagatca tcctgaagtg gaagcctcca tctgatccaa acggcaatat cacacattac 1980 ctggtgtttt gggagaggca ggctgaggac tccgagctgt tcgagctgga ttattgtctg 2040 aagggcctga agctgccatc caggacctgg tccccacctt tcgagagcga ggactctcag 2100 aagcacaacc agtccgagta cgaggatagc gccggcgagt gctgttcttg ccccaagaca 2160 gactcccaga tcctgaagga gctggaggag tcttccttta gaaagacctt cgaggattat 2220 ctgcataatg tggtgttcgt gcctcgcaag accagctctg gaacaggagc tgaggaccct 2280 aggccatcta ggaagaggcg gtccctgggc gatgtgggca acgtgacagt ggctgtgcca 2340 accgtggctg cttttcccaa tacctccagc acaagcgtgc ctacctctcc agaggagcac 2400 agaccttttg agaaggtggt gaacaaggag tccctggtca tcagcggcct gcgccatttc 2460 acaggctaca ggatcgagct gcaggcttgt aatcaggaca ccccagagga gagatgcagc 2520 gtggctgctt acgtgtccgc caggacaatg ccagaggcca aggctgacga tatcgtgggc 2580 cccgtgaccc acgagatctt tgagaacaat gtggtgcatc tgatgtggca ggagcccaag 2640 gagcctaacg gcctgatcgt gctgtacgag gtgtcctaca gacgctatgg cgatgaggag 2700 ctgcacctgt gcgtgtccag aaagcatttc gctctggaga ggggatgcag gctgagggga 2760 ctgagccctg gcaactactc tgtgagaatc cgcgccacat ccctggctgg caatggcagc 2820 tggaccgagc caacatactt ttatgtgacc gactatctgg atgtgccttc taacatcgcc 2880 aagatcatca tcggcccact gatcttcgtg tttctgttct ccgtggtcat cggcagcatc 2940 tatctgtttc tgaggaagag gcagccagac ggaccactgg gacctctgta cgcttcttcc 3000 aatccagagt atctgagcgc ctctgacgtg ttcccttgta gcgtgtacgt gccagatgag 3060 tgggaggtgt ctagggagaa gatcaccctg ctgagggagc tgggacaggg ctcctttggc 3120 atggtgtatg agggcaacgc cagggatatc atcaagggcg aggctgagac acgggtggcc 3180 gtgaagaccg tgaacgagtc cgctagcctg agagagcgca tcgagtttct gaatgaggcc 3240 tccgtgatga agggcttcac atgccaccat gtggtgagac tgctgggagt ggtgagcaag 3300 ggacagccca ccctggtggt catggagctg atggctcacg gcgacctgaa gtcttacctg 3360 cggtccctga gacccgaggc tgagaacaat cctggccgcc caccacctac actgcaggag 3420 atgatccaga tggccgctga gatcgccgac ggcatggctt atctgaacgc caagaagttc 3480 gtgcaccggg atctggccgc tagaaattgt atggtggccc atgactttac cgtgaagatc 3540 ggcgatttcg gcatgacaag ggacatctac gagaccgatt actatcggaa gggaggcaag 3600 ggactgctgc cagtgcggtg gatggctccc gagtccctga aggacggcgt gtttaccaca 3660 agctctgata tgtggtcctt cggcgtggtg ctgtgggaga tcaccagcct ggctgagcag 3720 ccataccagg gactgtctaa cgagcaggtg ctgaagtttg tgatggacgg cggctatctg 3780 gaccagcctg ataattgtcc agagcgggtg acagatctga tgagaatgtg ctggcagttt 3840 aaccccaaga tgagacctac cttcctggag atcgtgaatc tgctgaagga cgatctgcac 3900 ccctccttcc ccgaggtgag cttctttcat tctgaggaga acaaggctcc cgagtctgag 3960 gagctggaga tggagttcga ggacatggag aatgtgcctc tggatcgctc cagccactgc 4020 cagagggagg aggctggagg aagggacgga ggctcttccc tgggcttcaa gcgcagctac 4080 gaggagcaca tcccttatac acatatgaac ggcggcaaga agaatggcag aatcctgacc 4140 ctgccacgct ccaaccccag ctgagaattc 4170

Claims

1. A cell line expressing human INSR protein, CHO-K1-INSR-6B3, with accession number CGMCC No: 19200.

2. The use of the cell line of claim 1 for non-disease diagnostic purposes in determining the biological activity of human insulin or its analogues, wherein the human insulin analogues are insulin aspart, insulin detemir, or insulin glargine.

3. A method for non-disease diagnostic purposes of in vitro bioactivity assay of human insulin, characterized in that, The method includes assaying using the cell line expressing INSR protein as described in claim 1.

4. A method for non-disease diagnostic purposes of determining the insulin bioactivity in unknown samples in vitro using the cell line CHO-K1-INSR-6B3 as described in claim 1, characterized in that, The method involves adding the human insulin sample to be tested into the culture medium of the cell line, measuring the change in β-receptor phosphorylation within 0.5–3 hours, and determining the biological activity of human insulin based on the β-receptor phosphorylation level.

5. The method according to claim 4, characterized in that, The method includes the following steps: adding the human insulin sample to be tested into the culture medium of the cell line described in claim 1, adding antibody working solution, measuring the change in β receptor phosphorylation within 0.5 to 3 hours, plotting a working curve with concentration as the abscissa and signal value as the ordinate to determine the EC50 value; plotting a standard curve based on the β receptor phosphorylation level after stimulating CHO-K1-INSR-6B3 cells with standard, thereby determining the biological activity of insulin in the unknown sample.

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