Transfection reagent and use thereof

Abstract

Provided is a transfection reagent, comprising a solvent, organic substances, amino acids, vitamins and inorganic salts, wherein the organic substances comprise D-glucose, sodium pyruvate, i-inositol, phenol red and choline chloride; the amino acids comprise glycine, L-glutamine, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, L-phenylalanine, L-serine, L-tyrosine disodium salt, L-arginine hydrochloride, L-methionine, L-cystine dihydrochloride, L-histidine hydrochloride and L-tryptophan; the vitamins comprise nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate and riboflavin; and the inorganic salts comprise sodium chloride, sodium bicarbonate, potassium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium sulfate, hydrochloric acid and ferric nitrate. The transfection reagent is suitable for a wide range of applications, achieves the rapid onset of transfection, and has a good transfection effect.

Description

A transfection reagent and its application

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411564108.3, filed on November 4, 2024, entitled “A Transfection Reagent and Its Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to a transfection reagent and its application, belonging to the field of biotechnology. Background Technology

[0004] Transient transfection is one method of introducing a target nucleic acid into eukaryotic cells. In transient transfection, the target nucleic acid is introduced into a highly infectious cell line to obtain temporary but high-level expression of the target nucleic acid. The transfected target nucleic acid does not need to integrate into the host chromosome, and transfected cells can be harvested in a shorter time than with stable transfection, allowing for the detection of target gene expression in the lysate.

[0005] Currently, the most common products on the market for transient cell transfection are cationic liposomes or cationic polymer transfection reagents. However, these products have poor transfection effects on difficult-to-transfect cells such as primary cells. Besides using transfection reagents, electroporation transfection technology, which achieves transient cell transfection through electroporation, is also quite common. Its transfection principle involves using an instrument to stimulate cells with an electric field, creating transient pores in the cell membrane, allowing exogenous molecules to enter the cell and complete the transfection. While this method can transfect most cells, including difficult-to-transfect cells such as primary cells, it is more damaging to the cells, requires specialized equipment, and involves cumbersome procedures and high transfection costs.

[0006] Therefore, there is an urgent need to find a method for instantaneous cell transfection that has a wide range of applications, good transfection effect, does not require the purchase of special equipment, has simple operation steps, and low transfection cost. Summary of the Invention

[0007] To address the aforementioned problems, this application provides a transfection reagent comprising a solvent, organic matter, amino acids, vitamins, and inorganic salts; the organic matter includes D-glucose (glucose), sodium pyruvate, i-inositol, phenol red, and choline chloride; the amino acids include glycine, L-glutamine, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, L-phenylalanine, L-serine, and L-tyrosine disodium salt. L-arginine hydrochloride, L-methionine, L-cysteine ​​dihydrochloride (L-cysteine ​​2-hydrochloride), L-histidine hydrochloride, and L-tryptophan; the vitamins include nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, and riboflavin; the inorganic salts include sodium chloride, sodium bicarbonate (NaHCO3), potassium chloride, calcium chloride (CaCl2), sodium dihydrogen phosphate (NaH2PO4), magnesium sulfate (MgSO4), hydrochloric acid (HCl), and ferric nitrate (Fe(NO3)3).

[0008] In one embodiment of this application, the concentration of sodium chloride in the solvent is 3500–4500 mg / L; the concentration of glycine in the solvent is 3500–4000 mg / L; the concentration of sodium bicarbonate in the solvent is 2000–2500 mg / L; the concentration of D-glucose in the solvent is 2500–3000 mg / L; the concentration of potassium chloride in the solvent is 200–300 mg / L; the concentration of L-glutamine in the solvent is 300–400 mg / L; the concentration of calcium chloride in the solvent is 100–150 mg / L; and the concentration of sodium pyruvate in the solvent is… The concentration of sodium dihydrogen phosphate in the solvent is 70–85 mg / L; the concentration of magnesium sulfate in the solvent is 55–65 mg / L; the concentration of L-valine in the solvent is 55–65 mg / L; the concentration of L-isoleucine in the solvent is 60–70 mg / L; the concentration of L-leucine in the solvent is 60–70 mg / L; the concentration of L-threonine in the solvent is 50–65 mg / L; the concentration of L-lysine hydrochloride in the solvent is 80–95 mg / L; the concentration of hydrochloric acid in the solvent is 0.3–0.4 mM; the concentration of L-phenylalanine in the solvent is 0.3–0.4 mM. The concentration of the acid in the solvent is 35–45 mg / L; the concentration of L-serine in the solvent is 20–30 mg / L; the concentration of L-tyrosine disodium salt in the solvent is 60–70 mg / L; the concentration of L-arginine hydrochloride in the solvent is 45–60 mg / L; the concentration of L-methionine in the solvent is 15–25 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 35–45 mg / L; the concentration of L-histidine hydrochloride in the solvent is 20–30 mg / L; the concentration of L-tryptophan in the solvent is 8–15 mg / L; and the concentration of i-inositol in the solvent is… The concentration of the following substances is 3–8 mg / L: phenol red in solvent is 8–15 mg / L; nicotinamide in solvent is 1–5 mg / L; choline chloride in solvent is 1–5 mg / L; pyridoxine hydrochloride in solvent is 1–5 mg / L; thiamine hydrochloride in solvent is 1–5 mg / L; folic acid in solvent is 1–5 mg / L; D-calcium pantothenate in solvent is 1–5 mg / L; riboflavin in solvent is 0.1–0.5 mg / L; and ferric nitrate in solvent is 0.03–0.1 mg / L.

[0009] In one embodiment of this application, the concentration of sodium chloride in the solvent is 3900–4000 mg / L; the concentration of glycine in the solvent is 3700–3800 mg / L; the concentration of sodium bicarbonate in the solvent is 2200–2300 mg / L; the concentration of D-glucose in the solvent is 2700–2800 mg / L; the concentration of potassium chloride in the solvent is 240–250 mg / L; the concentration of L-glutamine in the solvent is 360–370 mg / L; the concentration of calcium chloride in the solvent is 120–130 mg / L; and the concentration of sodium pyruvate in the solvent is 65 mg / L. ~70 mg / L; the concentration of sodium dihydrogen phosphate in the solvent is 75-80 mg / L; the concentration of magnesium sulfate in the solvent is 58-63 mg / L; the concentration of L-valine in the solvent is 55-60 mg / L; the concentration of L-isoleucine in the solvent is 62-67 mg / L; the concentration of L-leucine in the solvent is 62-67 mg / L; the concentration of L-threonine in the solvent is 55-60 mg / L; the concentration of L-lysine hydrochloride in the solvent is 88-93 mg / L; the concentration of hydrochloric acid in the solvent is 0.3-0.4 mM (mmol / L); the L The concentration of L-phenylalanine in the solvent is 38–43 mg / L; the concentration of L-serine in the solvent is 24–29 mg / L; the concentration of L-tyrosine disodium salt in the solvent is 62–67 mg / L; the concentration of L-arginine hydrochloride in the solvent is 50–55 mg / L; the concentration of L-methionine in the solvent is 15–20 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 36–41 mg / L; the concentration of L-histidine hydrochloride in the solvent is 24–29 mg / L; the concentration of L-tryptophan in the solvent is 8–12 mg / L; and the concentration of i-inositol in the solvent is… The concentration of the agent is 4–6 mg / L; the concentration of phenol red in the solvent is 8–12 mg / L; the concentration of nicotinamide in the solvent is 2–3 mg / L; the concentration of choline chloride in the solvent is 2–3 mg / L; the concentration of pyridoxine hydrochloride in the solvent is 2–3 mg / L; the concentration of thiamine hydrochloride in the solvent is 2–3 mg / L; the concentration of folic acid in the solvent is 2–3 mg / L; the concentration of D-calcium pantothenate in the solvent is 2–3 mg / L; the concentration of riboflavin in the solvent is 0.2–0.3 mg / L; and the concentration of ferric nitrate in the solvent is 0.05–0.08 mg / L.

[0010] In one embodiment of this application, the concentration of sodium chloride in the solvent is 3968 mg / L; the concentration of glycine in the solvent is 3768.6 mg / L; the concentration of sodium bicarbonate in the solvent is 2294 mg / L; the concentration of D-glucose in the solvent is 2790 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of L-glutamine in the solvent is 362.08 mg / L; the concentration of calcium chloride in the solvent is 124 mg / L; the concentration of sodium pyruvate in the solvent is 68.2 mg / L; and the sodium dihydrogen phosphate is sodium phosphate monohydrate (NaH2PO4-H2O, sodium dihydrogen phosphate monohydrate), and the sodium phosphate monohydrate is... The concentration of the agent is 77.5 mg / L; the concentration of magnesium sulfate in the solvent is 60.5554 mg / L; the concentration of L-valine in the solvent is 58.28 mg / L; the concentration of L-isoleucine in the solvent is 65.1 mg / L; the concentration of L-leucine in the solvent is 65.1 mg / L; the concentration of L-threonine in the solvent is 58.9 mg / L; the concentration of L-lysine hydrochloride in the solvent is 90.52 mg / L; the concentration of hydrochloric acid in the solvent is 0.361 mM; the concentration of L-phenylalanine in the solvent is 40.92 mg / L; the concentration of L-serine in the solvent is 26.04 mg / L; and the concentration of L-tyrosine disodium is... The salt is L-tyrosine disodium salt dihydrate, and the concentration of L-tyrosine disodium salt dihydrate in the solvent is 64.48 mg / L; the concentration of L-arginine hydrochloride in the solvent is 52.08 mg / L; the concentration of L-methionine in the solvent is 18.6 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 39.06 mg / L; the L-histidine hydrochloride is L-histidine hydrochloride-H2O (L-histidine hydrochloride monohydrate), and the concentration of L-histidine hydrochloride-H2O in the solvent is 26.04 mg / L; the concentration of L-tryptophan in the solvent is 9.92 mg / L; the concentration of i-inositol in the solvent is 4.464 mg / L. / L; the concentration of phenol red in the solvent is 9.3 mg / L; the concentration of nicotinamide in the solvent is 2.48 mg / L; the concentration of choline chloride in the solvent is 2.48 mg / L; the concentration of pyridoxine hydrochloride in the solvent is 2.48 mg / L; the concentration of thiamine hydrochloride in the solvent is 2.48 mg / L; the concentration of folic acid in the solvent is 2.48 mg / L; the concentration of D-calcium pantothenate in the solvent is 2.48 mg / L; the concentration of riboflavin in the solvent is 0.248 mg / L; the ferric nitrate is ferric nitrate nonahydrate (Fe(NO3)3·9H2O, ferric nitrate nonahydrate), and the concentration of ferric nitrate nonahydrate in the solvent is 0.062 mg / L.

[0011] In one embodiment of this application, the transfection reagent is composed of a solvent, sodium chloride, glycine, sodium bicarbonate, D-glucose, potassium chloride, L-glutamine, calcium chloride, sodium pyruvate, sodium dihydrogen phosphate, magnesium sulfate, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, hydrochloric acid, L-phenylalanine, L-serine, L-tyrosine disodium salt, L-arginine hydrochloride, L-methionine, L-cysteine ​​dihydrochloride, L-histidine hydrochloride, L-tryptophan, i-inositol, phenol red, nicotinamide, choline chloride, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, riboflavin, and ferric nitrate.

[0012] In one embodiment of this application, the solvent includes water.

[0013] This application also provides a cell transfection method, which is not for the purpose of disease diagnosis and treatment, and the method includes: transfecting host cells using the above-mentioned transfection reagent.

[0014] In one embodiment of this application, the method includes the following steps:

[0015] Mixing step: Mix the target nucleic acid with the above transfection reagent to obtain a mixed solution;

[0016] Incubation steps: Add the mixed solution to the host cells for incubation. After incubation, remove the mixed solution to obtain the treated cells.

[0017] Transfection step: Add cell culture medium to the treated cells and culture them. After the culture is completed, transfected host cells are obtained.

[0018] In one embodiment of this application, during the mixing step, the concentration of the target nucleic acid in the transfection reagent is 150–200 pmol / mL.

[0019] In one embodiment of this application, the incubation step is performed by incubating at 20-25°C for at least 5 minutes.

[0020] In one embodiment of this application, the incubation step is performed by incubating at 20-25°C for 5-15 minutes.

[0021] In one embodiment of this application, the incubation step is performed by incubating at 20-25°C for 10 minutes.

[0022] In one embodiment of this application, the transfection step involves culturing at 35–40°C for at least 6 hours.

[0023] In one embodiment of this application, the transfection step involves culturing at 35–40°C for 6–48 hours.

[0024] In one embodiment of this application, the target nucleic acid includes siRNA and / or miRNA.

[0025] In one embodiment of this application, the host cells include HEK 293T cells, Hepa1-6 cells, primary hepatocytes, and / or C2C cells. 12 cell.

[0026] In one embodiment of this application, the transfection is transient transfection.

[0027] This application also provides a transfected cell, which is prepared by the above method.

[0028] This application also provides the application of the above-mentioned transfection reagents or cell transfection methods in cell transfection, wherein the application is not for the purpose of disease diagnosis and treatment.

[0029] This application also provides a transfection reagent, the components of which consist of a solvent, organic matter, amino acids, vitamins, and inorganic salts; the organic matter consists of D-glucose, sodium pyruvate, i-inositol, phenol red, and choline chloride; the amino acids consist of glycine, L-glutamine, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, L-phenylalanine, L-serine, L-tyrosine disodium salt, L-arginine hydrochloride, L-methionine, and L-cysteine. The product comprises glycine dihydrochloride, L-histidine hydrochloride, and L-tryptophan; the vitamin comprises nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, and riboflavin; the inorganic salt comprises sodium chloride, sodium bicarbonate, potassium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium sulfate, hydrochloric acid, and ferric nitrate; the concentration of sodium chloride in the solvent is 3500–4500 mg / L; the concentration of glycine in the solvent is 3500–4000 mg / L; the concentration of sodium bicarbonate in the solvent is… The concentration of the following components is specified: 2000–2500 mg / L; 2500–3000 mg / L of D-glucose in the solvent; 200–300 mg / L of potassium chloride in the solvent; 300–400 mg / L of L-glutamine in the solvent; 100–150 mg / L of calcium chloride in the solvent; 60–75 mg / L of sodium pyruvate in the solvent; and 70–85 mg / L of sodium dihydrogen phosphate in the solvent. / L; the concentration of magnesium sulfate in the solvent is 55-65 mg / L; the concentration of L-valine in the solvent is 55-65 mg / L; the concentration of L-isoleucine in the solvent is 60-70 mg / L; the concentration of L-leucine in the solvent is 60-70 mg / L; the concentration of L-threonine in the solvent is 50-65 mg / L; the concentration of L-lysine hydrochloride in the solvent is 80-95 mg / L; the concentration of hydrochloric acid in the solvent is 0.361mM; the concentration of L-phenylalanine in the solvent is 35-45 mg / L; the concentration of L-serine in the solvent is 20-30 mg / L; the concentration of L-tyrosine disodium salt in the solvent is 60-70 mg / L; the concentration of L-arginine hydrochloride in the solvent is 45-60 mg / L; the concentration of L-methionine in the solvent is 15-25 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 35-45 mg / L; the concentration of L-histidine hydrochloride in the solvent is 20-30 mg / L; the concentration of L-tryptophan in the solvent is 8-15 mg / L; the concentration of i-inositol in... The concentration of the following substances in the solvent is 3–8 mg / L: phenol red (8–15 mg / L), nicotinamide (1–5 mg / L), choline chloride (1–5 mg / L), pyridoxine hydrochloride (1–5 mg / L), thiamine hydrochloride (1–5 mg / L), folic acid (1–5 mg / L), D-calcium pantothenate (1–5 mg / L), riboflavin (0.1–0.5 mg / L), and ferric nitrate (0.03–0.1 mg / L). The solvent is water.

[0030] In one embodiment of this application, the concentration of sodium chloride in the solvent is 3900–4000 mg / L; the concentration of glycine in the solvent is 3700–3800 mg / L; the concentration of sodium bicarbonate in the solvent is 2200–2300 mg / L; the concentration of D-glucose in the solvent is 2700–2800 mg / L; the concentration of potassium chloride in the solvent is 240–250 mg / L; the concentration of L-glutamine in the solvent is 360–370 mg / L; the concentration of calcium chloride in the solvent is 120–130 mg / L; and the concentration of sodium pyruvate in the solvent is… The concentration of sodium dihydrogen phosphate in the solvent is 65–70 mg / L; the concentration of magnesium sulfate in the solvent is 75–80 mg / L; the concentration of L-valine in the solvent is 55–60 mg / L; the concentration of L-isoleucine in the solvent is 62–67 mg / L; the concentration of L-leucine in the solvent is 62–67 mg / L; the concentration of L-threonine in the solvent is 55–60 mg / L; the concentration of L-lysine hydrochloride in the solvent is 88–93 mg / L; the concentration of hydrochloric acid in the solvent is 0.361 mM; the concentration of L-phenylalanine... The concentration of the L-serine in the solvent is 38–43 mg / L; the concentration of the L-serine in the solvent is 24–29 mg / L; the concentration of the L-tyrosine disodium salt in the solvent is 62–67 mg / L; the concentration of the L-arginine hydrochloride in the solvent is 50–55 mg / L; the concentration of the L-methionine in the solvent is 15–20 mg / L; the concentration of the L-cysteine ​​dihydrochloride in the solvent is 36–41 mg / L; the concentration of the L-histidine hydrochloride in the solvent is 24–29 mg / L; the concentration of the L-tryptophan in the solvent is 8–12 mg / L; and the concentration of the i-inositol in the solvent is… The concentrations of the following components are as follows: 4–6 mg / L for the phenol red, 8–12 mg / L for the nicotinamide, 2–3 mg / L for the choline chloride, 2–3 mg / L for the pyridoxine hydrochloride, 2–3 mg / L for the thiamine hydrochloride, 2–3 mg / L for the folic acid, 2–3 mg / L for the D-calcium pantothenate, 0.2–0.3 mg / L for the riboflavin, and 0.05–0.08 mg / L for the ferric nitrate.

[0031] The technical solution of this application has the following advantages:

[0032] This application provides a transfection reagent comprising a solvent, organic matter, amino acids, vitamins, and inorganic salts; the organic matter includes D-glucose (glucose), sodium pyruvate, i-inositol, phenol red, and choline chloride; the amino acids include glycine, L-glutamine, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, L-phenylalanine, L-serine, L-tyrosine disodium salt, and L-arginine. The transfection reagent comprises L-methionine, L-cysteine ​​dihydrochloride (L-cysteine ​​2-hydrochloride), L-histidine hydrochloride, and L-tryptophan; the vitamins include nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, and riboflavin; the inorganic salts include sodium chloride, sodium bicarbonate (NaHCO3), potassium chloride, calcium chloride (CaCl2), sodium dihydrogen phosphate (NaH2PO4), magnesium sulfate (MgSO4), hydrochloric acid (HCl), and ferric nitrate (Fe(NO3)3). The transfection reagent has the following advantages:

[0033] First, studies have shown that the transfection reagent can effectively transfect the target nucleic acid into HEK 293T cells, Hepa1-6 cells, primary liver cells, and C2C cells. 12 It has the advantage of wide applicability in different types of difficult-to-transfect cells, such as cells.

[0034] Second, studies have shown that when the transfection reagent is used to transfect siRNA that inhibits the target gene in host cells, the expression of the target gene can be significantly reduced within 6 to 12 hours after transfection, and the effect of inhibiting the target gene is close to that of transfection using Lipofectamine 2000, with the advantages of fast transfection onset time and good transfection effect.

[0035] Third, the entire transfection process of transfecting the target nucleic acid in the host cell using the transfection reagent does not require special equipment, and the operation steps are simple and the transfection cost is low.

[0036] Fourth, the transfection reagent has good stability, which is beneficial for its market application;

[0037] Fifth, the components used in the transfection reagent are all inexpensive, further reducing transfection costs. Therefore, the transfection reagent has great application potential in cell transfection (especially transient cell transfection). Attached Figure Description

[0038] Figure 1: qPCR detection of the amount of mir-378a transfected at different concentration gradients into HEK 293T cell lines.

[0039] Figure 2: qPCR detection of the amount of mir-156a transfected at different concentration gradients into HEK 293T cell lines.

[0040] Figure 3: qPCR detection of the amount of si-errα transfected into HEK 293T cell lines at different concentration gradients.

[0041] Figure 4: qPCR detection of the amount of si-egfr transfected at different concentration gradients into the Hepa1-6 cell line.

[0042] Figure 5: Fluorescence microscopy detection of fluorescence signals in HEK 293T cell lines after transfection with Cy3-labeled miRNAs at different concentration gradients.

[0043] Figure 6: Western blotting analysis of GAPDH protein expression at different time points after transfection with si-gapdh in HEK 293T cell line using the transfection reagent of Example 1.

[0044] Figure 7: Quantitative analysis results of immunoblotting bands in Figure 6.

[0045] Figure 8: Western blot analysis of GAPDH protein expression at different time points after transfection with si-gapdh in HEK 293T cell line using the transfection reagent Lipofectamine 2000.

[0046] Figure 9: Quantitative analysis results of immunoblotting bands in Figure 8.

[0047] Figure 10: Western blotting analysis of GAPDH protein expression at different time points after primary liver cells were transfected with si-gapdh using the transfection reagent of Example 1.

[0048] Figure 11: Quantitative analysis results of immunoblotting bands in Figure 10.

[0049] Figure 12: Western blot analysis of GAPDH protein expression at different time points after primary liver cells were transfected with si-gapdh using the transfection reagent Lipofectamine 2000.

[0050] Figure 13: Quantitative analysis results of immunoblotting bands in Figure 12.

[0051] Figure 14: Western blotting detection of C2C cells after induction of differentiation using the transfection reagent from Example 1. 12 The expression effect of GAPDH protein at different time points after cell line transfection with si-gapdh.

[0052] Figure 15: Quantitative analysis results of immunoblotting bands in Figure 14.

[0053] Figure 16: Western blotting analysis of C2C cells after differentiation using Lipofectamine 2000 transfection reagent. 12 The expression effect of GAPDH protein at different time points after cell line transfection with si-gapdh.

[0054] Figure 17: Quantitative analysis results of immunoblotting bands in Figure 16.

[0055] Figure 18: Different cell lines (HEK 293T cell line, C2C) 12 Cell viability after co-incubation with the transfection reagent of Comparative Example 1 (cell lines, HepG2 cell line and 3T3L1 cell line) and Comparative Example 1).

[0056] Figure 19: qPCR detection of the amount of si-egfr transfected at 150 pmol / mL into the HEK 293T cell line.

[0057] Figure 20: qPCR detection of the amount of si-akt transfected at 150 pmol / mL into HEK 293T cell line. Detailed Implementation

[0058] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0059] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0060] Example 1: A transfection reagent

[0061] This embodiment provides a transfection reagent, the components of which are water, sodium chloride, glycine, sodium bicarbonate (NaHCO3), D-glucose (glucose), potassium chloride, L-glutamine, calcium chloride (CaCl2), sodium pyruvate, sodium monohydrate (NaH2PO4-H2O, sodium dihydrogen phosphate monohydrate), magnesium sulfate (MgSO4), L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, hydrochloric acid (HCl), L-phenylalanine, L-serine, and so on. The formula consists of amino acids, L-tyrosine disodium salt dihydrate, L-arginine hydrochloride, L-methionine, L-cystine dihydrochloride (L-cystine dihydrochloride), L-histidine hydrochloride-H2O (L-histidine hydrochloride monohydrate), L-tryptophan, i-inositol, phenol red, nicotinamide, choline chloride, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, riboflavin, and ferric nitrate nonahydrate (Fe(NO3)3·9H2O, ferric nitrate nonahydrate). The specific formula is shown in Table 1.

[0062] Table 1. Formulation of transfection reagent

[0063] Comparative Example 1: A transfection reagent

[0064] This comparative example provides a transfection reagent composed of water, sodium chloride, glycine, sodium bicarbonate (NaHCO3), D-glucose (glucose), potassium chloride, L-glutamine, calcium chloride (CaCl2), sodium pyruvate, sodium monohydrate (NaH2PO4-H2O, sodium dihydrogen phosphate monohydrate), magnesium sulfate (MgSO4), L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, hydrochloric acid (HCl), L-phenylalanine, and L-serine. It is composed of amino acids, L-tyrosine disodium salt dihydrate, L-arginine hydrochloride, L-methionine, L-cystine dihydrochloride (L-cystine dihydrochloride), L-histidine hydrochloride-H2O (L-histidine hydrochloride monohydrate), L-tryptophan, i-inositol, phenol red, nicotinamide, choline chloride, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, riboflavin, and ferric nitrate nonahydrate (Fe(NO3)3·9H2O, ferric nitrate nonahydrate). The specific formula is shown in Table 2.

[0065] Table 2 Formulation of Transfection Reagent

[0066] Comparative Example 2: A transfection reagent

[0067] This comparative example provides a transfection reagent composed of water, sodium chloride, glycine, sodium bicarbonate (NaHCO3), D-glucose (glucose), potassium chloride, L-glutamine, calcium chloride (CaCl2), sodium pyruvate, sodium monohydrate (NaH2PO4-H2O, sodium dihydrogen phosphate monohydrate), magnesium sulfate (MgSO4), L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, hydrochloric acid (HCl), L-phenylalanine, and L-serine. It is composed of amino acids, L-tyrosine disodium salt dihydrate, L-arginine hydrochloride, L-methionine, L-cystine dihydrochloride (L-cystine dihydrochloride), L-histidine hydrochloride-H2O (L-histidine hydrochloride monohydrate), L-tryptophan, i-inositol, phenol red, nicotinamide, choline chloride, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, riboflavin, and ferric nitrate nonahydrate (Fe(NO3)3·9H2O, ferric nitrate nonahydrate). The specific formula is shown in Table 3.

[0068] Table 3. Formulation of transfection reagent

[0069] Example 2: A cell transfection method

[0070] This embodiment provides a cell transfection method, which includes the following steps:

[0071] Mixing step: Mix the target nucleic acid with the transfection reagent of Example 1 to make the concentration of the target nucleic acid in the transfection reagent 150 pmol / mL to obtain a mixed solution;

[0072] Incubation steps: Add the mixed solution to the host cells until the host cells are submerged, and incubate at room temperature (25℃) for 10 min. After incubation, remove the mixed solution and wash three times with PBS buffer (pH 7.4, concentration 0.01M, purchased from Wuhan Sewell Biotechnology Co., Ltd.) to obtain the treated cells.

[0073] Transfection procedure: DMEM (base) cell culture medium (purchased from Gibco) containing 2% (v / v) fetal bovine serum (FBS, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) was added to the treated cells until the treated cells were submerged, and then cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 to obtain transfected host cells.

[0074] Comparative Example 1: A cell transfection method

[0075] This comparative example provides a cell transfection method, which is based on Example 2, by replacing the transfection reagent of Example 1 with the transfection reagent of Comparative Example 1.

[0076] Comparative Example 2: A cell transfection method

[0077] This comparative example provides a cell transfection method, which is based on Example 2, by replacing the transfection reagent of Example 1 with the transfection reagent of Comparative Example 2.

[0078] Experiment Example 1: Verification of the Transfection Performance of Transfection Reagent

[0079] This experimental example verifies the transfection performance of the transfection reagent in Example 1. The verification process is as follows:

[0080] 1. Synthesis of target nucleic acid

[0081] The following nucleotide sequences were synthesized as target nucleic acids: mir-378a (5'-CUGGACUUGGAGUCAGAAGG-3') as shown in SEQ ID NO.1 (5'-CUGGACUUGGAGUCAGAAGG-3'), mir-156a (5'-UGACAGAAGAGAGUGAGCAC-CY3-3') as shown in SEQ ID NO.2 (5'-UGACAGAAGAGAGAGAGCAC-CY3-3'), si-errα (5'-UGCACAUUGAAGAUGCUGA-3') as shown in SEQ ID NO.4 (5'-GGAACUGGAUAUUCUGAAAdTdT-3', where dTdT is a dangling fragment at the 3' end of the siRNA), and si-gapdh (5'-CAGAAGACUGUGGAUGGCC-3') as shown in SEQ ID NO.5 (5'-CAGAAGACUGUGGAUGGCC-3').

[0082] 2. Transfection of target nucleic acid

[0083] 2.1 Transfection with mir-378a, mir-156a and si-errα

[0084] HEK 293T cell line (purchased from the U.S. Tissue Culture Collection) was cultured at 1 × 10⁻⁶ cells per well. 6Cells were seeded onto each well and each well was supplemented with 2 mL of 10% (v / v) fetal bovine serum (FBS, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and 1% (w / v, g / 100 mL) penicillin antibiotics. STREPTOMYCIN, purchased from Gibco, was cultured in 6-well plates with DMEM (base) cell culture medium (also purchased from Gibco) at 37°C and 5% (v / v) CO2 for 12 hours until the cell density reached over 70%. The cell culture medium was then removed, and the cells were washed three times with PBS buffer to obtain the cells to be transfected. mir-378a, mir-156a, and si-errα were added to the transfection reagent in Example 1 to concentrations of 40, 80, 160, 240, 400, and 640 pmol / ml, respectively, to obtain mixed solutions of mir-378a, mir-156a, and si-errα at different concentration gradients. The cells were then cultured in wells of DMEM (base) cell culture medium without the target nucleic acid. As a blank control (Blank group), wells containing DMEM (base) cell culture medium with 200 pmol / mL target nucleic acid were used as negative controls (Ctrl group). Different concentration gradients of mir-378a mixed solutions, different concentration gradients of mir-156a mixed solutions, and different concentration gradients of si-errα mixed solutions were added to 6-well plates at a volume of 1 mL per well. After incubation at room temperature (25℃) for 10 min, the mixed solutions were removed, and the cells were washed three times with PBS buffer to obtain treated cells. DMEM (base) cell culture medium containing 2% (v / v) fetal bovine serum was added to 6-well plates at a volume of 2 mL per well and cultured in a cell culture incubator at 37℃ and 5% (v / v) CO2 to obtain HEK 293T cells transfected with different concentration gradients of mir-378a, different concentration gradients of mir-156a, and different concentration gradients of si-errα.

[0085] 2.2 Transfection of si-egfr

[0086] Si-egfr was added to the transfection reagent of Example 1 to concentrations of 40, 80, 160, 240, 400, and 640 pmol / mL to obtain si-egfr mixed solutions with different concentration gradients. Following the transfection of miR-378a, miR-156a, and si-errα, the different concentration gradients of si-egfr were transfected into the Hepa1-6 cell line (purchased from the U.S. Tissue Culture Collection) to obtain Hepa1-6 cells transfected with different concentration gradients of si-egfr.

[0087] 2.3 Transfection of si-gapdh

[0088] si-gapdh was added to the transfection reagent of Example 1 to a concentration of 150 pmol / mL to obtain a si-gapdh mixed solution; using Lipofectamine 2000 (purchased from Thermo Fisher Scientific) as a transfection control, si-gapdh was transfected into the Hepa1-6 cell line (purchased from the U.S. Tissue Culture Collection) according to the transfection of mir-378a, mir-156a and si-errα to obtain Hepa1-6 cells transfected with si-gapdh.

[0089] si-gapdh was added to the transfection reagent in Example 1 to a concentration of 150 pmol / mL to obtain a si-gapdh mixed solution. Using Lipofectamine 2000 (purchased from Thermo Fisher Scientific) as a transfection control, si-gapdh was transfected into primary liver cells (primary liver cells were obtained from the livers of C57 mice, purchased from Jicui Pharmaceutical Co., Ltd.; the extraction method of primary liver cells is described in the literature "Cabral, Fatima; Miller, Colton M.; Kudrna, Katrina M.; Hass, Blake E.; Daubendiek, Jocelyn G.; Kellar, Brianna M.; Harris, Edward N. (2018). Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion. Journal of Visualized In Experiments, (132), primary liver cells transfected with si-gapdh were obtained.

[0090] si-gapdh was added to the transfection reagent of Example 1 to a concentration of 150 pmol / mL to obtain a si-gapdh mixed solution; using Lipofectamine 2000 (purchased from Thermo Fisher Scientific) as a transfection control, si-gapdh was transfected into C2C cells that had been induced to differentiate into myotube cells, following the transfection procedures of miR-378a, miR-156a, and si-errα. 12 C2C cell line 12 The cell line was purchased from Wuhan Sewell Biotechnology Co., Ltd., C2C. 12 For the cell line induction differentiation method, please refer to the literature "Zhang Lin, Liu Yan, Liu Yulan, & Zhang Jing. (2017). Effects of different concentrations of EPA and DHA on C2C".12 Effects of si-gapdh on myoblast proliferation and apoptosis. Chinese Journal of Animal Husbandry, 53(1), 5. 12 cell.

[0091] In the transfection control experiments, when using Lipofectamine 2000 transfection reagent, si-gapdh was added to a mixture of 6 μL of Lipofectamine 2000 and 2 mL of serum-free medium (purchased from Gibco) to obtain a si-gapdh mixed solution. In the transfection control experiments, wells containing serum-free medium without si-gapdh served as blank controls, and wells containing serum-free medium containing 50 pmol / mL NC sequence (nucleotide sequence as shown in SEQ ID NO. 6, 5'-UUCUCCGAACGUGUCACGUdTdT-3', where dTdT is the overhanging fragment at the 3' end of the siRNA) served as negative controls.

[0092] 3. Detection of different host cells

[0093] HEK 293T cells transfected with different concentration gradients of miR-378a, miR-156a, si-errα, and si-egfr were cultured for 1 hour and then subjected to qPCR (qPCR method see reference "Tan P, Pepin"). Lavoie JL. Mouse Adipose Tissue Collection and Processing for RNA Analysis. J Vis Exp. 2018 Jan 31;(131):57026.”) The amount of different target nucleic acids entering cells at different concentration gradients was detected, and the results are shown in Figures 1 to 4. As can be seen from Figures 1 to 4, as the amount of target nucleic acid transfected increases, the amount of target nucleic acid entering the cells also increases in a gradient.

[0094] HEK 293T cells were incubated for 10 min in different concentration gradients of miR-156a (marked with Cy3) and the mixed solution was removed. The cells were washed three times with PBS buffer to obtain the treated cells. DMEM (base) cell culture medium containing 0.2 mg / mL RNase A (Thermo Fisher Scientific, Cat#12091021) and 2% (v / v) fetal bovine serum was added to 6-well plates at a rate of 2 mL per well. The cells were cultured at 37°C and 5% (v / v) CO2 for 1 h to obtain HEK 293T cells transfected with different concentration gradients of miR-156a (with added RNase A). The purpose of step A was to remove any target nucleic acids that might adhere to the cell surface. The cell culture medium was removed, and the cells were washed three times with PBS buffer to obtain cultured cells. DAPI staining solution (purchased from Beyotime Biotechnology Co., Ltd.) was added to each well of a 6-well plate at a rate of 2 mL, and the cells were incubated at room temperature (25°C) in the dark for 15 min to stain the cell nuclei, resulting in stained cells. The fluorescence signal intensity of the stained cells was detected using a confocal microscope, and the results are shown in Figure 5. Figure 5 also confirms that as the amount of transfected target nucleic acid increases, the amount of target nucleic acid entering the cells also increases in a gradient.

[0095] Hepa1-6 cells transfected with si-gapdh, primary liver cells transfected with si-gapdh, and C2C cells transfected with si-gapdh were obtained after culturing for 6h, 12h, 24h, or 48h. 12 Cells were transfected with si-gapdh, and the expression of GAPDH protein at different time points was detected using Western blotting (the antibodies used in Western blotting were GAPDH Rab Cat#A19056 and HSP90α / β Rab Cat#A5027; the Western blotting method can be found in the literature "Hnasko TS, Hnasko RM. The Western Blot. Methods Mol Biol. 2015; 1318:87-96.", where HSP90 was the reference protein). ImageJ was used to perform grayscale analysis of the immunoblotting bands to visually represent the expression levels. The results are shown in Figures 6-17. As shown in Figures 6-17, the expression levels of GAPDH protein were detected in HEK 293T cells, Hepa1-6 cells, primary liver cells, and C2C cells induced to differentiate into myotube cells. 12In four different cell lines, si-gapdh cells were transfected using the transfection reagent of Example 1. Western blotting was used to detect the transfection effect on different cell lines. It was found that the expression of the target gene was significantly reduced within 6 to 12 hours using the transfection reagent of Example 1, and the target gene inhibition effect was close to that of transfection using the transfection reagent (Lipofectamine 2000). At this time, the total cost of using the transfection reagent of Example 1 was only half that of transfection using the transfection reagent (Lipofectamine 2000).

[0096] Experiment Example 2: Verification of the Transfection Performance of the Transfection Reagent

[0097] This experimental example verifies the transfection performance of the transfection reagent in Example 1. The verification process is as follows:

[0098] Based on Experiment 1, HEK 293T cell line was used at a density of 1×10⁶ cells per well. 6 Cells were seeded onto each well and each well was supplemented with 2 mL of 10% (v / v) fetal bovine serum (FBS, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and 1% (w / v, g / 100 mL) penicillin antibiotics. STREPTOMYCIN cells (purchased from Gibco) were cultured in 6-well plates with DMEM (base) cell culture medium (purchased from Gibco) at 37°C and 5% (v / v) CO2 for 12 hours until the cell density reached 70% or higher. The cell culture medium was then removed, and the cells were washed three times with PBS buffer to obtain the cells to be transfected. Using the transfection reagent from Example 1 as a control, 1 mL of the transfection reagent from Example 1 was added to each well of the 6-well plate and incubated at room temperature (25°C) for 10 minutes. The transfection reagent was then removed, and the cells were washed three times with PBS buffer to obtain the treated cells. 2 mL of trypan blue (4 mg / mL) and PBS buffer were added to each well of the 6-well plate and incubated at room temperature (25°C) for 5 minutes to stain the cells, obtaining the stained cells. The staining was observed using an inverted microscope, and the results are shown in Figure 18.

[0099] Using the transfection reagent from Example 1 as a control, and following the treatment method for the HEK 293T cell line, the cells were transfected into myotube cells at C2C sites. 12 The same experiment was performed on the cell lines, HepG2 cell line (purchased from the U.S. Tissue Culture Collection), and 3T3L1 cell line (purchased from the U.S. Tissue Culture Collection) to detect cell viability. The results are shown in Figure 18.

[0100] The results in Figure 18 show that a large number of cells died after treatment with Comparative Example 1, so Comparative Example 1 is not suitable for cell transfection.

[0101] Experiment Example 3: Verification of the Transfection Performance of the Transfection Reagent

[0102] This experiment verified the transfection performance of the transfection reagent in Example 2. The verification process is as follows:

[0103] Based on Experiment 1, HEK 293T cell line was used at a density of 1×10⁶ cells per well. 6 The cells were seeded into 6-well plates containing 2 mL of DMEM (base) cell culture medium (Gibco) with 10% (v / v) fetal bovine serum (FBS, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and 1% (w / v, g / 100 mL) penicillin streptomycin (PENICILLIN STREPTOMYCIN, purchased from Gibco) per well. The cells were incubated at 37°C and 5% (v / v) CO2 for 12 h until the cell density reached over 70%. The cell culture medium was then removed, and the cells were washed three times with PBS buffer to obtain the cells for transfection. si-egfr (nucleotide sequence as shown in SEQ ID NO.4, 5'-GGAACUGGAUAUUCUGAAAdTdT-3') and si-akt (nucleotide sequence as shown in SEQ ID NO.4) were then transfected. As shown in NO.7, 5'-CUGACCAAGAUGACAGCAU-3') was added to the transfection reagent of Comparative Example 1 to a concentration of 150 pmol / ml to obtain si-egfr mixed solution and si-akt mixed solution. Wells containing DMEM (base) cell culture medium without the target nucleic acid served as blank controls (Blank group), and wells containing DMEM (base) cell culture medium with different concentration gradients of the target nucleic acid served as negative controls (Ctrl group). The si-egfr mixed solution and si-akt mixed solution were added to 6-well plates at a volume of 1 mL per well. After incubation at room temperature (25℃) for 10 min, the mixed solutions were removed, and the cells were washed three times with PBS buffer to obtain treated cells. DMEM (base) cell culture medium containing 2% (v / v) fetal bovine serum was added to 6-well plates at a volume of 2 mL per well and cultured in a cell culture incubator at 37℃ and 5% (v / v) CO2 to obtain HEK cells transfected with si-egfr. 293T cells and HEK 293T cells transfected with si-akt.

[0104] HEK 293T cells transfected with si-EGFR and si-AKT were cultured for 24 hours. The amount of different target nucleic acids entering the cells was detected by qPCR. The results are shown in Figures 19 and 20. As can be seen from Figures 19 and 20, the knockout of both EGFR and AKT was poor, indicating that the transfection effect of the transfection reagent in Comparative Example 2 was poor.

[0105] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A transfection reagent, characterized in that, The transfection reagent comprises solvents, organic matter, amino acids, vitamins, and inorganic salts; the organic matter includes D-glucose, sodium pyruvate, i-inositol, phenol red, and choline chloride; the amino acids include glycine, L-glutamine, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, L-phenylalanine, L-serine, L-tyrosine disodium salt, L-arginine hydrochloride, L-methionine, L-cysteine ​​dihydrochloride, and L-histidine. The product contains hydrochloride and L-tryptophan; the vitamins include nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, and riboflavin; the inorganic salts include sodium chloride, sodium bicarbonate, potassium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium sulfate, hydrochloric acid, and ferric nitrate; the concentration of sodium chloride in the solvent is 3500–4500 mg / L; the concentration of glycine in the solvent is 3500–4000 mg / L; the concentration of sodium bicarbonate in the solvent is 2000–2500 mg / L. The concentration of D-glucose in the solvent is 2500–3000 mg / L; the concentration of potassium chloride in the solvent is 200–300 mg / L; the concentration of L-glutamine in the solvent is 300–400 mg / L; the concentration of calcium chloride in the solvent is 100–150 mg / L; the concentration of sodium pyruvate in the solvent is 60–75 mg / L; the concentration of sodium dihydrogen phosphate in the solvent is 70–85 mg / L; the concentration of magnesium sulfate is... The concentration of the L-valine in the solvent is 55–65 mg / L; the concentration of the L-isoleucine in the solvent is 60–70 mg / L; the concentration of the L-leucine in the solvent is 60–70 mg / L; the concentration of the L-threonine in the solvent is 50–65 mg / L; the concentration of the L-lysine hydrochloride in the solvent is 80–95 mg / L; and the concentration of the hydrochloric acid in the solvent is 0.3–0.4mM; the concentration of L-phenylalanine in the solvent is 35-45 mg / L; the concentration of L-serine in the solvent is 20-30 mg / L; the concentration of L-tyrosine disodium salt in the solvent is 60-70 mg / L; the concentration of L-arginine hydrochloride in the solvent is 45-60 mg / L; the concentration of L-methionine in the solvent is 15-25 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 35-45 mg / L; the concentration of L-histidine hydrochloride in the solvent is 20-30 mg / L; the concentration of L-tryptophan in the solvent is 8-15 mg / L; the i- The concentrations of inositol, phenol red, nicotinamide, choline chloride, pyridoxine, thiamine, and ferric nitrate in the solvent are as follows: inositol is 3–8 mg / L; phenol red is 8–15 mg / L; nicotinamide is 1–5 mg / L; choline chloride is 1–5 mg / L; pyridoxine hydrochloride is 1–5 mg / L; thiamine hydrochloride is 1–5 mg / L; folic acid is 1–5 mg / L; calcium D-pantothenate is 1–5 mg / L; riboflavin is 0.1–0.5 mg / L; and ferric nitrate is 0.03–0.1 mg / L.

2. The transfection reagent as described in claim 1, characterized in that, The concentration of sodium chloride in the solvent is 3900–4000 mg / L; the concentration of glycine in the solvent is 3700–3800 mg / L; the concentration of sodium bicarbonate in the solvent is 2200–2300 mg / L; the concentration of D-glucose in the solvent is 2700–2800 mg / L; the concentration of potassium chloride in the solvent is 240–250 mg / L; the concentration of L-glutamine in the solvent is 360–370 mg / L; the concentration of calcium chloride in the solvent is 120–130 mg / L; and the concentration of sodium pyruvate in the solvent is 65–70 mg / L. The concentration of sodium dihydrogen phosphate in the solvent is 75–80 mg / L; the concentration of magnesium sulfate in the solvent is 58–63 mg / L; the concentration of L-valine in the solvent is 55–60 mg / L; the concentration of L-isoleucine in the solvent is 62–67 mg / L; the concentration of L-leucine in the solvent is 62–67 mg / L; the concentration of L-threonine in the solvent is 55–60 mg / L; the concentration of L-lysine hydrochloride in the solvent is 88–93 mg / L; the concentration of hydrochloric acid in the solvent is 0.3–0.4 mM; the concentration of L-phenylalanine in the solvent is… The concentration of L-serine in the solvent is 38–43 mg / L; the concentration of L-serine in the solvent is 24–29 mg / L; the concentration of L-tyrosine disodium salt in the solvent is 62–67 mg / L; the concentration of L-arginine hydrochloride in the solvent is 50–55 mg / L; the concentration of L-methionine in the solvent is 15–20 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 36–41 mg / L; the concentration of L-histidine hydrochloride in the solvent is 24–29 mg / L; the concentration of L-tryptophan in the solvent is 8–12 mg / L; the concentration of i-inositol in the solvent is… The concentrations of the following components are specified: 4–6 mg / L; phenol red in the solvent is 8–12 mg / L; nicotinamide in the solvent is 2–3 mg / L; choline chloride in the solvent is 2–3 mg / L; pyridoxine hydrochloride in the solvent is 2–3 mg / L; thiamine hydrochloride in the solvent is 2–3 mg / L; folic acid in the solvent is 2–3 mg / L; D-calcium pantothenate in the solvent is 2–3 mg / L; riboflavin in the solvent is 0.2–0.3 mg / L; and ferric nitrate in the solvent is 0.05–0.08 mg / L.

3. The transfection reagent as described in claim 2, characterized in that, The concentration of sodium chloride in the solvent is 3968 mg / L; the concentration of glycine in the solvent is 3768.6 mg / L; the concentration of sodium bicarbonate in the solvent is 2294 mg / L; the concentration of D-glucose in the solvent is 2790 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of L-glutamine in the solvent is 362.08 mg / L; the concentration of calcium chloride in the solvent is 124 mg / L; the concentration of sodium pyruvate in the solvent is 68.2 mg / L; the sodium dihydrogen phosphate is sodium dihydrogen phosphate monohydrate, and the concentration of sodium dihydrogen phosphate monohydrate in the solvent is 77.5 mg / L. L; the concentration of magnesium sulfate in the solvent is 60.5554 mg / L; the concentration of L-valine in the solvent is 58.28 mg / L; the concentration of L-isoleucine in the solvent is 65.1 mg / L; the concentration of L-leucine in the solvent is 65.1 mg / L; the concentration of L-threonine in the solvent is 58.9 mg / L; the concentration of L-lysine hydrochloride in the solvent is 90.52 mg / L; the concentration of hydrochloric acid in the solvent is 0.361 mM; the concentration of L-phenylalanine in the solvent is 40.92 mg / L; the concentration of L-serine in the solvent is 26.04 mg / L; the concentration of L-tyrosine... The disodium salt of L-tyrosine is a disodium salt dihydrate, and the concentration of the L-tyrosine disodium salt dihydrate in the solvent is 64.48 mg / L; the concentration of L-arginine hydrochloride in the solvent is 52.08 mg / L; the concentration of L-methionine in the solvent is 18.6 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 39.06 mg / L; the L-histidine hydrochloride is a L-histidine hydrochloride monohydrate, and the concentration of the L-histidine hydrochloride monohydrate in the solvent is 26.04 mg / L; the concentration of L-tryptophan in the solvent is 9.92 mg / L; and the concentration of I-inositol in the solvent is 4.4 mg / L. The concentrations of the following components are specified: 64 mg / L; phenol red in the solvent is 9.3 mg / L; nicotinamide in the solvent is 2.48 mg / L; choline chloride in the solvent is 2.48 mg / L; pyridoxine hydrochloride in the solvent is 2.48 mg / L; thiamine hydrochloride in the solvent is 2.48 mg / L; folic acid in the solvent is 2.48 mg / L; D-calcium pantothenate in the solvent is 2.48 mg / L; riboflavin in the solvent is 0.248 mg / L; and ferric nitrate is ferric nitrate nonahydrate, with a concentration of 0.062 mg / L in the solvent.

4. The transfection reagent according to any one of claims 1 to 3, characterized in that, The transfection reagent is composed of solvent, sodium chloride, glycine, sodium bicarbonate, D-glucose, potassium chloride, L-glutamine, calcium chloride, sodium pyruvate, sodium dihydrogen phosphate, magnesium sulfate, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, hydrochloric acid, L-phenylalanine, L-serine, L-tyrosine disodium salt, L-arginine hydrochloride, L-methionine, L-cysteine ​​dihydrochloride, L-histidine hydrochloride, L-tryptophan, i-inositol, phenol red, nicotinamide, choline chloride, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, riboflavin, and ferric nitrate.

5. A cell transfection method, characterized in that, The method includes: transfecting host cells using the transfection reagent according to any one of claims 1 to 4.

6. The cell transfection method as described in claim 5, characterized in that, The method includes the following steps: Mixing step: Mix the target nucleic acid with the transfection reagent according to any one of claims 1 to 4 to obtain a mixed solution; Incubation steps: Add the mixed solution to the host cells for incubation. After incubation, remove the mixed solution to obtain the treated cells. Transfection step: Add cell culture medium to the treated cells and culture them. After the culture is completed, transfected host cells are obtained.

7. The cell transfection method as described in claim 6, characterized in that, In the mixing step, the concentration of the target nucleic acid in the transfection reagent is 150–200 pmol / mL.

8. The cell transfection method as described in claim 6 or 7, characterized in that, In the incubation step, the incubation is carried out at 20-25°C for at least 5 minutes; in the transfection step, the culture is carried out at 35-40°C for at least 6 hours.

9. A transfected cell, characterized in that, The transfected cells are prepared by the method according to any one of claims 5 to 8.

10. The use of the transfection reagent according to any one of claims 1 to 4 or the cell transfection method according to any one of claims 5 to 8 in cell transfection.

11. A transfection reagent, characterized in that, The transfection reagent consists of a solvent, organic matter, amino acids, vitamins, and inorganic salts; the organic matter consists of D-glucose, sodium pyruvate, i-inositol, phenol red, and choline chloride; the amino acids consist of glycine, L-glutamine, L-valine, L-isoleucine, L-leucine, L-threonine, L-lysine hydrochloride, L-phenylalanine, L-serine, L-tyrosine disodium salt, L-arginine hydrochloride, L-methionine, L-cysteine ​​dihydrochloride, and L-histamine. The product is composed of glycine and L-tryptophan; the vitamin is composed of nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate, and riboflavin; the inorganic salt is composed of sodium chloride, sodium bicarbonate, potassium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium sulfate, hydrochloric acid, and ferric nitrate; the concentration of sodium chloride in the solvent is 3500–4500 mg / L; the concentration of glycine in the solvent is 3500–4000 mg / L; the concentration of sodium bicarbonate in the solvent is 2000– The concentration of D-glucose in the solvent is 2500 mg / L; the concentration of potassium chloride in the solvent is 200–300 mg / L; the concentration of L-glutamine in the solvent is 300–400 mg / L; the concentration of calcium chloride in the solvent is 100–150 mg / L; the concentration of sodium pyruvate in the solvent is 60–75 mg / L; and the concentration of sodium dihydrogen phosphate in the solvent is 70–85 mg / L. The concentration of magnesium sulfate in the solvent is 55–65 mg / L; the concentration of L-valine in the solvent is 55–65 mg / L; the concentration of L-isoleucine in the solvent is 60–70 mg / L; the concentration of L-leucine in the solvent is 60–70 mg / L; the concentration of L-threonine in the solvent is 50–65 mg / L; the concentration of L-lysine hydrochloride in the solvent is 80–95 mg / L; and the concentration of hydrochloric acid in the solvent is 0.361mM; the concentration of L-phenylalanine in the solvent is 35-45 mg / L; the concentration of L-serine in the solvent is 20-30 mg / L; the concentration of L-tyrosine disodium salt in the solvent is 60-70 mg / L; the concentration of L-arginine hydrochloride in the solvent is 45-60 mg / L; the concentration of L-methionine in the solvent is 15-25 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 35-45 mg / L; the concentration of L-histidine hydrochloride in the solvent is 20-30 mg / L; the concentration of L-tryptophan in the solvent is 8-15 mg / L; the concentration of i-inositol in... The concentration of the following substances in the solvent is 3–8 mg / L: phenol red (8–15 mg / L), nicotinamide (1–5 mg / L), choline chloride (1–5 mg / L), pyridoxine hydrochloride (1–5 mg / L), thiamine hydrochloride (1–5 mg / L), folic acid (1–5 mg / L), D-calcium pantothenate (1–5 mg / L), riboflavin (0.1–0.5 mg / L), and ferric nitrate (0.03–0.1 mg / L). The solvent is water.

12. The transfection reagent as described in claim 11, characterized in that, The concentration of sodium chloride in the solvent is 3900–4000 mg / L; the concentration of glycine in the solvent is 3700–3800 mg / L; the concentration of sodium bicarbonate in the solvent is 2200–2300 mg / L; the concentration of D-glucose in the solvent is 2700–2800 mg / L; the concentration of potassium chloride in the solvent is 240–250 mg / L; the concentration of L-glutamine in the solvent is 360–370 mg / L; the concentration of calcium chloride in the solvent is 120–130 mg / L; and the concentration of sodium pyruvate in the solvent is 65–70 mg / L. The concentration of sodium dihydrogen phosphate in the solvent is 75–80 mg / L; the concentration of magnesium sulfate in the solvent is 58–63 mg / L; the concentration of L-valine in the solvent is 55–60 mg / L; the concentration of L-isoleucine in the solvent is 62–67 mg / L; the concentration of L-leucine in the solvent is 62–67 mg / L; the concentration of L-threonine in the solvent is 55–60 mg / L; the concentration of L-lysine hydrochloride in the solvent is 88–93 mg / L; the concentration of hydrochloric acid in the solvent is 0.361 mM; the concentration of L-phenylalanine in the solvent is… The concentration of L-serine in the solvent is 38–43 mg / L; the concentration of L-serine in the solvent is 24–29 mg / L; the concentration of L-tyrosine disodium salt in the solvent is 62–67 mg / L; the concentration of L-arginine hydrochloride in the solvent is 50–55 mg / L; the concentration of L-methionine in the solvent is 15–20 mg / L; the concentration of L-cysteine ​​dihydrochloride in the solvent is 36–41 mg / L; the concentration of L-histidine hydrochloride in the solvent is 24–29 mg / L; the concentration of L-tryptophan in the solvent is 8–12 mg / L; the concentration of i-inositol in the solvent is… The concentrations of the following components are specified: 4–6 mg / L; phenol red in the solvent is 8–12 mg / L; nicotinamide in the solvent is 2–3 mg / L; choline chloride in the solvent is 2–3 mg / L; pyridoxine hydrochloride in the solvent is 2–3 mg / L; thiamine hydrochloride in the solvent is 2–3 mg / L; folic acid in the solvent is 2–3 mg / L; D-calcium pantothenate in the solvent is 2–3 mg / L; riboflavin in the solvent is 0.2–0.3 mg / L; and ferric nitrate in the solvent is 0.05–0.08 mg / L.

Images