阅读说明：本技术 重组人纤连蛋白及其制备、活性测定和稳定性实验方法 (Recombinant human fibronectin and preparation, activity determination and stability experimental method thereof ) 是由 刘传玉 王曼宇 李泽鹏 李晓晖 张俊梅 于 2021-06-23 设计创作，主要内容包括：本发明提供了一种重组人纤连蛋白及其制备、活性测定和稳定性实验方法,包括：利用大肠杆菌密码子的偏好性对重组人纤连蛋白进行密码子优化,构建重组表达载体。利用基因工程方法进行表达纯化,通过AKTA纯化仪使用PMB柱除细菌内毒素。经通过本发明的技术方案,重组人纤连蛋白纯化时间短,表达量高,纯度可达94%-96%,细菌内毒素含量低,更具安全性,同时稳定性较好,也具有促进细胞增殖活性。(The invention provides recombinant human fibronectin and a preparation, activity determination and stability test method thereof, wherein the preparation, activity determination and stability test method comprises the following steps: and (3) carrying out codon optimization on the recombinant human fibronectin by using the codon preference of escherichia coli to construct a recombinant expression vector. The expression and purification are carried out by using a genetic engineering method, and bacterial endotoxin is removed by using a PMB column through an AKTA purifier. Through the technical scheme of the invention, the recombinant human fibronectin has the advantages of short purification time, high expression quantity, purity of 94-96%, low content of bacterial endotoxin, safety, good stability and cell proliferation promoting activity.)

1. A recombinant human fibronectin (rhFN) having an amino acid sequence as shown in SEQ ID NO. 1.

2. The method of claim 1, wherein the method specifically comprises the steps of:

s1, expression; transferring the recombinant plasmid rhFN-pET28a (+) into Escherichia coli BL21(DE3) to obtain positive genetically engineered bacterium BL21(DE3)/pET28 a-rhFN; inoculating the positive transformant screened by the kanamycin-resistant LB plate into 10mL of kanamycin-resistant LB culture medium for overnight culture;

s2: inducing; transferring the strain the next day, culturing until logarithmic phase, adding inducer IPTG for induced fermentation, inducing at 20 deg.C for 16 hr, and centrifuging to collect thallus;

s3, identification; s2, respectively sampling 1.5mL of non-induced bacterial liquid and induced bacterial liquid in the induction process, centrifugally collecting thalli at 4 ℃, 12000 Xg and 5min, redissolving the thalli by 1.5mL of LPBS, ultrasonically crushing the thalli at low temperature, centrifugally separating supernatant and sediment at 4 ℃, 12000 Xg and 5min after completing ultrasonication, and redissolving the sediment by 1.5mL of LPBS; taking 32 mu L of non-induced bacterial liquid, crushed centrifugal supernatant and crushed centrifugal sediment samples, adding 5 times of protein loading buffer solution, and identifying the expression condition of the protein by SDS-PAGE;

s4: purifying; connecting an AKTA purifier with a Ni-NTA column, purifying by a step-by-step elution method, crushing thalli at low temperature, centrifugally collecting supernatant, and purifying by affinity chromatography to obtain recombinant human fibronectin;

s5: removing toxins; and (3) connecting a PMB column to remove bacterial endotoxin by using an AKTA purifier.

3. The method of claim 2, wherein step S2 comprises the following steps: transferring the strain to a 700 mLLB/bottle culture medium according to a final OD of 0.04 for culture until an OD600 reaches 0.6-0.8, adding isopropyl thiogalactoside (IPTG) with a final concentration of 0.5mmol/L for induction fermentation, wherein the IPTG is an inducer, inducing for 16 hours at 20 ℃, centrifuging and collecting thalli at 4 ℃ or more than 3000 Xg for 40min to obtain bacterial sludge, and the bacterial sludge obtaining amount is 3 g/L.

4. The method of claim 2, wherein step S4 comprises the following steps:

s41: carrying out heavy suspension on the bacterial sludge, and crushing the bacterial suspension by using an ultrasonic crusher or a high-pressure homogenizer after heavy suspension;

s42: adding the crushed suspension into a centrifuge tube, balancing the weight, centrifuging at 4 ℃ for 30min at a temperature of more than or equal to 25000 Xg, and collecting the supernatant;

s43: filtering the centrifuged supernatant of the bacterial liquid by using a suction filtration device, and filtering the supernatant by using a 0.22-micron filter membrane to obtain a protein stock solution for subsequent tests;

s44: connecting AKTA and Ni-NTA columns, and flushing the columns by using ultrapure water with the volume of 5-10 times of the column volume at the flow rate which is 50-80% of the maximum flow rate that the column material can bear;

s45, after the flow-through liquid conductivity value is close to 0 and the ultraviolet absorption value has no obvious change, using liquid A with 5-8 times of column volume to balance the column until the flow-through liquid conductivity value and the ultraviolet absorption value are stable and unchanged, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s46, after the column is well balanced, the sample loading is started, the flow rate is 10% -30% of the maximum flow rate that the column material can bear, if the protein stock solution is small in volume and high in concentration, the flow rate is properly reduced or the repeated sample loading times are increased, and when the ultraviolet absorption value of the flow-through solution starts to change, the sample loading flow-through solution is collected and sampled for SDS-PAGE electrophoresis;

s47: after the sample loading is finished, continuing to balance by using the solution A until the conductivity value and the ultraviolet absorption value of the flow-through liquid are close to the values before the sample loading and are stable, and stopping balancing; the balance flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s48: after the column is balanced, eluting by adopting a step-by-step elution mode; in the first step, 2% B and 98% A are used for elution, and the flow rate during elution is 30% -50% of the maximum flow rate which can be borne by the column material; collecting eluent when the ultraviolet absorption value begins to change, and sampling for SDS-PAGE electrophoresis;

s49: when the ultraviolet absorption value in the first step of elution in the step S48 does not change any more, performing the second step of elution by using 5% B and 95% A, wherein the flow rate in the elution is 30% -50% of the maximum flow rate which can be borne by the column material, collecting the eluent when the ultraviolet absorption value begins to change, and sampling for SDS-PAGE electrophoresis;

s410: when the ultraviolet absorption value in the second step of elution in the step S49 does not change any more, performing the third step of elution by using 30% B and 70% A, wherein the flow rate in the elution is 30% -50% of the maximum flow rate that the column material can bear, collecting the eluent when the ultraviolet absorption value begins to change, and sampling for performing SDS-PAGE electrophoresis; s411: when the ultraviolet absorption value at the time of elution in the third step in step S410 does not change any more, column washing is performed using 100% B, and proteins that have not yet been washed are washed away; the flow rate during elution is 30-50% of the maximum flow rate which can be borne by the column material;

s412: performing grey scale analysis on the SDS-PAGE electrophoresis result by using ClinXImageAnalysis, and calculating the protein purity;

s413: the concentration of the purified protein is measured by using an ultraviolet spectrophotometer;

s414: concentrating and replacing the purified protein by using a PALL30KD ultrafiltration tube; centrifuging at 4 deg.C for 4min at 3800 Xg, and replacing buffer with PBS (pH7.4); the concentrated protein is subjected to concentration determination by using an ultraviolet spectrophotometer again;

s415: and (3) taking one part of the concentrated protein for stability test, dividing the part into two parts, adding 2-5% trehalose and mannitol into one part, adding no protective agent into the other part, filtering and sterilizing the protein by using a 0.22 mu m filter, placing the protein in a refrigerator at 4 ℃ for overnight storage, and measuring the concentration the next day.

5. The method of claim 4, wherein step S5 comprises the following steps:

s51: connecting the PMB bacterial endotoxin removal purification column to AKTA, and washing the column by using a buffer solution C with 5 times of the column volume, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s52: using ultrapure water with 20 times of column volume to flush the column, wherein the flow rate is 50% -80% of the maximum flow rate which can be borne by the column material;

s53: when the conductance is close to 0 and the ultraviolet absorption value is not changed any more, using PBS with 3 times of column volume to balance the column, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s54: after the column is balanced, starting to load the sample, and circulating the sample protein overnight to remove bacterial endotoxin in the protein, wherein the flow rate is 10% -30% of the maximum flow rate which can be borne by the column material;

s55: washing out residual protein in the column by using PBS (phosphate buffer solution) until the electric conductivity and the ultraviolet absorption value are not changed any more, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s56: and (3) measuring the concentration of the protein after removing the bacterial endotoxin by using an ultraviolet spectrophotometer, calculating the recovery rate, adding trehalose and mannitol with the final concentration of 2-5%, and then subpackaging and freeze-drying.

6. The method of claim 4 or 5, wherein the buffer A is 20mM Tris, 50mM NaCl, pH 8.0; buffer B was 20mM Tris, 50mM NaCl, 500mM imidazole pH 8.0; buffer C was 20mMPB, 1% sodium deoxycholate, pH 7.0.

7. The method of claims 1-6, wherein the method for measuring the cell proliferation-promoting activity of recombinant human fibronectin comprises the steps of:

m1: BALB/c3T3 cells were seeded at 5000 cells/well in 96-well cell culture plates (complete medium) and cultured at 37 ℃ in a 5% CO2 cell incubator for 24 hours;

m2: replacing the maintenance culture medium and continuing to culture for 24 hours;

m3: adding recombinant human fibronectin and PBS (negative control group) with different concentrations, and culturing for 24 hr;

m4, adding 10 mu LCCK-8 reagent into each hole, incubating in a 5% CO2 cell incubator at 37 ℃ for 1 hour, and taking out;

m5, reading the absorbance value of the 96-well plate at 450nm by using a microplate reader, and recording the measurement result.

8. The method of claims 1-6, which specifically comprises the following steps:

p1: adding trehalose and mannitol as protective agents into the purified protein solution to enable the protein concentration to be 1mg/mL, standing at 4 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days, detecting the protein concentration and recording the result;

p2: adding trehalose and mannitol into the purified protein solution as protective agents, freeze-drying the protein solution on the same day, re-dissolving the freeze-dried protein to ensure that the protein concentration is 1mg/mL, respectively placing the protein solution at 25 ℃ and 37 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days, detecting the protein concentration and recording the result;

p3: trehalose, mannitol and 20% glycerol were added to the purified protein solution as a protective agent to give a protein concentration of 1mg/mL, and the protein concentration was measured and recorded after standing at 25 ℃ and 37 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days, respectively.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to recombinant human fibronectin and preparation, activity determination and stability test methods thereof.

Background

Fibronectin (FN, acronym) is a key adhesion protein found in blood, extracellular and pericellular matrices, and is a macromolecular glycoprotein with a molecular weight of about 440KD linked by almost two identical monomers via C-terminal disulfide bonds. Three types are mainly included: type I, type II, type III. Wherein type I and type II contain intrachain disulfide bonds and type III does not contain disulfide bonds, so that the disulfide bonds can be partially unfolded under the action of external force. Fibronectin (FN), whose main function is to enhance intercellular adhesion and adhesion between cells and a matrix, plays an important role in regulating cell adhesion, migration, proliferation, etc.

Most of the existing FN is natural fibronectin extracted from blood or tissues of animals, has limited yield and high cost, and limits the production and medical and cosmetic application of the fibronectin. FN is too large in molecular weight (consisting of more than 2000 amino acids) to be absorbed by the skin. The gene engineering technology is utilized to construct the micromolecule recombinant fibronectin which is similar to natural fibronectin in function, thereby effectively solving the defect that the natural fibronectin is overlarge in molecule and difficult to produce, simultaneously retaining the activity of the fibronectin and reducing the production cost.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides a recombinant human fibronectin and a preparation, activity determination and stability experimental method thereof.

The invention is realized by the following technical scheme: 1. a recombinant human fibronectin (rhFN) having an amino acid sequence as shown in SEQ ID NO. 1.

A preparation method of recombinant human fibronectin is characterized by comprising the following steps:

s1, expression; transferring the recombinant plasmid rhFN-pET28a (+) into Escherichia coli BL21(DE3) to obtain positive genetically engineered bacterium BL21(DE3)/pET28 a-rhFN; inoculating the positive transformant screened by the kanamycin-resistant LB plate into 10mL of kanamycin-resistant LB culture medium for overnight culture;

s2: inducing; transferring the strain the next day, culturing until logarithmic phase, adding inducer IPTG for induced fermentation, inducing at 20 deg.C for 16 hr, and centrifuging to collect thallus;

s3, identification; s2, respectively sampling 1.5mL of non-induced bacterial liquid and induced bacterial liquid in the induction process, centrifugally collecting thalli at 4 ℃, 12000 Xg and 5min, redissolving the thalli by 1.5mL of LPBS, ultrasonically crushing the thalli at low temperature, centrifugally separating supernatant and sediment at 4 ℃, 12000 Xg and 5min after completing ultrasonication, and redissolving the sediment by 1.5mL of LPBS; taking 32 mu L of non-induced bacterial liquid, crushed centrifugal supernatant and crushed centrifugal sediment samples, adding 5 times of protein loading buffer solution, and identifying the expression condition of the protein by SDS-PAGE;

s4: purifying; connecting an AKTA purifier with a Ni-NTA column, purifying by a step-by-step elution method, crushing thalli at low temperature, centrifugally collecting supernatant, and purifying by affinity chromatography to obtain recombinant human fibronectin;

s5: removing toxins; and (3) connecting a PMB column to remove bacterial endotoxin by using an AKTA purifier.

Preferably, the specific steps in step S2 are as follows: transferring the strain to a 700 mLLB/bottle culture medium according to a final OD of 0.04 for culture until an OD600 reaches 0.6-0.8, adding isopropyl thiogalactoside (IPTG) with a final concentration of 0.5mmol/L for induction fermentation, wherein the IPTG is an inducer, inducing for 16 hours at 20 ℃, centrifuging and collecting thalli at 4 ℃ or more than 3000 Xg for 40min to obtain bacterial sludge, and the bacterial sludge obtaining amount is 3 g/L.

Preferably, the specific steps in step S4 are as follows:

s41: carrying out heavy suspension on the bacterial sludge, and crushing the bacterial suspension by using an ultrasonic crusher or a high-pressure homogenizer after heavy suspension;

s42: adding the crushed suspension into a centrifuge tube, balancing the weight, centrifuging at 4 ℃ for 30min at a temperature of more than or equal to 25000 Xg, and collecting the supernatant;

s43: filtering the centrifuged supernatant of the bacterial liquid by using a suction filtration device, and filtering the supernatant by using a 0.22-micron filter membrane to obtain a protein stock solution for subsequent tests;

s44: connecting AKTA and Ni-NTA columns, and flushing the columns by using ultrapure water with the volume of 5-10 times of the column volume at the flow rate which is 50-80% of the maximum flow rate that the column material can bear;

s45, after the flow-through liquid conductivity value is close to 0 and the ultraviolet absorption value has no obvious change, using liquid A with 5-8 times of column volume to balance the column until the flow-through liquid conductivity value and the ultraviolet absorption value are stable and unchanged, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s46, after the column is well balanced, the sample loading is started, the flow rate is 10% -30% of the maximum flow rate that the column material can bear, if the protein stock solution is small in volume and high in concentration, the flow rate is properly reduced or the repeated sample loading times are increased, and when the ultraviolet absorption value of the flow-through solution starts to change, the sample loading flow-through solution is collected and sampled for SDS-PAGE electrophoresis;

s47: after the sample loading is finished, continuing to balance by using the solution A until the conductivity value and the ultraviolet absorption value of the flow-through liquid are close to the values before the sample loading and are stable, and stopping balancing; the balance flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s48: after the column is balanced, eluting by adopting a step-by-step elution mode; the first step was performed using 2% B and 98% a for elution with the aim of eluting the hetero-proteins. The flow rate during elution is 30-50% of the maximum flow rate which can be borne by the column material; collecting eluent when the ultraviolet absorption value begins to change, and sampling for SDS-PAGE electrophoresis;

s49: when the ultraviolet absorption value at the time of the first elution in step S48 did not change any more, a second elution was performed using 5% B and 95% A in order to elute hetero-proteins. The flow rate during elution is 30-50% of the maximum flow rate that the column material can bear. The eluate was collected at the beginning of the change in the UV absorbance and sampled for SDS-PAGE electrophoresis.

S410: when the ultraviolet absorption value at the time of the second elution in step S49 did not change any more, the third elution was carried out using 30% B and 70% A in order to elute the target protein. The flow rate during elution is 30-50% of the maximum flow rate that the column material can bear. Collecting eluent when the ultraviolet absorption value begins to change, and sampling for SDS-PAGE electrophoresis; as shown in fig. 4.

S411: when the ultraviolet absorption value at the time of elution in the third step in step S410 does not change any more, column washing is performed using 100% B, and proteins that have not yet been washed are washed away; the flow rate during elution is 30-50% of the maximum flow rate which can be borne by the column material;

s412: performing grey scale analysis on the SDS-PAGE electrophoresis result by using ClinXImageAnalysis, and calculating the protein purity; protein band analysis is shown in FIG. 5, and protein purity analysis is shown in FIG. 6.

S413: the concentration of the purified protein is measured by using an ultraviolet spectrophotometer;

s414: the purified protein was concentrated and changed using a PALL30KD ultrafilter tube. Centrifuging at 4 deg.C for 4min at 3800 Xg, and replacing buffer with PBS (pH7.4); the concentrated protein is subjected to concentration determination by using an ultraviolet spectrophotometer again;

s415: and (3) taking one part of the concentrated protein for stability test, dividing the part into two parts, adding 2-5% trehalose and mannitol into one part, adding no protective agent into the other part, filtering and sterilizing the protein by using a 0.22 mu m filter, placing the protein in a refrigerator at 4 ℃ for overnight storage, and measuring the concentration the next day.

Preferably, the specific steps in step S5 are as follows:

s51: connecting the PMB bacterial endotoxin removal purification column to AKTA, and washing the column by using a buffer solution C with 5 times of the column volume, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s52: using ultrapure water with 20 times of column volume to flush the column, wherein the flow rate is 50% -80% of the maximum flow rate which can be borne by the column material;

s53: when the conductance is close to 0 and the ultraviolet absorption value is not changed any more, using PBS with 3 times of column volume to balance the column, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s54: after the column is balanced, starting to load the sample, and circulating the sample protein overnight to remove bacterial endotoxin in the protein, wherein the flow rate is 10% -30% of the maximum flow rate which can be borne by the column material;

s55: washing out residual protein in the column by using PBS (phosphate buffer solution) until the electric conductivity and the ultraviolet absorption value are not changed any more, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material;

s56: and (3) measuring the concentration of the protein after removing the bacterial endotoxin by using an ultraviolet spectrophotometer, calculating the recovery rate, adding trehalose and mannitol with the final concentration of 2-5%, and then subpackaging and freeze-drying.

Preferably, buffer A is 20mM Tris, 50mM NaCl, pH 8.0; buffer B was 20mM Tris, 50mM NaCl, 500mM imidazole pH 8.0; buffer C was 20mMPB, 1% sodium deoxycholate, pH 7.0.

A method for measuring the cell proliferation promoting activity of recombinant human fibronectin comprises the following steps:

m1: BALB/c3T3 cells were plated at 5000 cells/well in 96-well cell culture plates (complete medium), 37 ℃ with 5% CO₂Culturing in a cell culture box for 24 hours;

m2: replacing the maintenance culture medium and continuing to culture for 24 hours;

m3: adding recombinant human fibronectin and PBS (negative control group) with different concentrations, and culturing for 24 hr;

m4, adding 10 mu LCCK-8 reagent into each hole, incubating in a 5% CO2 cell incubator at 37 ℃ for 1 hour, and taking out;

m5, reading the absorbance value of the 96-well plate at 450nm by using a microplate reader, and recording the measurement result.

A stability experiment method of recombinant human fibronectin specifically comprises the following steps:

p1: adding trehalose and mannitol as protective agents into the purified protein solution to enable the protein concentration to be 1mg/mL, standing at 4 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days, detecting the protein concentration and recording the result;

p2: adding trehalose and mannitol into the purified protein solution as protective agents, freeze-drying the protein solution on the same day, re-dissolving the freeze-dried protein to ensure that the protein concentration is 1mg/mL, respectively placing the protein solution at 25 ℃ and 37 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days, detecting the protein concentration and recording the result;

p3: trehalose, mannitol and 20% glycerol were added to the purified protein solution as a protective agent to give a protein concentration of 1mg/mL, and the protein concentration was measured and recorded after standing at 25 ℃ and 37 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days, respectively.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following beneficial effects: the recombinant fibronectin provided by the invention adopts an escherichia coli expression system, is soluble expression, and has the advantages of convenient preparation, simple purification process, lower cost and shorter production period.

1. The purification method provided by the invention adopts an ultrafiltration liquid exchange mode, can effectively shorten the time required by purification, and greatly shortens the production period.

The protein concentration of the rhFN purified protein can reach more than 1mg/ml, and the purity of the ClinxImageAnalyzis subjected to gray scale analysis is between 94% and 96%.

3. The purification method provided by the invention can effectively remove bacterial endotoxin in the protein, the recovery rate of the protein after the bacterial endotoxin removal is over 90 percent, the content of the bacterial endotoxin is lower than 1 EU/mu g, and the stimulation to the skin can be effectively reduced.

And 4, the rhFN has good stability after purification, the recovery rate of the protein can reach more than 97 percent when the rhFN is placed in PBS without any protective agent at 4 ℃ for one night, and the recovery rate can reach 99.5 percent when the rhFN is placed in the protective agent with trehalose and mannitol at 4 ℃ for one night.

5. The recombinant fibronectin provided by the invention can effectively promote cell proliferation and differentiation.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a map of the constructed recombinant plasmid rhFN-pET28a (+);

FIG. 2 is a graph showing the results of analysis of the phase expression of recombinant human fibronectin;

FIG. 3 is a graph showing the results of a soluble expression assay of recombinant human fibronectin;

FIG. 4 is a graph showing the results of purification of recombinant human fibronectin;

FIG. 5 is a graph of protein band analysis after rhFN purification;

FIG. 6 is a graph showing protein purity analysis after rhFN purification;

FIG. 7 is a graph showing the results of cell proliferation promotion by recombinant human fibronectin;

FIG. 8 is a graph showing the results of a stability experiment for recombinant human fibronectin, in which 1: standing the purified protein at 4 ℃; 2: standing the freeze-dried reconstituted protein at 25 ℃; 3: standing the freeze-dried reconstituted protein at 37 ℃; 4: preserving the protein with glycerol, and standing at 25 ℃; 5: glycerol preserved protein was left at 37 ℃.

Detailed Description

In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and thus the scope of the present invention is not limited by the specific embodiments disclosed below.

The recombinant human fibronectin and the methods for preparing, measuring the activity and testing the stability thereof according to the embodiments of the present invention will be described in detail with reference to FIGS. 1 to 8.

Example 1

Expression and identification of recombinant human fibronectin

Expressing: transferring the recombinant plasmid rhFN-pET28a (+) into escherichia coli BL21(DE3) to obtain positive genetic engineering bacteria BL21(DE3)/pET28a-rhFN, inoculating a positive transformant screened by a kanamycin-resistant LB flat plate into 10mL of kanamycin-resistant LB culture medium for overnight culture, transferring the positive transformant into 700 mLLB/bottle culture medium according to the final OD of 0.04 on the next day until the OD600 reaches 0.6-0.8, adding isopropyl thiogalactoside (IPTG) with the final concentration of 0.5mmol/L for induction fermentation, taking IPTG as an inducer, inducing for 16 hours at 20 ℃, centrifuging and collecting thalli at 4 ℃ of more than or equal to 3000 Xg for 40min to obtain bacterial sludge, wherein the bacterial sludge obtaining amount is 3 g/L. The expression phase is shown in FIG. 2, and the expression amount of the target protein increases with the increase of the induction time.

And (3) identification: sampling the non-induced bacterial liquid and the induced bacterial liquid respectively by 1.5mL in the induction process, centrifugally collecting thalli at 4 ℃, 12000 Xg and 5min, redissolving the thalli by 1.5mL of LPBS, carrying out low-temperature ultrasonic crushing on the thalli, centrifugally separating supernatant and sediment at 4 ℃, 12000 Xg and 5min after ultrasonic treatment, and redissolving the sediment by 1.5mL of LPBS. Taking 32 mu L of each of the non-induced bacterial liquid, the crushed centrifugal supernatant and the crushed centrifugal sediment sample, adding 5 Xprotein loading buffer solution, and identifying the expression condition of the protein by SDS-PAGE, wherein the target protein is mainly soluble expression as shown in figure 3.

Example 2

Purification of recombinant human fibronectin

Solution preparation

And (3) buffer solution A: 20mM Tris, 50mM NaCl, pH8.0

And (3) buffer solution B: 20mM Tris, 50mM NaCl, 500mM imidazole pH8.0

And (3) buffer C: 20mMPB, 1% sodium deoxycholate, pH7.0

During purification, an AKTA purifier is connected with a Ni-NTA column and purified by a step-by-step elution method.

(1) And (4) carrying out heavy suspension on the bacterial sludge, and crushing the bacterial suspension by using an ultrasonic crusher or a high-pressure homogenizer after the heavy suspension.

(2) Adding the crushed suspension into a centrifuge tube, balancing the weight, centrifuging at 4 ℃ for 30min at a temperature of more than or equal to 25000 Xg, and collecting the supernatant.

(3) And filtering the centrifuged supernatant of the bacterial liquid by using a suction filtration device, and filtering the supernatant by using a 0.22-micron filter membrane to obtain a protein stock solution for subsequent experiments.

(4) AKTA and Ni-NTA columns were connected, and the column was rinsed with 5-10 column volumes of ultrapure water. The flow rate is 50% -80% of the maximum flow rate that the column material can bear.

(5) And after the flow-through liquid conductivity value is close to 0 and the ultraviolet absorption value is not obviously changed, balancing the column by using the liquid A with the volume of 5-8 times of the column volume until the flow-through liquid conductivity value and the ultraviolet absorption value are stable and unchanged. The flow rate is 30-50% of the maximum flow rate that the column can withstand.

(6) And (3) after the column is balanced, starting to load the sample, wherein the flow rate is 10% -30% of the maximum flow rate which can be borne by the column material. If the protein stock solution is small in volume and high in concentration, the flow rate should be reduced or the number of times of repeated loading should be increased appropriately. When the UV absorbance of the flow-through began to change, the sample flow-through was collected and sampled for SDS-PAGE electrophoresis.

(7) And after the sample loading is finished, continuously using the liquid A for balancing until the conductivity value and the ultraviolet absorption value of the flow-through liquid are close to the values before the sample loading and are stable, and stopping balancing. The equilibrium flow rate is 30-50% of the maximum flow rate that the column can bear.

(8) After the column is balanced, elution is carried out in a step-by-step elution mode. The first step was performed using 2% B and 98% a for elution with the aim of eluting the hetero-proteins. The flow rate during elution is 30-50% of the maximum flow rate that the column material can bear. The eluate was collected at the beginning of the change in the UV absorbance and sampled for SDS-PAGE electrophoresis.

(9) When the UV absorbance of the first elution step did not change, a second elution step was performed using 5% B and 95% A in order to elute the contaminating proteins. The flow rate during elution is 30-50% of the maximum flow rate that the column material can bear. The eluate was collected at the beginning of the change in the UV absorbance and sampled for SDS-PAGE electrophoresis.

(10) When the ultraviolet absorption value at the second elution was not changed any more, the third elution was carried out using 30% B and 70% A in order to elute the target protein. The flow rate during elution is 30-50% of the maximum flow rate that the column material can bear. When the ultraviolet absorption value begins to change, the eluent is collected, and a sample is taken for SDS-PAGE electrophoresis, as shown in figure 4, the target protein is eluted in a large amount at 150mM imidazole, and the purity reaches more than 95%.

(11) When the UV absorbance at the third elution step did not change, the column was washed with 100% B to wash out the remaining proteins. The flow rate during elution is 30-50% of the maximum flow rate that the column material can bear.

(12) The protein purity was calculated by performing a gray scale analysis on the results of SDS-PAGE electrophoresis using ClinXImageAnalysis. The protein band analysis is shown in FIG. 5, and the software analysis result shows that 4 protein bands exist in the lane, wherein the 3 rd protein band is the target protein; the protein purity analysis is shown in FIG. 6, and the purity result of the target protein is 96%.

(13) The concentration of the purified protein was determined using an ultraviolet spectrophotometer.

(14) The purified protein was concentrated and changed using a PALL30KD ultrafilter tube. The centrifugation procedure was 4 ℃ at 3800 Xg for 4min, PBS was replaced with buffer, pH 7.4. And (4) measuring the concentration of the concentrated protein by using an ultraviolet spectrophotometer again.

(15) And (3) taking one part of the concentrated protein for stability test, dividing the part into two parts, adding 2-5% trehalose and mannitol into one part, adding no protective agent into the other part, filtering and sterilizing the protein by using a 0.22 mu m filter, placing the protein in a refrigerator at 4 ℃ for overnight storage, and measuring the concentration the next day.

Example 3

And (3) connecting a PMB column to remove bacterial endotoxin by using an AKTA purifier.

(1) The PMB bacterial endotoxin removal purification column was connected to AKTA and the column was washed with 5 column volumes of buffer C at a flow rate of 30-50% of the maximum flow rate that the column material can withstand.

(2) The column was rinsed with 20 column volumes of ultrapure water at a flow rate of 50% -80% of the maximum flow rate that the column material can withstand.

(3) When the conductance is close to 0 and the ultraviolet absorption value is not changed any more, the column is balanced by PBS with 3 times of the column volume, and the flow rate is 30-50% of the maximum flow rate which can be borne by the column material.

(4) After the column is equilibrated, the loading is started and the protein is cycled overnight to remove bacterial endotoxin from the protein at a flow rate of 10% to 30% of the maximum flow rate that the column can withstand.

(5) And (3) flushing out residual protein in the column by using PBS (phosphate buffer solution) until the electric conductivity and the ultraviolet absorption value are not changed any more, wherein the flow rate is 30-50% of the maximum flow rate which can be borne by the column material.

(6) And (3) measuring the concentration of the protein after removing the bacterial endotoxin by using an ultraviolet spectrophotometer, calculating the recovery rate, adding trehalose and mannitol with the final concentration of 2-5%, and then subpackaging and freeze-drying.

The recovery rate of the high-load metal ion chelating chromatography filler to the His-tag label protein is over 95 percent, and the recovery purity is over 90 percent. The His-Tag label improves the expression quantity and the solubility of the target protein and greatly simplifies the purification process of the human recombinant fibronectin.

Example 4

Recombinant human fibronectin cell proliferation-promoting Activity assay

The specific implementation process comprises the following steps:

(1) BALB/c3T3 cells were plated at 5000 cells/well in 96-well cell culture plates (complete medium), 37 ℃ with 5% CO₂The cell culture box was cultured for 24 hours.

(2) The culture was continued for 24 hours with replacement of the maintenance medium.

(3) Recombinant human fibronectin and PBS (negative control group) were added at different concentrations, respectively, and the culture was continued for 24 hours.

(4) Add 10. mu. LCCK-8 reagent to each well at 37 ℃ with 5% CO₂The cell incubator was incubated for 1 hour and then removed.

(5) And reading the light absorption value of the 96-well plate at 450nm by using a microplate reader, and recording the measurement result.

The results of the specific experiments are shown in FIG. 7, and the recombinant human fibronectin which is subjected to soluble expression by Escherichia coli has the function of cell proliferation after the nucleotide sequence is optimized, and the half effective concentration EC₅₀It was 0.01107 ng/mL.

Example 5

Recombinant human fibronectin stability assay

The specific implementation process comprises the following steps:

(1) trehalose and mannitol are added into the purified protein solution as protective agents to enable the protein concentration to be 1mg/mL, and the protein concentration is detected and recorded after the protein solution is placed at 4 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days.

(2) Trehalose and mannitol are added into the purified protein solution as protective agents, the protein solution is freeze-dried on the same day, the freeze-dried protein is redissolved to enable the protein concentration to be 1mg/mL, and the protein concentration is detected and recorded after the protein solution is respectively placed at 25 ℃ and 37 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days.

(3) Trehalose, mannitol and 20% glycerol were added to the purified protein solution as a protective agent to give a protein concentration of 1mg/mL, and the protein concentration was measured and recorded after standing at 25 ℃ and 37 ℃ for 0 day, 1 day, 3 days, 5 days and 7 days, respectively.

The specific result is shown in fig. 8, the protein of the present invention is stable in concentration and good in stability within 7 days when the protein is placed under various conditions.

Sequence listing

The amino acid sequence of recombinant human fibronectin is: (SEQ ID NO. 1):

MGEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVTTTPKNGPGPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTT

the nucleotide sequence which is not optimized is as follows (SEQ ID NO. 2):

ccatgggcgaaattgacaaaccatcccagatgcaagtgaccgatgttcaggacaacagcattagtgtcaagtggctgccttcaagttcccctgttactggttacagagtaaccaccactcccaaaaatggaccaggaccaacaaaaactaaaactgcaggtccagatcaaacagaaatgactattgaaggcttgcagcccacagtggagtatgtggttagtgtctatgctcagaatccaagcggagagagtcagcctctggttcagactgcagtaaccaacattgatcgccctaaaggactggcattcactgatgtggatgtcgattccatcaaaattgcttgggaaagcccacaggggcaagtttccaggtacagggtgacctactcgagccctgaggatggaatccatgagctattccctgcacctgatggtgaagaagacactgcagagctgcaaggcctcagaccgggttctgagtacacagtcagtgtggttgccttgcacgatgatatggagagccagcccctgattggaacccagtccacagctattcctgcaccaactgacctgaagttcactcaggtcacacccacaagcctgagcgcccagtggacaccacccaatgttcagctcactggatatcgagtgcgggtgacccccaaggagaagaccggaccaatgaaagaaatcaaccttgctcctgacagctcatccgtggttgtatcaggacttatggtggccaccaaatatgaagtgagtgtctatgctcttaaggacactttgacaagcagaccagctcagggagttgtcaccactctcgag

the optimized nucleotide sequence is as follows (SEQ ID NO. 3):

CCATGGGCGAAATTGATAAACCGAGTCAGATGCAGGTTACCGATGTGCAGGATAATAGCATTAGTGTTAAATGGCTGCCGAGCAGCAGCCCGGTGACCGGTTATCGTGTTACCACCACCCCGAAAAATGGCCCGGGTCCGACCAAAACCAAAACCGCCGGTCCGGATCAGACCGAAATGACCATTGAAGGCCTGCAGCCGACCGTGGAATATGTTGTTAGTGTGTATGCCCAGAATCCGAGCGGCGAAAGTCAGCCGCTGGTTCAGACCGCAGTTACCAATATTGATCGCCCGAAAGGTCTGGCATTCACTGATGTGGATGTTGATAGCATTAAGATTGCATGGGAAAGCCCGCAGGGTCAGGTTAGTCGCTATCGCGTGACCTATAGCAGCCCGGAAGATGGCATTCATGAACTGTTTCCGGCCCCGGATGGCGAAGAAGATACCGCCGAACTGCAGGGTCTGCGCCCGGGTAGCGAATATACCGTGAGTGTTGTGGCACTGCATGATGATATGGAAAGTCAGCCTCTGATTGGCACCCAGAGCACCGCCATTCCGGCACCGACCGATCTGAAATTCACTCAGGTTACCCCGACCAGTCTGAGCGCCCAGTGGACCCCGCCGAATGTTCAGCTGACCGGTTATCGCGTTCGCGTGACCCCGAAAGAAAAAACCGGCCCGATGAAAGAAATTAATCTGGCCCCGGATAGTAGTAGTGTTGTTGTTAGTGGTCTGATGGTTGCAACCAAATATGAAGTGAGTGTGTATGCGCTGAAAGATACCCTGACCAGTCGTCCGGCCCAGGGTGTTGTTACCACCCTCGAG

in the description herein, the description of the terms "one embodiment," "some embodiments," "specific embodiments," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Nicotikes biological Limited liability company

<120> recombinant human fibronectin and preparation, activity determination and stability experimental method thereof

<130> 3

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 272

<212> PRT

<213> Homo sapiens

<400> 1

Met Gly Glu Ile Asp Lys Pro Ser Gln Met Gln Val Thr Asp Val Gln

1 5 10 15

Asp Asn Ser Ile Ser Val Lys Trp Leu Pro Ser Ser Ser Pro Val Thr

20 25 30

Gly Tyr Arg Val Thr Thr Thr Pro Lys Asn Gly Pro Gly Pro Thr Lys

35 40 45

Thr Lys Thr Ala Gly Pro Asp Gln Thr Glu Met Thr Ile Glu Gly Leu

50 55 60

Gln Pro Thr Val Glu Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser

65 70 75 80

Gly Glu Ser Gln Pro Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg

85 90 95

Pro Lys Gly Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile

100 105 110

Ala Trp Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr

115 120 125

Ser Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly

130 135 140

Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr

145 150 155 160

Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro Leu

165 170 175

Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe

180 185 190

Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn

195 200 205

Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr

210 215 220

Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val

225 230 235 240

Val Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala

245 250 255

Leu Lys Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr

260 265 270

<210> 2

<211> 824

<212> DNA

<213> Homo sapiens

<400> 2

ccatgggcga aattgacaaa ccatcccaga tgcaagtgac cgatgttcag gacaacagca 60

ttagtgtcaa gtggctgcct tcaagttccc ctgttactgg ttacagagta accaccactc 120

ccaaaaatgg accaggacca acaaaaacta aaactgcagg tccagatcaa acagaaatga 180

ctattgaagg cttgcagccc acagtggagt atgtggttag tgtctatgct cagaatccaa 240

gcggagagag tcagcctctg gttcagactg cagtaaccaa cattgatcgc cctaaaggac 300

tggcattcac tgatgtggat gtcgattcca tcaaaattgc ttgggaaagc ccacaggggc 360

aagtttccag gtacagggtg acctactcga gccctgagga tggaatccat gagctattcc 420

ctgcacctga tggtgaagaa gacactgcag agctgcaagg cctcagaccg ggttctgagt 480

acacagtcag tgtggttgcc ttgcacgatg atatggagag ccagcccctg attggaaccc 540

agtccacagc tattcctgca ccaactgacc tgaagttcac tcaggtcaca cccacaagcc 600

tgagcgccca gtggacacca cccaatgttc agctcactgg atatcgagtg cgggtgaccc 660

ccaaggagaa gaccggacca atgaaagaaa tcaaccttgc tcctgacagc tcatccgtgg 720

ttgtatcagg acttatggtg gccaccaaat atgaagtgag tgtctatgct cttaaggaca 780

ctttgacaag cagaccagct cagggagttg tcaccactct cgag 824

<210> 3

<211> 824

<212> DNA

<213> Homo sapiens

<400> 3

ccatgggcga aattgataaa ccgagtcaga tgcaggttac cgatgtgcag gataatagca 60

ttagtgttaa atggctgccg agcagcagcc cggtgaccgg ttatcgtgtt accaccaccc 120

cgaaaaatgg cccgggtccg accaaaacca aaaccgccgg tccggatcag accgaaatga 180

ccattgaagg cctgcagccg accgtggaat atgttgttag tgtgtatgcc cagaatccga 240

gcggcgaaag tcagccgctg gttcagaccg cagttaccaa tattgatcgc ccgaaaggtc 300

tggcattcac tgatgtggat gttgatagca ttaagattgc atgggaaagc ccgcagggtc 360

aggttagtcg ctatcgcgtg acctatagca gcccggaaga tggcattcat gaactgtttc 420

cggccccgga tggcgaagaa gataccgccg aactgcaggg tctgcgcccg ggtagcgaat 480

ataccgtgag tgttgtggca ctgcatgatg atatggaaag tcagcctctg attggcaccc 540

agagcaccgc cattccggca ccgaccgatc tgaaattcac tcaggttacc ccgaccagtc 600

tgagcgccca gtggaccccg ccgaatgttc agctgaccgg ttatcgcgtt cgcgtgaccc 660

cgaaagaaaa aaccggcccg atgaaagaaa ttaatctggc cccggatagt agtagtgttg 720

ttgttagtgg tctgatggtt gcaaccaaat atgaagtgag tgtgtatgcg ctgaaagata 780

ccctgaccag tcgtccggcc cagggtgttg ttaccaccct cgag 824