Method for high-throughput detection of recombinant collagen expression level in engineering bacterium fermentation broth

Abstract

The invention provides a method for high throughput detection of recombinant collagen expression level in engineering bacteria fermentation broth, engineering bacteria and control bacteria for secretory expression of recombinant collagen are cultured, light absorption value detection of protein content in fermentation broth supernatant is directly carried out by using a Tianji red staining method, and detection liquid comprises Tianji red and acetic acid; subtracting the light absorption value of the control bacteria from the light absorption value of the engineering bacteria, and substituting into the standard curve to obtain the expression quantity of the target recombinant collagen. According to the method disclosed by the invention, the content of the protein in the supernatant of the fermentation liquor is directly and quantitatively detected by using a Tianqi red staining method, the expression level of the high-yield engineering strain of the recombinant collagen can be rapidly and efficiently detected, and the method has the advantages of simple steps, low cost, no need of large-scale equipment, high flux, high accuracy and no need of being limited to medicine control.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for high-throughput detection of recombinant collagen expression levels in engineered bacteria fermentation broth. Background Art

[0002] Collagen accounts for 25-30% of the total protein in the body and plays an important role in maintaining the normal physiological functions of cells, tissues, and organs. Collagen is widely used in biomedicine, cosmetics, biotechnology and other industries, but its source has become the main reason for the limited application of collagen. There are three main sources of collagen: (1) Traditional extraction method. Although the extraction process is mature and the activity is high, there is a risk of pathogenicity and immune rejection; (2) Chemical synthesis method solves the hidden dangers of immune rejection and virus, but the synthesis technology is relatively complex, costly, and has no biological activity; (3) Genetic engineering method. Recombinant collagen produced by genetic engineering technology has the advantages of high safety, batch stability, high activity, and no immune rejection, and has become an important research direction for collagen production.

[0003] The production of recombinant collagen usually relies on a variety of host systems, including but not limited to Escherichia coli, Pichia pastoris, mammalian cells, and transgenic plants and animals. However, no matter what host is used, the accurate detection of collagen expression levels becomes an indispensable part of the entire process. Especially when Pichia pastoris is used as the expression host, due to the integration of exogenous genes into the Pichia pastoris genome, strains with different gene doses may be produced, and the gene dose significantly affects the protein expression level of Pichia pastoris. However, the two are not a simple linear relationship, but show nonlinear dynamic changes. This complexity increases the workload of screening engineered bacteria with higher protein expression levels, resulting in a lot of time, manpower and material resources required to detect the expression level of recombinant collagen produced by Pichia pastoris.

[0004] At present, the quantitative detection methods for collagen mainly include Kjeldahl nitrogen determination, hydroxyproline spectrophotometry, SDS-PAGE, high performance liquid chromatography, mass spectrometry, enzyme-linked immunosorbent assay, collagen mimetic peptide hybridization, etc. However, most of the existing detection methods are for the identification of purified collagen, and have defects such as cumbersome determination steps, high cost, and dependence on large equipment, which makes it difficult to use for direct quantitative detection of large-scale production of recombinant collagen fermentation broth.

[0005] Sirius red is a strongly acidic anionic dye. Each molecule contains 6 sulfonic acid groups. It is easy to bind tightly to the basic amino acid groups of collagen. The slender dye molecules are attached to the collagen fibers in a parallel manner. After staining, it does not fade and has specificity. In 1964, Sweat first reported picric acid Sirius red (Sirius Red F3BA) as a histological specific staining method. Subsequently, Junqueira et al. improved this method and developed it into a classic collagen qualitative detection technology in the field of histology. There are currently two commercial kits based on Sirius red on the market, namely Sircol Soluble Collagen Assay and Sirius Red Total Collagen Assay. However, these kits have some inevitable defects, such as the need for time-consuming pre-treatment steps for samples, the high price of the kits and the need to improve the accuracy of the test results. In addition, these kits use picric acid as a solvent, and picric acid, as a controlled drug, has obstacles in promotion and application, and is not suitable for use in laboratory environments. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for high-throughput detection of recombinant collagen expression levels in engineered bacterial fermentation broth. The method uses Sirius red staining to directly quantitatively detect the amount of protein contained in the supernatant of the fermentation broth, and can quickly and efficiently detect the expression level of high-yield engineered strains of recombinant collagen protein. It also has the advantages of simple steps, low cost, no need for large equipment, high throughput, high accuracy, and no need to be restricted by controlled drugs.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A method for high-throughput detection of recombinant collagen expression levels in engineered bacteria fermentation broth, wherein the engineered bacteria and control bacteria used to secrete and express recombinant collagen are cultured, and the protein content in the fermentation broth supernatant is directly detected by the absorbance value using the Sirius red staining method, wherein the detection solution includes Sirius red and acetic acid; the absorbance value of the engineered bacteria is deducted from the absorbance value of the control bacteria, and the result is substituted into a standard curve to obtain the expression level of the target recombinant collagen.

[0009] As one of the preferred embodiments of the present invention, the engineered bacteria used for secreting and expressing recombinant collagen refers to a strain containing a target recombinant collagen expression vector; the control bacteria refers to a corresponding strain containing an empty vector.

[0010] As one of the preferred embodiments of the present invention, in the detection solution of the Sirius red staining method, the concentration of Sirius red is 50 μmol / L to 350 μmol / L.

[0011] As one of the preferred embodiments of the present invention, in the detection solution of the Sirius red staining method, the concentration of acetic acid is 0.05 mol / L to 2 mol / L.

[0012] As one of the preferred embodiments of the present invention, when the sample is detected by the Sirius red staining method, the volume ratio of the sample to the detection solution is 1:(4-12).

[0013] As one of the preferred embodiments of the present invention, when the sample is detected by the Sirius red staining method, the incubation time of the sample and the detection solution is 15 min to 360 min.

[0014] As one of the preferred embodiments of the present invention, the standard curve is obtained in the following manner: the absorbance value of the collagen standard is detected by the Sirius red staining method, and a standard curve is prepared according to the absorbance value and the concentration gradient.

[0015] As one of the preferred embodiments of the present invention, the collagen standard is bovine type I collagen.

[0016] As one of the preferred embodiments of the present invention, the method comprises the following specific steps:

[0017] (1) Preparing the detection solution: preparing the detection solution;

[0018] (2) Sample preparation: culturing the engineered bacteria for secreting and expressing recombinant collagen and the control bacteria, centrifuging after the fermentation, and saving the supernatant for later use;

[0019] (3) dyeing: the solutions obtained in steps (1) and (2) are mixed evenly and allowed to stand;

[0020] (4) Centrifugation: After staining is completed, centrifuge, discard the supernatant, and collect the complex precipitate;

[0021] (5) Washing: Rinse the precipitate, centrifuge, discard the supernatant, and wash away the unbound dye;

[0022] (6) Redissolution: Add NaOH solution to the collected complex precipitate for redissolution until the precipitate is completely dissolved;

[0023] (7) Detection: Detect absorbance;

[0024] (8) Calculation: Substitute the absorbance value of the engineered bacteria test into the standard curve after deducting the absorbance value of the control bacteria test. After calculation, the recombinant collagen concentration of the sample to be tested is obtained.

[0025] As one of the preferred embodiments of the present invention, the calculation formula is:

[0026] Y = a*X+b;

[0027] Wherein, "Y" represents the absorbance value; "X" represents the collagen concentration, mg / L; "a" represents the slope of the standard curve; and "b" represents the intercept of the standard curve.

[0028] As one of the preferred embodiments of the present invention, the recombinant collagen in the sample to be tested includes but is not limited to type I, type II, and type III collagen. Meanwhile, the sample to be tested in the present invention can also be collagen extracted from animal tissues.

[0029] The advantages of the present invention compared to the prior art are:

[0030] (1) The present invention can directly and quantitatively detect the protein content of the fermentation supernatant of the engineered bacteria expressing recombinant collagen (so far, there is no method that can directly and quantitatively detect the expression amount of recombinant collagen in the fermentation broth of the engineered bacteria), and can quickly and efficiently detect the expression level of the high-yield engineered strain of recombinant collagen, thus solving the problems of difficulty in detecting the high-yield strain of exogenous recombinant collagen expression and the large workload of detection;

[0031] (2) The Sirius red staining solution (Sirius red, acetic acid) used in the detection method of the present invention is inexpensive and highly specific, and does not require complicated pretreatment and purification steps for the collagen sample; compared with the cumbersome process of traditional SDS-PAGE detection, the present invention greatly saves manpower, material resources, and time investment; at the same time, the centrifuge and microplate reader used in the detection process are also two conventional laboratory instruments, and the present invention can achieve high-throughput, quantitative detection of the expression level of exogenous recombinant collagen;

[0032] (3) In the Sirius red staining method of the present invention, there is no need to use traditional picric acid controlled drugs, which is convenient for promotion and application;

[0033] (4) In the detection method of the present invention, the standard curve of Sirius Red for detecting collagen has good linear correlation in the range of 0 to 1000 mg / L, the detection result has high accuracy and is not affected by subjective factors. At the same time, there is a low coefficient of variation between parallel samples;

[0034] (5) The method of the present invention has a wide range of applications. The test samples that can be detected are collagen extracted from animal tissues or recombinant collagen expressed by various host systems. It is not limited by the collagen sequence and peptide chain length, and is not affected by the high viscosity of the collagen system. It can be widely used in the collagen industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a flow chart of the method for high-throughput detection of recombinant collagen expression level in the fermentation broth of engineered bacteria in Examples 1 to 3;

[0036] Figure 2is the standard curve of Sirius red for detecting collagen in Example 4;

[0037] Figure 3 This is the SDS-PAGE detection diagram of the fermentation supernatant of the recombinant strain GS115 / pPIC9K-II at a concentration of 0.5 mg / mL G418 in Experimental Example 1;

[0038] Figure 4 This is the SDS-PAGE detection diagram of the fermentation supernatant of the recombinant strain GS115 / pPIC9K-II at a concentration of 0.75 mg / mL G418 in Experimental Example 2;

[0039] Figure 5 This is the SDS-PAGE detection diagram of the fermentation supernatant of the recombinant strain GS115 / pPIC9K-II at a concentration of 1 mg / mL G418 in Experimental Example 3;

[0040] Figure 6 This is the SDS-PAGE detection diagram of the supernatant of the fermentation broth of the recombinant strain GS115 / pPIC9K-II at a concentration of 2 mg / mL G418 in Experimental Example 4;

[0041] Figure 7 This is the SDS-PAGE detection diagram of the supernatant of the fermentation broth of the recombinant strain GS115 / pPIC9K-II at a concentration of 3 mg / mL G418 in Experimental Example 5;

[0042] Figure 8 This is the SDS-PAGE detection diagram of the supernatant of the fermentation broth of the recombinant strain GS115 / pPIC9K-II at a concentration of 4 mg / mL G418 in Experimental Example 6. DETAILED DESCRIPTION

[0043] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0044] The host bacteria used in the following examples are Pichia pastoris GS115, the expression vector is pPIC9K, the engineered bacteria capable of secreting and expressing recombinant collagen are "Pichia pastoris recombinant type II collagen engineered bacteria (GS115 / pPIC9K-II)", and the control bacteria are "Pichia pastoris strain containing an empty plasmid (GS115 / pPIC9K)".

[0045] Among them, the construction process of Pichia pastoris recombinant type II collagen engineering bacteria is as follows: select the 2250bp to 2708bp of the cDNA encoding the human COL2A1 gene (see ncbi: NM_033150.3), add a 6×His tag sequence to the C-terminus, and then hand it over to a biological company for synthesis and construction on the expression vector pPIC9K, and then linearize the recombinant plasmid and introduce it into Pichia pastoris GS115 competent cells.

[0046] Construction of Pichia pastoris strain containing empty plasmid: pPIC9K plasmid was linearized and introduced into Pichia pastoris GS115 competent cells.

[0047] At the same time, among the materials used in the following examples, the collagen standard "bovine type I collagen" was purchased from the China Food and Drug Inspection Institute; other reagents or instruments used without indicating the manufacturer are conventional products that can be purchased through regular channels.

[0048] Example 1

[0049] A method for high-throughput detection of recombinant collagen expression level in an engineered bacterial fermentation broth in this embodiment is described in detail in Figure 1 , including the following steps:

[0050] (1) Preparation of detection solution: Prepare detection solution (50 μmol / L Sirius Red + 0.05 mol / L acetic acid) and precool it at 4°C.

[0051] (2) Sample preparation: The bacterial solution to be fermented (Pichia pastoris recombinant type II collagen engineering bacteria "GS115 / pPIC9K-II" and control bacteria "GS115 / pPIC9K") were inoculated into BMGY medium at a 2% inoculation rate, and cultured at 30°C, 240 rpm for 18 h; centrifuged at 5000 rpm, discarded the supernatant, and resuspended with an equal volume of BMMY, and fermented at 28°C, 240 rpm for 48 h; during the fermentation period, 100% methanol was added every 24 h to a final concentration of 1.5%; after 48 h of fermentation, the sample was collected, centrifuged at 5000 rpm, and the supernatant was stored for later use.

[0052] (3) Staining: 100 μL of sample was mixed with 400 μL of detection solution (volume ratio of 1:4) and incubated for 15 min. During this process, the dye and collagen formed a complex precipitate.

[0053] (4) Centrifugation: After staining is completed, centrifuge at 12000 rpm for 30 min, discard the supernatant, and collect the complex precipitate.

[0054] (5) Washing: Add 1000 μL of washing solution (0.5 mol / L acetic acid) to each tube to rinse the precipitate, centrifuge at 12000 rpm for 30 min, discard the supernatant, and wash away the unbound dye.

[0055] (6) Redissolution: Add 0.5 mol / L NaOH solution to the collected complex precipitate for redissolution until the precipitate is completely dissolved.

[0056] (7) Detection: The reconstituted liquid was transferred to a 96-well plate and the absorbance was detected at a wavelength of 540 nm using an ELISA reader.

[0057] (8) Calculation: Substitute the absorbance value of the engineered bacteria test into the standard curve after deducting the absorbance value of the control bacteria test. After calculation, the recombinant collagen concentration of the sample to be tested is obtained.

[0058] The calculation formula is:

[0059] Y = a*X+b;

[0060] Wherein, "Y" represents the absorbance value; "X" represents the collagen concentration, mg / L; "a" represents the slope of the standard curve; and "b" represents the intercept of the standard curve.

[0061] Example 2

[0062] A method for high-throughput detection of recombinant collagen expression level in an engineered bacterial fermentation broth in this embodiment is described in detail in Figure 1 , including the following steps:

[0063] (1) Preparation of detection solution: Prepare detection solution (300 μmol / L Sirius Red + 0.5 mol / L acetic acid) and precool it at 4°C.

[0064] (2) Sample preparation: The bacterial solution to be fermented (Pichia pastoris recombinant type II collagen engineering bacteria "GS115 / pPIC9K-II" and control bacteria "GS115 / pPIC9K") were inoculated into BMGY medium at a 2% inoculation rate, and cultured at 30°C, 240 rpm for 18 h; centrifuged at 5000 rpm, discarded the supernatant, and resuspended with an equal volume of BMMY, and fermented at 28°C, 240 rpm for 48 h; during the fermentation period, 100% methanol was added every 24 h to a final concentration of 1.5%; after 48 h of fermentation, the sample was collected, centrifuged at 5000 rpm, and the supernatant was stored for later use.

[0065] (3) Staining: Mix 100 μL of sample with 1000 μL of detection solution (volume ratio of 1:10) and incubate for 30 min. During this process, the dye and collagen form a complex precipitate.

[0066] (4) Centrifugation: After staining is completed, centrifuge at 12000 rpm for 30 min, discard the supernatant, and collect the complex precipitate.

[0067] (5) Washing: Add 1000 μL of washing solution (0.5 mol / L acetic acid) to each tube to rinse the precipitate, centrifuge at 12000 rpm for 30 min, discard the supernatant, and wash away the unbound dye.

[0068] (6) Redissolution: Add 0.5 mol / L NaOH solution to the collected complex precipitate for redissolution until the precipitate is completely dissolved.

[0069] (7) Detection: The reconstituted liquid was transferred to a 96-well plate and the absorbance was detected at a wavelength of 540 nm using an ELISA reader.

[0070] (8) Calculation: Substitute the absorbance value of the engineered bacteria test into the standard curve after deducting the absorbance value of the control bacteria test. After calculation, the recombinant collagen concentration of the sample to be tested is obtained.

[0071] The calculation formula is:

[0072] Y = a*X+b;

[0073] Wherein, "Y" represents the absorbance value; "X" represents the collagen concentration, mg / L; "a" represents the slope of the standard curve; and "b" represents the intercept of the standard curve.

[0074] Example 3

[0075] A method for high-throughput detection of recombinant collagen expression level in an engineered bacterial fermentation broth in this embodiment is described in detail in Figure 1 , including the following steps:

[0076] (1) Preparation of detection solution: Prepare detection solution (350 μmol / L Sirius Red + 2 mol / L acetic acid) and precool it at 4°C.

[0077] (2) Sample preparation: The bacterial solution to be fermented (Pichia pastoris recombinant type II collagen engineering bacteria "GS115 / pPIC9K-II" and control bacteria "GS115 / pPIC9K") were inoculated into BMGY medium at a 2% inoculation rate, and cultured at 30°C, 240 rpm for 18 h; centrifuged at 5000 rpm, discarded the supernatant, and resuspended with an equal volume of BMMY, and fermented at 28°C, 240 rpm for 48 h; during the fermentation period, 100% methanol was added every 24 h to a final concentration of 1.5%; after 48 h of fermentation, the sample was collected, centrifuged at 5000 rpm, and the supernatant was stored for later use.

[0078] (3) Staining: 100 μL of sample was mixed with 1200 μL of detection solution (volume ratio of 1:12) and incubated for 360 min. During this process, the dye and collagen formed a complex precipitate.

[0079] (4) Centrifugation: After staining is completed, centrifuge at 12000 rpm for 30 min, discard the supernatant, and collect the complex precipitate.

[0080] (5) Washing: Add 1000 μL of washing solution (0.5 mol / L acetic acid) to each tube to rinse the precipitate, centrifuge at 12000 rpm for 30 min, discard the supernatant, and wash away the unbound dye.

[0081] (6) Redissolution: Add 0.5 mol / L NaOH solution to the collected complex precipitate for redissolution until the precipitate is completely dissolved.

[0082] (7) Detection: The reconstituted liquid was transferred to a 96-well plate and the absorbance was detected at a wavelength of 540 nm using an ELISA reader.

[0083] (8) Calculation: Substitute the absorbance value of the engineered bacteria test into the standard curve after deducting the absorbance value of the control bacteria test. After calculation, the recombinant collagen concentration of the sample to be tested is obtained.

[0084] The calculation formula is:

[0085] Y = a*X+b;

[0086] Wherein, "Y" represents the absorbance value; "X" represents the collagen concentration, mg / L; "a" represents the slope of the standard curve; and "b" represents the intercept of the standard curve.

[0087] Example 4

[0088] A method of drawing a standard curve in the above-mentioned embodiments 1 to 3 of this embodiment:

[0089] The collagen standard is bovine type I collagen, and the standard is configured to concentrations of 0 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, and 1000 mg / L. 1000 μL of detection solution is added to each 100 μL of standard solution of different concentrations for staining (staining, washing, re-dissolving and detection steps are the same as in Example 2). A standard curve is drawn with the absorbance value as the ordinate and the concentration of the collagen standard as the abscissa, as shown in FIG. Figure 2 shown.

[0090] Experimental Example 1

[0091] Protein quantification of recombinant strain GS115 / pPIC9K-II at 0.5 mg / mL G418 concentration:

[0092] 1. Experimental methods:

[0093] Referring to the method of Example 2, the Pichia pastoris engineered bacteria obtained by G418 resistance screening was fermented and cultured. After the culture was completed, the culture was centrifuged and the supernatant was collected.

[0094] Mix the supernatant and protein loading buffer in a ratio of 4:1, and denature at 98℃ or in a boiling water bath for 10 minutes. Then, install the electrophoresis tank and gel plate, pour in the electrophoresis buffer, fill the inner tank to overflow, and the liquid level in the outer tank is higher than the electrode wire. Slowly remove the hole-making comb, and after the sample is loaded, close the tank cover, turn on the power, and electrophoresed at 90V for 35 minutes until the sample is compressed into a line. Increase the voltage to 190V and continue electrophoresis until the indicator band approaches or reaches the bottom of the gel. Stain the gel after electrophoresis with Coomassie Brilliant Blue. After decolorization with clean water, transfer the gel to the gel imaging system for photography, and use image J for quantification (1μg BSA as a control).

[0095] The steps of the Sirius Red detection method refer to Example 2, subtract the detection value of the control group from the detection value of the experimental group, and then substitute the value into the standard curve in Example 4 to obtain the detection concentration value.

[0096] 2. Experimental results:

[0097] The SDS-PAGE results of the supernatant of Pichia pastoris engineered strains screened at a concentration of 0.5 mg / mL G418 are as follows: Figure 3 As shown. A1 to A12 are sample numbers, the quantitative concentration (mg / L) is converted from the image J quantitative result, and the detection concentration (mg / L) is converted from the Sirius Red collagen detection result into the standard curve. It can be seen from the quantitative results, the detection results and the SDS-PAGE gel image that the present invention can directly quantitatively detect the protein content of the fermentation supernatant of Pichia pastoris expressing recombinant collagen.

[0098] Table 1 Sirius red detection and image J quantitative results of collagen at 0.5 mg / mL G418 concentration (mg / L)

[0099]

[0100] Experimental Example 2

[0101] Protein quantification of recombinant strain GS115 / pPIC9K-II at 0.75 mg / mL G418 concentration:

[0102] 1. Experimental methods:

[0103] Refer to Experimental Example 1.

[0104] 2. Experimental results:

[0105] The SDS-PAGE results of the supernatant of Pichia pastoris engineered bacteria screened at a concentration of 0.75 mg / mL G418 are as follows: Figure 4 As shown. Wherein, B1 to B12 are sample numbers, the quantitative concentration (mg / L) is converted from the image J quantitative result, and the detection concentration (mg / L) is converted from the Sirius Red collagen detection result into the standard curve. It can be seen from the quantitative results, the detection results and the SDS-PAGE gel image that the present invention can directly quantitatively detect the protein content of the fermentation supernatant of Pichia pastoris expressing recombinant collagen.

[0106] Table 2 Sirius red detection and image J quantitative results of collagen at 0.75 mg / mL G418 concentration (mg / L)

[0107]

[0108] Experimental Example 3

[0109] Protein quantification of recombinant strain GS115 / pPIC9K-II at 1 mg / mL G418 concentration:

[0110] 1. Experimental methods:

[0111] Refer to Experimental Example 1.

[0112] 2. Experimental results:

[0113] The SDS-PAGE results of the supernatant of Pichia pastoris engineered strains screened at a concentration of 1 mg / mL G418 are as follows: Figure 5 As shown. Wherein, C1-C12 are sample numbers, the quantitative concentration (mg / L) is converted from the image J quantitative result, and the detection concentration (mg / L) is converted from the Sirius Red collagen detection result into the standard curve. It can be seen from the quantitative results, the detection results and the SDS-PAGE gel image that the present invention can directly quantitatively detect the protein content of the fermentation supernatant of Pichia pastoris expressing recombinant collagen.

[0114] Table 3 Sirius red detection and image J quantitative results of collagen at 1 mg / mL G418 concentration (mg / L)

[0115]

[0116]

[0117] Experimental Example 4

[0118] Protein quantification of recombinant strain GS115 / pPIC9K-II at 2 mg / mL G418 concentration:

[0119] 1. Experimental methods:

[0120] Refer to Experimental Example 1.

[0121] 2. Experimental results:

[0122] The SDS-PAGE results of the supernatant of Pichia pastoris engineered bacteria screened at a concentration of 2 mg / mL G418 are as follows: Figure 6 As shown. Wherein, D1 to D12 are sample numbers, the quantitative concentration (mg / L) is converted from the image J quantitative result, and the detection concentration (mg / L) is converted from the Sirius Red collagen detection result into the standard curve. It can be seen from the quantitative results, the detection results and the SDS-PAGE gel image that the present invention can directly quantitatively detect the protein content of the fermentation supernatant of Pichia pastoris expressing recombinant collagen.

[0123] Table 4 Sirius red detection and image J quantitative results of collagen at 2 mg / mL G418 concentration (mg / L)

[0124]

[0125] Experimental Example 5

[0126] Protein quantification of recombinant strain GS115 / pPIC9K-II at 3 mg / mL G418 concentration:

[0127] 1. Experimental methods:

[0128] Refer to Experimental Example 1.

[0129] 2. Experimental results:

[0130] The SDS-PAGE results of the supernatant of Pichia pastoris engineered bacteria screened at a concentration of 3 mg / mL G418 are as follows: Figure 7 As shown. Among them, E1 to E12 are sample numbers, the quantitative concentration (mg / L) is converted from the image J quantitative result, and the detection concentration (mg / L) is converted from the Sirius Red collagen detection result into the standard curve. It can be seen from the quantitative results, the detection results and the SDS-PAGE gel image that the present invention can directly quantitatively detect the protein content of the fermentation supernatant of Pichia pastoris expressing recombinant collagen.

[0131] Table 5 Sirius red detection and image J quantitative results of collagen at 3 mg / mL G418 concentration (mg / L)

[0132]

[0133] Experimental Example 6

[0134] Protein quantification of recombinant strain GS115 / pPIC9K-II at 4 mg / mL G418 concentration:

[0135] 1. Experimental methods:

[0136] Refer to Experimental Example 1.

[0137] 2. Experimental results:

[0138] The SDS-PAGE results of the supernatant of Pichia pastoris engineered strains screened at a concentration of 4 mg / mL G418 are as follows: Figure 8 As shown. Wherein F1 to F12 are sample numbers, the quantitative concentration (mg / L) is converted from the image J quantitative result, and the detection concentration (mg / L) is converted from the Sirius Red collagen detection result into the standard curve. It can be seen from the quantitative results and the detection results as well as the SDS-PAGE gel image that the present invention can directly quantitatively detect the protein content of the fermentation supernatant of Pichia pastoris expressing recombinant collagen.

[0139] Table 6 Sirius red detection and image J quantitative results of collagen at 4 mg / mL G418 concentration (mg / L)

[0140]

[0141] Based on the above experimental examples, it can be seen from the quantitative results, the detection results and the SDS-PAGE gel image that the present invention can directly quantitatively detect the protein content of the fermentation supernatant of Pichia pastoris expressing recombinant collagen, and has high throughput, high accuracy, simple process, and time saving, and can quickly and efficiently detect the expression level of the recombinant collagen high-yield strain.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for high-throughput detection of recombinant collagen expression levels in engineered bacterial fermentation broth, characterized in that: The engineered bacteria and control bacteria used to secrete and express recombinant collagen are cultured, and the Sirius red staining method is used to directly detect the absorbance value of the protein content in the fermentation broth supernatant. The detection solution includes Sirius red and acetic acid. The absorbance value of the engineered bacteria is subtracted from the absorbance value of the control bacteria and substituted into the standard curve to obtain the expression amount of the target recombinant collagen.

2. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 1, characterized in that: The engineered bacteria used for secreting and expressing recombinant collagen refers to a strain containing a target recombinant collagen expression vector; the control bacteria refers to a corresponding strain containing an empty vector.

3. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 1, characterized in that: In the detection solution of the Sirius red staining method, the concentration of Sirius red is 50 μmol / L to 350 μmol / L.

4. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 1, characterized in that: In the detection solution of the Sirius red staining method, the concentration of acetic acid is 0.05 mol / L to 2 mol / L.

5. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 1, characterized in that: When the sample is detected by the Sirius red staining method, the volume ratio of the sample to the detection solution is 1:(4-12).

6. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 1, characterized in that: When the Sirius red staining method is used to detect samples, the incubation time of the sample and the detection solution is 15 minutes to 360 minutes.

7. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 1, characterized in that: The standard curve is obtained in the following manner: the absorbance value of the collagen standard is detected by using the Sirius red staining method, and the standard curve is prepared according to the absorbance value and the concentration gradient.

8. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 7, characterized in that: The collagen standard is bovine type I collagen.

9. The method for high-throughput detection of recombinant collagen expression level in an engineered bacterial fermentation broth according to any one of claims 1 to 8, characterized in that: The specific steps include: (1) Preparing the detection solution: preparing the detection solution; (2) Sample preparation: culturing the engineered bacteria for secreting and expressing recombinant collagen and the control bacteria, centrifuging after the fermentation, and saving the supernatant for later use; (3) dyeing: the solutions obtained in steps (1) and (2) are mixed evenly and allowed to stand; (4) Centrifugation: After staining is completed, centrifuge, discard the supernatant, and collect the complex precipitate; (5) Washing: Rinse the precipitate, centrifuge, discard the supernatant, and wash away the unbound dye; (6) Redissolution: Add NaOH solution to the collected complex precipitate for redissolution until the precipitate is completely dissolved; (7) Detection: Detect absorbance; (8) Calculation: Substitute the absorbance value of the engineered bacteria test into the standard curve after deducting the absorbance value of the control bacteria test. After calculation, the recombinant collagen concentration of the sample to be tested is obtained.

10. The method for high-throughput detection of recombinant collagen expression level in engineered bacteria fermentation broth according to claim 9, characterized in that: The calculation formula is: Y = a*X+b; In the formula, "Y" represents the absorbance value; "X" represents the collagen concentration, mg / L; "a" represents the slope of the standard curve; and "b" represents the intercept of the standard curve.

Images