A transfection reagent and its application

Abstract

The present invention relates to a transfection reagent and its application, belonging to the field of biotechnology. The present invention provides a transfection reagent, the components of which include solvent, organic matter, amino acid, vitamin and inorganic salt; organic matter includes D-glucose, sodium pyruvate, i-inositol, phenol red and choline chloride; amino acid includes 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; vitamins include nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate and riboflavin; inorganic salt includes sodium chloride, sodium bicarbonate, potassium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium sulfate, hydrochloric acid and ferric nitrate. This transfection reagent has a wide range of applications, fast transfection onset time and good transfection effect.

Description

Technical Field

[0001] The invention relates to a transfection reagent and application thereof, belonging to the field of biotechnology. Background Art

[0002] Transient transfection is one of the ways to introduce target nucleic acids 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 have to be integrated into the host chromosome, and the transfected cells can be harvested in a shorter time than stable transfection, and the expression of the target gene in the lysate can be detected.

[0003] At present, the common products used for transient cell transfection on the market are mainly cationic liposomes or cationic polymer transfection reagents. However, such products have poor transfection effects on primary cells and other difficult-to-transfect cells. In addition to using transfection reagents, electroporation transfection technology that achieves transient cell transfection through electroporation is also relatively common. The transfection principle is to use an instrument to form transient holes on the cell membrane when the cell is stimulated by an electric field, allowing exogenous molecules to enter the cell to complete the transfection. Although this method can achieve transfection of most cells including primary cells and other difficult-to-transfect cells, it is more harmful to the cells and requires the purchase of special equipment. The operation steps are cumbersome and the transfection cost is high.

[0004] Therefore, there is an urgent need to find a method for achieving transient cell transfection that has a wide range of applications, good transfection effects, does not require the purchase of special equipment, has simple operating steps, and has low transfection costs. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a transfection reagent, the components of which include solvent, organic matter, amino acid, vitamin and inorganic salt; the organic matter includes D-glucose (glucose), sodium pyruvate, i-inositol (i-Inositol), phenol red and choline chloride; the amino acid includes 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-cystine 2 hydrochloride), L-histidine hydrochloride and L-tryptophan; the vitamins include niacinamide, 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).

[0006] In one embodiment of the present invention, 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 The concentration of the sodium dihydrogen phosphate in the solvent is 70-85 mg / L; the concentration of the magnesium sulfate in the solvent is 55-65 mg / L; 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; the concentration of the hydrochloric acid in the solvent is 0.3-0.4 mM; the concentration of the L-phenylalanine in the solvent is 100-200 mg / L ... The concentration of the acid in the solvent is 35-45 mg / L; the concentration of the L-serine in the solvent is 20-30 mg / L; the concentration of the L-tyrosine disodium salt in the solvent is 60-70 mg / L; the concentration of the L-arginine hydrochloride in the solvent is 45-60 mg / L; the concentration of the L-methionine in the solvent is 15-25 mg / L; the concentration of the L-cystine dihydrochloride in the solvent is 35-45 mg / L; the concentration of the L-histidine hydrochloride in the solvent is 20-30 mg / L; the concentration of the L-tryptophan in the solvent is 8-15 mg / L; the concentration of the i-inositol in the solvent is 10-25 mg / L. The concentration of the phenol red in the solvent is 3-8 mg / L; the concentration of the nicotinamide in the solvent is 8-15 mg / L; the concentration of the choline chloride in the solvent is 1-5 mg / L; the concentration of the pyridoxine hydrochloride in the solvent is 1-5 mg / L; the concentration of the thiamine hydrochloride in the solvent is 1-5 mg / L; the concentration of the folic acid in the solvent is 1-5 mg / L; the concentration of the D-calcium pantothenate in the solvent is 1-5 mg / L; the concentration of the riboflavin in the solvent is 0.1-0.5 mg / L; and the concentration of the ferric nitrate in the solvent is 0.03-0.1 mg / L.

[0007] In one embodiment of the present invention, 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; the concentration of sodium pyruvate in the solvent is 65 ~70mg / L; the concentration of sodium dihydrogen phosphate in the solvent is 75~80mg / L; the concentration of magnesium sulfate in the solvent is 58~63mg / L; the concentration of L-valine in the solvent is 55~60mg / L; the concentration of L-isoleucine in the solvent is 62~67mg / L; the concentration of L-leucine in the solvent is 62~67mg / L; the concentration of L-threonine in the solvent is 55~60mg / L; the concentration of L-lysine hydrochloride in the solvent is 88~93mg / L; the concentration of hydrochloric acid in the solvent is 0.3~0.4mM (mmol / L); the L -phenylalanine concentration in the solvent is 38~43mg / L; L-serine concentration in the solvent is 24~29mg / L; L-tyrosine disodium concentration in the solvent is 62~67mg / L; L-arginine hydrochloride concentration in the solvent is 50~55mg / L; L-methionine concentration in the solvent is 15~20mg / L; L-cystine dihydrochloride concentration in the solvent is 36~41mg / L; L-histidine hydrochloride concentration in the solvent is 24~29mg / L; L-tryptophan concentration in the solvent is 8~12mg / L; i-inositol concentration in the solvent is 10~20mg / L; The concentration of the phenol red in the solvent is 4-6 mg / L; the concentration of the nicotinamide in the solvent is 8-12 mg / L; the concentration of the choline chloride in the solvent is 2-3 mg / L; the concentration of the pyridoxine hydrochloride in the solvent is 2-3 mg / L; the concentration of the thiamine hydrochloride in the solvent is 2-3 mg / L; the concentration of the folic acid in the solvent is 2-3 mg / L; the concentration of the D-calcium pantothenate in the solvent is 2-3 mg / L; the concentration of the riboflavin in the solvent is 0.2-0.3 mg / L; and the concentration of the ferric nitrate in the solvent is 0.05-0.08 mg / L.

[0008] In one embodiment of the present invention, 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 phosphate monobasic (NaH2PO4-H2O, sodium dihydrogen phosphate monohydrate), and the concentration of sodium phosphate monobasic in the solvent is 248 ... potassium chloride in the solvent is 248 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the concentration of potassium chloride in the solvent is 248 mg / L; the The concentration of the magnesium sulfate in the solvent is 77.5 mg / L; the concentration of the magnesium sulfate in the solvent is 60.5554 mg / L; the concentration of the L-valine in the solvent is 58.28 mg / L; the concentration of the L-isoleucine in the solvent is 65.1 mg / L; the concentration of the L-leucine in the solvent is 65.1 mg / L; the concentration of the L-threonine in the solvent is 58.9 mg / L; the concentration of the L-lysine hydrochloride in the solvent is 90.52 mg / L; the concentration of the hydrochloric acid in the solvent is 0.361 mM; the concentration of the L-phenylalanine in the solvent is 40.92 mg / L; the concentration of the L-serine in the solvent is 26.04 mg / L; the L-tyrosine disodium The salt is L-tyrosine disodium salt dihydrate, and the concentration of the L-tyrosine disodium salt dihydrate in the solvent is 64.48 mg / L; the concentration of the L-arginine hydrochloride in the solvent is 52.08 mg / L; the concentration of the L-methionine in the solvent is 18.6 mg / L; the concentration of the L-cystine 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 the L-histidine hydrochloride-H2O in the solvent is 26.04 mg / L; the concentration of the L-tryptophan in the solvent is 9.92 mg / L; the concentration of the i-inositol in the solvent is 4.464 mg / 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

[0009] In one embodiment of the present invention, the components of the transfection reagent are 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-cystine dihydrochloride, L-histidine hydrochloride, L-tryptophan, i-inositol, phenol red, nicotinamide, choline chloride, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, calcium D-pantothenate, riboflavin and ferric nitrate.

[0010] In one embodiment of the present invention, the solvent comprises water.

[0011] The present invention also provides a cell transfection method, which is not for the purpose of disease diagnosis and treatment, and comprises: using the above transfection reagent to transfect host cells.

[0012] In one embodiment of the present invention, the method comprises the following steps:

[0013] Mixing step: mixing the target nucleic acid with the above transfection reagent to obtain a mixed solution;

[0014] Incubation step: adding the mixed solution to the host cells for incubation, and after the incubation, removing the mixed solution to obtain treated cells;

[0015] Transfection step: adding cell culture medium to the treated cells for culturing, and after the culturing is completed, transfected host cells are obtained.

[0016] In one embodiment of the present invention, in the mixing step, the concentration of the target nucleic acid in the transfection reagent is 150-200 pmol / mL.

[0017] In one embodiment of the present invention, in the incubation step, the incubation is: incubating at 20-25° C. for at least 5 minutes.

[0018] In one embodiment of the present invention, in the incubation step, the incubation is: incubating at 20-25° C. for 5-15 min.

[0019] In one embodiment of the present invention, in the incubation step, the incubation is: incubating at 20-25° C. for 10 min.

[0020] In one embodiment of the present invention, in the transfection step, the culturing is: culturing at 35-40° C. for at least 6 hours.

[0021] In one embodiment of the present invention, in the transfection step, the culturing is: culturing at 35-40° C. for 6-48 hours.

[0022] In one embodiment of the present invention, the target nucleic acid includes siRNA and / or miRNA.

[0023] In one embodiment of the present invention, the host cells include HEK 293T cells, Hepa1-6 cells, primary liver cells and / or C2C 12 cell.

[0024] In one embodiment of the present invention, the transfection is a transient transfection.

[0025] The present invention also provides a transfected cell, wherein the transfected cell is prepared by the above method.

[0026] The present invention also provides the use of the above transfection reagent or the above cell transfection method in cell transfection, and the use is not for the purpose of disease diagnosis and treatment.

[0027] The technical solution of the present invention has the following advantages:

[0028] The invention provides a transfection reagent, the components of which include a solvent, an organic matter, an amino acid, a vitamin and an inorganic salt; the organic matter includes D-glucose (glucose), sodium pyruvate, i-inositol (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-cystine dihydrochloride (L-cystine 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:

[0029] First, the study showed that the transfection reagent can effectively transfect the target nucleic acid into HEK 293T cells, Hepa1-6 cells, primary liver cells and C2C 12 It has the advantage of a wide range of applications in different types of difficult-to-transfect cells such as cells;

[0030] Second, the study showed that when the transfection reagent was used to transfect siRNA that inhibited the target gene in the host cells, the expression of the target gene could be observed to be significantly reduced 6 to 12 hours after transfection, and the target gene inhibition effect was close to that of transfection using Lipofectamine 2000, with the advantages of fast transfection onset time and good transfection effect;

[0031] Third, the entire transfection process of transfecting the target nucleic acid in the host cell using the transfection reagent does not require the use of special equipment, the operation steps are simple, and the transfection cost is low;

[0032] Fourth, the transfection reagent has good stability, which is conducive to market application;

[0033] Fifth, the components used in the transfection reagent are all inexpensive, further reducing the transfection cost. Therefore, the transfection reagent has great application prospects in cell transfection (especially transient cell transfection). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : qPCR was used to detect the amount of mir-378a transfected into HEK 293T cell line with different concentration gradients.

[0035] Figure 2 :qPCR was used to detect the amount of mir-156a transfected into HEK 293T cell line with different concentration gradients.

[0036] Figure 3 : qPCR was used to detect the amount of si-errα transfected into HEK 293T cell line with different concentration gradients.

[0037] Figure 4 :qPCR was used to detect the amount of si-egfr transfected with different concentration gradients into the Hepa1-6 cell line.

[0038] Figure 5 :The fluorescence signals of HEK 293T cells transfected with different concentration gradients of Cy3-labeled miRNA were detected by fluorescence microscopy.

[0039] Figure 6 : Western Blot was used to detect the expression of GAPDH protein at different time points after HEK 293T cell line was transfected with si-gapdh using the transfection reagent of Example 1.

[0040] Figure 7 : Figure 6 Quantitative analysis results of immunoblot bands.

[0041] Figure 8:Western Blot was used to detect the expression of GAPDH protein at different time points after HEK 293T cell line was transfected with si-gapdh using the transfection reagent Lipofectamine 2000.

[0042] Fig. 9 : Figure 8 Quantitative analysis results of immunoblot bands.

[0043] Fig.10 : Western Blot was used to detect the expression of GAPDH protein at different time points after primary liver cells were transfected with si-gapdh using the transfection reagent of Example 1.

[0044] Fig.11 : Fig.10 Quantitative analysis results of immunoblot bands.

[0045] Fig.12 :Western Blot was used to detect the expression of GAPDH protein at different time points after primary liver cells were transfected with si-gapdh using the transfection reagent Lipofectamine 2000.

[0046] Fig.13 : Fig.12 Quantitative analysis results of immunoblot bands.

[0047] Fig.14 :Western Blot detection of C2C cells after differentiation induced by using the transfection reagent of Example 1 12 The expression effect of GAPDH protein at different time points after cell lines were transfected with si-gapdh.

[0048] Fig.15 : Fig.14 Quantitative analysis results of immunoblot bands.

[0049] Fig.16 :Western Blot detection of C2C cells after differentiation using the transfection reagent Lipofectamine 2000 12 The expression effect of GAPDH protein at different time points after cell lines were transfected with si-gapdh.

[0050] Fig.17 : Fig.16 Quantitative analysis results of immunoblot bands.

[0051] Fig.18 :Different cell lines (HEK 293T cell line, C2C 12 Cell activity after co-incubation of the cell lines, HepG2 cell lines and 3T3L1 cell lines with the transfection reagent of Comparative Example 1.

[0052] Fig.19 : qPCR detection of the amount of 150pmol / mL si-egfr transfected into HEK 293T cell line.

[0053] Fig. 20 : qPCR detection of the amount of 150pmol / mL si-akt transfected into HEK 293T cell line. DETAILED DESCRIPTION

[0054] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0055] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0056] Example 1: A transfection reagent

[0057] The present 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 phosphate monobasic (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-silanol The invention comprises amino acid, L-tyrosine disodium salt dihydrate, L-arginine hydrochloride, L-methionine, L-cystine dihydrochloride (L-cystine 2 hydrochloride), L-histidine hydrochloride-H2O (L-histidine hydrochloride monohydrate), L-tryptophan, i-inositol (i-Inositol), phenol red, niacinamide, 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.

[0058] Table 1 Transfection reagent formula

[0059]

[0060] Comparative Example 1: A transfection reagent

[0061] The present comparative example 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 phosphate monobasic (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-silanol The formula is as shown in Table 2.

[0062] Table 2 Transfection reagent formula

[0063]

[0064] Comparative Example 2: A transfection reagent

[0065] The present comparative example 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 phosphate monobasic (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-silanol The formula is as shown in Table 3.

[0066] Table 3 Transfection reagent formula

[0067]

[0068] Example 2: A cell transfection method

[0069] This embodiment provides a cell transfection method, which comprises the following steps:

[0070] Mixing step: mixing the target nucleic acid with the transfection reagent of Example 1 so that the concentration of the target nucleic acid in the transfection reagent is 150 pmol / mL to obtain a mixed solution;

[0071] Incubation step: After adding the mixed solution to the host cells until the host cells are immersed, incubate at room temperature (25°C) for 10 minutes. After the 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 treated cells;

[0072] Transfection step: 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 immersed, and then cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 to obtain transfected host cells.

[0073] Comparative Example 1: A cell transfection method

[0074] This comparative example provides a cell transfection method, which is based on Example 2, except that the transfection reagent in Example 1 is replaced by the transfection reagent in Comparative Example 1.

[0075] Comparative Example 2: A cell transfection method

[0076] This comparative example provides a cell transfection method, which is based on Example 2, except that the transfection reagent in Example 1 is replaced by the transfection reagent in Comparative Example 2.

[0077] Experimental Example 1: Verification of transfection performance of transfection reagent

[0078] This experimental example verifies the transfection performance of the transfection reagent in Example 1, and the verification process is as follows:

[0079] 1. Synthesis of target nucleic acid

[0080] The mir-378a with a nucleotide sequence as shown in SEQ ID NO.1 (5'-CUGGACUUGGAGUCAGAAGG-3'), the mir-156a with a nucleotide sequence as shown in SEQ ID NO.2 (5'-UGACAGAAGAGAGUGAGCAC-CY3-3'), the si-errα with a nucleotide sequence as shown in SEQ ID NO.3 (5'-UGCACAUUGAAGAUGCUGA-3'), the si-egfr with a nucleotide sequence as shown in SEQ ID NO.4 (5'-GGAACUGGAUAUUCUGAAAdTdT-3', wherein dTdT is an overhanging fragment at the 3' end of siRNA), and the si-gapdh with a nucleotide sequence as shown in SEQ ID NO.5 (5'-CAGAAGACUGUGGAUGGCC-3') were used as target nucleic acids.

[0081] 2. Transfection of target nucleic acid

[0082] 2.1 Transfection of mir-378a, mir-156a and si-errα

[0083] HEK 293T cells (purchased from American Tissue Culture Collection) were cultured at 1 × 10 6The cells were plated in an amount of 100 cells / well and 2 mL of 10% (v / v) fetal bovine serum (FBS, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and 1% (w / v, g / 100 mL) double antibody (PENICILLIN STREPTOMYCIN, purchased from Gibco) in a 6-well plate containing DMEM (base) cell culture medium (purchased from Gibco), cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 12 h until the cell density reached more than 70%, then the cell culture medium was removed and washed three times with PBS buffer to obtain cells to be transfected; mir-378a, mir-156a and si-errα were added to the transfection reagent of Example 1 to concentrations of 40, 80, 160, 240, 400 and 640 pmol / ml, respectively, to obtain mir-378a mixed solutions with different concentration gradients, mir-156a mixed solutions with different concentration gradients and si-errα mixed solutions with different concentration gradients; the wells containing DMEM (base) cell culture medium without the target nucleic acid were added and the culture medium was removed. As blank control (Blank group), wells added with DMEM (base) cell culture medium containing 200 pmol / mL target nucleic acid were used as negative control (Ctrl group). 1 mL of mixed solutions of mir-378a with different concentration gradients, mir-156a with different concentration gradients, and si-errα with different concentration gradients were added to 6-well plates respectively. After incubation at room temperature (25°C) for 10 min, the mixed solutions were removed and washed three times with PBS buffer to obtain treated cells; 2 mL of DMEM (base) cell culture medium containing 2% (v / v) fetal bovine serum was added to 6-well plates at each well, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 to obtain HEK 293T cells transfected with different concentration gradients of mir-378a, HEK 293T cells transfected with different concentration gradients of mir-156a, and HEK 293T cells transfected with different concentration gradients of si-errα.

[0084] 2.2 Transfection of si-EGFR

[0085] The si-egfr was added to the transfection reagent of Example 1 to concentrations of 40, 80, 160, 240, 400, and 640 pmol / mL, respectively, to obtain si-egfr mixed solutions with different concentration gradients; referring to the transfection of mir-378a, mir-156a, and si-errα, si-egfr with different concentration gradients was transfected into the Hepa1-6 cell line (purchased from the American Tissue Culture Collection) to obtain Hepa1-6 cells transfected with si-egfr with different concentration gradients.

[0086] 2.3 Transfection of si-GAPDH

[0087] 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 the transfection reagent Lipofectamine 2000 (purchased from Thermo Fisher Scientific) as a transfection control, si-gapdh was transfected into the Hepa1-6 cell line (purchased from the American Tissue Culture Collection) with reference to the transfection of mir-378a, mir-156a and si-errα to obtain Hepa1-6 cells transfected with 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; the transfection reagent Lipofectamine 2000 (purchased from Thermo Fisher Scientific) was used as a transfection control, and si-gapdh was transfected into primary hepatocytes (primary hepatocytes were obtained from the liver of C57 mice, and C57 mice were purchased from Jicui Pharmaceutical Company. The extraction method of primary hepatocytes can be found 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 Experiments, (132).”) to obtain primary liver cells transfected with si-GAPDH.

[0089] 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; the transfection reagent Lipofectamine 2000 (purchased from Thermo Fisher Scientific) was used as a transfection control, and si-gapdh was transfected into C2C cells that had been induced to differentiate into myotubes with reference to the transfection of mir-378a, mir-156a, and si-errα. 12 Cell line (C2C 12 Cell lines were purchased from Wuhan Sewell Biotechnology Co., Ltd. 12The cell line differentiation induction method can be found in 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.

[0090] In the transfection control related experiments, when the transfection reagent Lipofectamine 2000 was used for transfection, si-gapdh was added to a mixed system of 6 μL of transfection reagent Lipofectamine 2000 and 2 mL of serum-free culture medium (purchased from Gibco) to obtain a si-gapdh mixed solution. In addition, the wells to which the serum-free culture medium without si-gapdh was added were used as blank controls, and the wells to which the serum-free culture medium containing 50 pmol / mL of the NC sequence (the nucleotide sequence is shown in SEQ ID NO. 6, 5'-UUCUCCGAACGUGUCACGUdTdT-3', wherein dTdT is the overhang fragment at the 3' end of the siRNA) was added were used as negative controls.

[0091] 3. Detection of different host cells

[0092] HEK 293T cells transfected with mir-378a at different concentrations, HEK 293T cells transfected with mir-156a at different concentrations, HEK 293T cells transfected with si-errα at different concentrations, and Hepa1-6 cells transfected with si-egfr at different concentrations were cultured for 1 hour, and qPCR (for the qPCR method, see the reference “Tan P, Pepin É, Lavoie JL. Mouse Adipose Tissue Collection and Processing for RNA Analysis. J Vis Exp. 2018 Jan 31;(131):57026.”) was used to detect the amount of different target nucleic acids entering the cells at different concentrations. The test results are shown in Figure 1~Figure 4 .Depend on Figure 1~Figure 4 It can be seen that as the amount of transfected target nucleic acid increases, the target nucleic acid entering the cell also increases in a gradient.

[0093] HEK 293T cells were incubated for 10 min in different concentration gradients of mir-156a (mir-156a was labeled with Cy3), the mixed solution was removed, and the cells were washed three times with PBS buffer to obtain 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 an addition volume of 2 mL per well, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 1 h to obtain HEK 293T cells transfected with different concentration gradients of mir-156a (addition of RNase A). The purpose of A is to remove the target nucleic acid that may adhere to the outside of the cell); remove the cell culture medium, wash with PBS buffer for 3 times to obtain cultured cells; add DAPI staining solution (purchased from Bio-Tech Biotech Co., Ltd.) to the 6-well plate at an amount of 2 mL per well, incubate at room temperature (25°C) in the dark for 15 minutes to stain the cell nucleus, and obtain stained cells; use a confocal microscope to detect the fluorescence signal intensity of the stained cells. The detection results are shown in Figure 5 . Figure 5 The results also confirmed that as the amount of target nucleic acid transfected increased, the target nucleic acid entering the cells also increased in a gradient.

[0094] Hepa1-6 cells transfected with si-gapdh, primary liver cells transfected with si-gapdh, and C2C cells transfected with si-gapdh obtained by culturing for 6 h, 12 h, 24 h, or 48 h were used. 12 The cells were transfected with si-gapdh and the expression of GAPDH protein at different time points was detected by Western Blot (the antibodies used in Western Blot were GAPDHRab Cat#A19056 and HSP90α / β Rab Cat#A5027. For the Western Blot method, please refer to the literature “Hnasko TS, Hnasko RM. The Western Blot. Methods Mol Biol. 2015;1318:87-96.”, where HSP90 was the reference protein). The grayscale analysis of the immunoblot bands was performed using image J to intuitively reflect the expression level. The detection results are shown in Figure 6~Figure 17 .like Figure 6~Figure 17 As shown, HEK 293T cells, Hepa1-6 cells, primary liver cells, and C2C cells induced to differentiate into myotubes 12The si-gapdh was transfected with the transfection reagent of Example 1 on the four different cell lines, and the transfection effect on different cell lines was detected by Western Blot. It can be found that the expression of the target gene can be observed to be significantly reduced in 6 to 12 hours using the transfection reagent of Example 1, and the target gene inhibition effect is close to the effect of transfection using the transfection reagent (Lipofectamine 2000). At this time, the total cost required for transfection using the transfection reagent of Example 1 is only half of that required for transfection using the transfection reagent (Lipofectamine 2000).

[0095] Experimental Example 2: Verification of transfection performance of transfection reagent

[0096] This experimental example verifies the transfection performance of the transfection reagent in Example 1, and the verification process is as follows:

[0097] Based on Experimental Example 1, HEK 293T cells were cultured at 1×10 6 The cells were plated in an amount of 100 cells / well and 2 mL of 10% (v / v) fetal bovine serum (FBS, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and 1% (w / v, g / 100 mL) double antibody (PENICILLIN STREPTOMYCIN, purchased from Gibco) in a 6-well plate containing DMEM (base) cell culture medium (purchased from Gibco), cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 12 hours until the cell density reaches more than 70%, then the cell culture medium is removed, and the cells are washed three times with PBS buffer to obtain cells to be transfected; using the transfection reagent of Example 1 as a control, the transfection reagent of Comparative Example 1 is added to the 6-well plate in an amount of 1 mL per well, incubated at room temperature (25°C) for 10 minutes, then the transfection reagent is removed, and the cells are washed three times with PBS buffer to obtain treated cells; 4 mg / mL trypan blue and PBS buffer are added to the 6-well plate in an amount of 2 mL per well, incubated at room temperature (25°C) for 5 minutes to stain the cells, and stained cells are obtained; the cell staining is observed using an inverted microscope, and the observation results are shown in FIG. Fig.18 .

[0098] Using the transfection reagent of Example 1 as a control, referring to the treatment method of HEK 293T cell line, C2C cells induced to differentiate into myotube cells were respectively 12 The same experiment was performed on the cell line, HepG2 cell line (purchased from the American Tissue Culture Collection), and 3T3L1 cell line (purchased from the American Tissue Culture Collection) to detect cell survival. The test results are shown in Fig.18 .

[0099] Fig.18The results showed that a large number of cells died after being treated with Comparative Example 1, so Comparative Example 1 is not suitable for cell transfection.

[0100] Experimental Example 3: Verification of transfection performance of transfection reagent

[0101] This experimental example verifies the transfection performance of the transfection reagent in Example 2, and the verification process is as follows:

[0102] Based on Experimental Example 1, HEK 293T cells were cultured at 1×10 6 The cells were plated in a 6-well plate with 2 mL of DMEM (base) cell culture medium (purchased from Gibco) containing 10% (v / v) fetal bovine serum (FBS, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and 1% (w / v, g / 100 mL) of double antibody (PENICILLIN STREPTOMYCIN, purchased from Gibco) added to each well. The cells were cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 12 h until the cell density reached more than 70%. The cell culture medium was removed and the cells were washed three times with PBS buffer to obtain the cells to be transfected. The 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. NO.7, 5'-CUGACCAAGAUGACAGCAU-3') were added to the transfection reagent of Comparative Example 1 to a concentration of 150pmol / ml to obtain si-egfr mixed solution and si-akt mixed solution; the wells added with DMEM (base) cell culture medium without the target nucleic acid were used as blank controls (Blank group), and the wells added with DMEM (base) cell culture medium containing target nucleic acids of different concentration gradients were used as negative controls (Ctrl group). The si-egfr mixed solution and the si-akt mixed solution were added to 6-well plates at an amount of 1mL per well, respectively, and incubated at room temperature (25°C) for 10min, then the mixed solution was 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 an amount of 2mL per well, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 to obtain HEK cells transfected with si-egfr. 293T cells and HEK 293T cells transfected with si-akt.

[0103] HEK 293T cells transfected with si-egfr and HEK 293T cells transfected with si-akt obtained after 24 h of culture were used to detect the amount of different target nucleic acids entering the cells. The test results are shown in Figure 19~Figure 20 .Depend on Figure 19~Figure 20It can be seen that the knockout of EGFR and AKT is poor, which shows that the transfection effect of the transfection reagent in Comparative Example 2 is poor.

[0104] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A transfection reagent, characterized in that The components of the transfection reagent are composed of solvent, organic matter, amino acid, vitamin and inorganic salt; the organic matter is composed of D-glucose, sodium pyruvate, i-inositol, phenol red and choline chloride; the amino acid is composed 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, L-histamine The invention relates to a novel nanostructured carbonyl chloride solvent comprising: a nanostructured carbonyl chloride salt and L-tryptophan; the vitamins comprise nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, folic acid, D-calcium pantothenate and riboflavin; the inorganic salts comprise 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- 2500mg / L; the concentration of D-glucose in the solvent is 2500~3000mg / L; the concentration of potassium chloride in the solvent is 200~300mg / L; the concentration of L-glutamine in the solvent is 300~400mg / L; the concentration of calcium chloride in the solvent is 100~150mg / L; the concentration of sodium pyruvate in the solvent is 60~75mg / L; the concentration of sodium dihydrogen phosphate in the solvent is 70~85mg / 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 35-45mg / L; the concentration of L-serine in the solvent is 20-30mg / L; the concentration of L-tyrosine disodium salt in the solvent is 60-70mg / L; the concentration of L-arginine hydrochloride in the solvent is 45-60mg / L; the concentration of L-methionine in the solvent is 15-25mg / L; the concentration of L-cystine dihydrochloride in the solvent is 35-45mg / L; the concentration of L-histidine hydrochloride in the solvent is 20-30mg / L; the concentration of L-tryptophan in the solvent is 8-15mg / L; the concentration of i-inositol in the solvent is The concentration in the solvent is 3-8 mg / L; the concentration of phenol red in the solvent is 8-15 mg / L; the concentration of nicotinamide in the solvent is 1-5 mg / L; the concentration of choline chloride in the solvent is 1-5 mg / L; the concentration of pyridoxine hydrochloride in the solvent is 1-5 mg / L; the concentration of thiamine hydrochloride in the solvent is 1-5 mg / L; the concentration of folic acid in the solvent is 1-5 mg / L; the concentration of D-calcium pantothenate in the solvent is 1-5 mg / L; the concentration of riboflavin in the solvent is 0.1-0.5 mg / L; the concentration of ferric nitrate in the solvent is 0.03-0.1 mg / L; the solvent is water. .

2. The transfection reagent according to 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; 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 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-cystine 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 4~6mg / L; the concentration of phenol red in the solvent is 8~12mg / L; the concentration of nicotinamide in the solvent is 2~3mg / L; the concentration of choline chloride in the solvent is 2~3mg / L; the concentration of pyridoxine hydrochloride in the solvent is 2~3mg / L; the concentration of thiamine hydrochloride in the solvent is 2~3mg / L; the concentration of folic acid in the solvent is 2~3mg / L; the concentration of D-calcium pantothenate in the solvent is 2~3mg / L; the concentration of riboflavin in the solvent is 0.2~0.3mg / L; the concentration of ferric nitrate in the solvent is 0.05~0.08mg / L.

3. The transfection reagent according to 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 acid is L-tyrosine disodium salt dihydrate, and the concentration of the L-tyrosine disodium salt dihydrate in the solvent is 64.48 mg / L; the concentration of the L-arginine hydrochloride in the solvent is 52.08 mg / L; the concentration of the L-methionine in the solvent is 18.6 mg / L; the concentration of the L-cystine dihydrochloride in the solvent is 39.06 mg / L; the L-histidine hydrochloride is L-histidine hydrochloride monohydrate, and the concentration of the L-histidine hydrochloride monohydrate in the solvent is 26.04 mg / L; the concentration of the L-tryptophan in the solvent is 9.92 mg / L; the concentration of the i-inositol in the solvent is 4.4 64mg / L; the concentration of phenol red in the solvent is 9.3mg / L; the concentration of nicotinamide in the solvent is 2.48mg / L; the concentration of choline chloride in the solvent is 2.48mg / L; the concentration of pyridoxine hydrochloride in the solvent is 2.48mg / L; the concentration of thiamine hydrochloride in the solvent is 2.48mg / L; the concentration of folic acid in the solvent is 2.48mg / L; the concentration of D-calcium pantothenate in the solvent is 2.48mg / L; the concentration of riboflavin in the solvent is 0.248mg / L; the ferric nitrate is ferric nitrate nonahydrate, and the concentration of ferric nitrate nonahydrate in the solvent is 0.062mg / L.

4. A cell transfection method, characterized in that: The method comprises: transfecting a host cell using the transfection reagent according to any one of claims 1 to 3.

5. The cell transfection method according to claim 4, characterized in that The method comprises the following steps: Mixing step: mixing the target nucleic acid with the transfection reagent according to any one of claims 1 to 3 to obtain a mixed solution; Incubation step: adding the mixed solution to the host cells for incubation, and after the incubation, removing the mixed solution to obtain treated cells; Transfection step: adding cell culture medium to the treated cells for culturing, and after the culturing is completed, transfected host cells are obtained.

6. The cell transfection method according to claim 5, characterized in that In the mixing step, the concentration of the target nucleic acid in the transfection reagent is 150-200 pmol / mL.

7. The cell transfection method according to claim 5 or 6, characterized in that: In the incubation step, the incubation is: incubating at 20-25°C for at least 5 minutes; in the transfection step, the culture is: culturing at 35-40°C for at least 6 hours.

8. Use of the transfection reagent according to any one of claims 1 to 3 or the cell transfection method according to any one of claims 4 to 7 in cell transfection.

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