CN118978583A

CN118978583A - A key factor for organoid culture R-spondin 1 and its preparation method and application

Info

Publication number: CN118978583A
Application number: CN202411459262.4A
Authority: CN
Inventors: 孙益军; 张玥; 张峰; 杨文骏; 李利云; 王琪玉
Original assignee: Jiangsu Keygen Biotech Corp ltd
Current assignee: Jiangsu Keygen Biotech Corp ltd
Priority date: 2024-10-18
Filing date: 2024-10-18
Publication date: 2024-11-19

Abstract

The present invention relates to the field of biotechnology, and discloses an organoid culture key factor R-spondin 1, a preparation method and an application thereof. The method comprises: constructing a nucleic acid for preparing the key factor R-spondin 1, optimizing the nucleic acid, and then connecting the nucleic acid with a plasmid by enzyme cutting to obtain a recombinant plasmid, transforming the recombinant plasmid into an Escherichia coli BL21 (DE3) strain, and culturing to obtain the organoid culture key factor R-spondin 1 for human colon organoid culture.

Description

Organ-like culture key factor R-spondin 1 and preparation method and application thereof

Technical Field

The invention particularly relates to an organoid culture key factor R-spondin 1, and a preparation method and application thereof, and belongs to the technical field of biology.

Background

The spondin 1 belongs to the Rspo family of Wnt modulators, which currently consists of four structurally related secretory ligands Rspo 1-4, all of which contain a furin-like domain and a thrombospondin domain. Rspo-1 is expressed in certain areas of the developing central nervous system, adrenal gland, ovary, testis, thyroid and trachea. Rspo interacts with the Frizzled/LRP6 receptor complex by stimulating the Wnt/β -catenin signaling pathway.

The organoids have wide application prospects in various fields such as stem cells and development, regenerative medicine, disease research, drug development, accurate medicine and the like, and at present, the organoids cultured in vitro can be continuously cultured for a plurality of weeks without passage. R-spondin 1 is taken as an activating factor of a classical Wnt signal path, is a key component of an organoid culture medium and supports sustainable long-term culture of organoids. By expressing a large amount of R-Spondin 1, a stable and high-purity protein sample can be provided, which is helpful for developing related experimental study, drug screening and clinical test. R-Spondin 1 was produced by recombinant techniques in order to investigate its signaling process and potential therapeutic value. Currently, the commercial recombinant human R-Spondin 1 protein is mainly derived from CHO cells and HEK293 cell lines. The major drawbacks of mammalian cell lines for production are high cost and low yield.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a preparation method of an organoid culture key factor R-spondin 1, and the organoid culture key factor R-spondin 1 with better functionality and higher stability is obtained.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, the present invention provides a method for preparing an organoid culture key factor R-spondin 1, comprising:

Constructing nucleic acid for preparing a key factor R-spondin 1, optimizing the nucleic acid, and enabling the optimized nucleotide sequence to be shown as SEQ ID NO. 4;

Connecting the optimized nucleic acid with the plasmid through enzyme digestion to obtain a recombinant plasmid;

transforming the recombinant plasmid into an escherichia coli BL21 (DE 3) strain;

culturing the transformed escherichia coli BL21 (DE 3) strain, and inducing the target protein to express to obtain the organoid culture key factor R-spondin 1, wherein the amino acid sequence of the organoid culture key factor R-spondin 1 is shown as SEQ ID NO. 1.

In combination with the first aspect, further, the optimized nucleic acid is connected with the plasmid through two restriction sites of NcoI and XhoI to obtain the recombinant plasmid.

Further, the plasmid is pET-28a (+), and the adopted promoter is T7 promoter.

Further, optimizing the nucleic acid, comprising:

The 5 'end of the nucleic acid is added with NcoI restriction enzyme site, and the 3' end is added with a stop codon and XhoI restriction enzyme site.

Further, the method further comprises: and adding a poly His tag between the 3' -end of the nucleic acid and a stop codon, wherein the amino acid sequence of the poly His tag is shown as SEQ ID NO. 2.

Further, transformation of the recombinant plasmid into E.coli BL21 (DE 3) strain comprises:

Mixing the recombinant plasmid with BL21 (DE 3) competent cells, and adding the mixture into an LB culture medium; wherein, the ratio of BL21 (DE 3) competent cells to recombinant plasmid number is not less than 250:1.

Further, the transformed escherichia coli BL21 (DE 3) strain is cultured, and the conditions for inducing the expression of the target protein are as follows: culturing at 25-28deg.C, not more than 220rpm, for at least 12 hr.

Further, after the target protein expression is induced, the organoid culture key factor R-spondin 1 is obtained through breaking thalli, separating and purifying, and His-tag affinity chromatography column is adopted for separating and purifying.

In a second aspect, the invention provides an organoid culture key factor R-spondin 1, prepared by any one of the methods described above.

In a third aspect, the invention provides the use of an organoid culture key factor R-Spondin 1, with the addition of R-Spondin 1 for human colonocytes culture.

Compared with the prior art, the invention has the beneficial effects that:

(1) Compared with R-spondin 1 prepared by using a eukaryotic expression system, the method for preparing the R-spondin 1 by using the escherichia coli expression system is simple, a large amount of R-spondin 1 can be obtained in a short time, the purity is up to more than 95%, and the expression amount is up to 1280mg/L and more;

(2) The R-spondin 1 prepared by using the escherichia coli expression system has lower cost and higher yield; the R-spondin 1 expression and purification process is simple to operate, good in repeatability and capable of being put into a mass production process.

Drawings

FIG. 1 is a diagram of plasmid pET-28a (+) provided by an embodiment of the present invention;

FIG. 2 is a schematic diagram of an optimized nucleic acid provided by an embodiment of the present invention;

FIG. 3 is a SDS-PAGE diagram of recombinant plasmid transformed E.coli BL21 (DE 3) strain and expressing R-Spondin 1 provided in the examples of the present invention;

FIG. 4 is a SDS-PAGE chart showing the mass expression and separation of R-Spondin 1 according to the embodiment of the present invention;

FIG. 5 is a SDS-PAGE diagram of R-Spondin 1 purified according to the present invention;

FIG. 6 is a SDS-PAGE of post-dialysis R-Spondin 1 according to an embodiment of the present invention;

FIG. 7 is a microscopic image of the application of R-Spondin 1 at different concentrations to organoid culture provided in the examples of the present invention.

Detailed Description

The invention is further described below. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.

Aiming at the problems of high cost and low yield caused by commercial recombinant human R-Spondin 1 protein mainly derived from CHO cells and HEK293 cell lines, the invention provides a preparation method of organ-like culture key factor R-Spondin 1, which utilizes escherichia coli to efficiently express R-Spondin 1, wherein the escherichia coli cell is BL21 (DE 3); the amino acid sequence of R-spondin 1 is shown as SEQ ID NO. 1.

The invention provides a organoid culture key factor R-spondin 1 and a preparation method and application thereof, and the organoid culture key factor R-spondin 1 comprises the following steps:

(1) Constructing and preparing a recombinant plasmid of R-spondin 1;

(2) Transforming the recombinant plasmid into escherichia coli competent BL21 (DE 3);

(3) R-spondin 1 recombinant protein is expressed in escherichia coli;

(4) Harvesting the recombinantly expressed R-spondin 1 from the e.coli inclusion bodies;

(5) Human colon organoid cultures after addition of R-Spondin 1.

The coding gene is constructed to an escherichia coli BL21 (DE 3) strain and is used for guiding the secretory expression of N protein.

Example 1: construction of recombinant plasmids.

Synthesizing a nucleotide for encoding R-spondin 1 by general biology (Anhui) Co., ltd, and adding a restriction enzyme NcoI cleavage site at the 5' -end of the nucleotide; the 3 'end is added with a stop codon TAA and a restriction enzyme XhoI restriction enzyme cleavage site, the 3' end of the nucleotide for synthesizing the R-spondin 1 is added with a poly His tag, the amino acid sequence of the obtained synthesized product is shown as SEQ ID NO. 3, the nucleotide sequence is shown as SEQ ID NO. 4, and the nucleotide sequence is shown as FIG. 2, and is a schematic diagram of the optimized nucleic acid.

The amino acid sequence of the poly His tag is shown as SEQ ID NO. 2.

The synthesized product is digested with restriction enzymes NcoI and XhoI, and then subjected to agarose gel electrophoresis, and the target fragment is recovered by gel cutting.

The target fragment is respectively connected with pET-28a (+) which is subjected to double digestion by restriction enzymes NcoI and XhoI to construct a recombinant plasmid, and the recombinant plasmid is named pET-28a-R-Spondin 1, his and Human as shown in figure 1.

The promoter employed may be a T7 promoter.

E.coli DH5 alpha is transformed by pET-28a-R-Spondin 1 and His, cultured for 16 hours at 37 ℃, single colony is selected for plasmid extraction, the extracted plasmid is subjected to agarose gel electrophoresis identification after double enzyme digestion by restriction enzymes NcoI and XhoI, positive clones are sent to general biology (Anhui) stock company for sequencing, clones with correct sequencing are selected for amplified extraction of the plasmid, the extracted plasmid is subjected to aseptic filtration, and the plasmid is preserved at-20 ℃ for standby.

Example 2: the recombinant plasmid transformed E.coli competent BL21 (DE 3) and expressed R-Spondin 1.

And (3) carrying out recombinant expression of the R-Spondin 1 by transforming the constructed recombinant plasmid pET-28a-R-Spondin 1, his and human into escherichia coli competent BL21 (DE 3). Wherein the ratio of BL21 (DE 3) competence to recombinant plasmid number is not less than 250:1, preferably 250:1.

Plasmids were transformed into BL21 competence and plated on Kana-resistant plates, overnight at 37℃and then each plate was inoculated with 31 mL of Kana-resistant LB liquid medium, cultured at 37℃and 220rpm until turbidity. Sampling before induction: taking 1mL of turbid bacterial liquid in an EP tube, centrifuging, discarding supernatant, and adding 1mLPBS for later use. Adding 0.5mM IPTG with final concentration into the culture solution of 3 tubes, respectively culturing at 16 deg.C and 28 deg.C and 37 deg.C for 12h, respectively taking 1mL of the induced culture solution, adding into an EP tube of 1.5mL, centrifuging, discarding the supernatant, adding 1mL of PBS, mixing uniformly, taking 500 μl of bacterial liquid from the test tube, adding into a centrifuge tube of 1.5mL, centrifuging at 6000r/min, removing the supernatant, and leaving precipitate. 500. Mu.L of 1xPBS was added to each centrifuge tube, the pellet was suspended, centrifuged at 6000r/min for 5min, and the supernatant was removed, leaving the pellet. 400. Mu.L of 1XPBS and 100. Mu.L of 5 XP Loading Buffer suspension sediment were added to each centrifuge tube and placed in a 95℃cooker for 10min. 10. Mu.L of the sample was loaded, and 3. Mu.L of the sample was added to the Maker, and the sample was subjected to SDS-PAGE under a constant pressure of 180V for 40 min. And (5) finishing gel electrophoresis, dyeing, decoloring and analyzing the result.

All samples were added to 10ul 5 x loading buffer, metal bath at 95 ℃ for 7min, running with 4% -20% pre-made gum. The results are shown in FIG. 3, wherein lane 1 shows untransformed whole bacteria, blank control, lane 2 shows whole bacteria after overnight induction disruption at 16 ℃, lane 3 shows whole bacteria after overnight induction disruption at 28 ℃, and lane 4 shows whole bacteria after overnight induction disruption at 37 ℃.

As can be seen from fig. 3: R-Spondin 1 was identified as having a distinct band of interest in E.coli, expressed at 16℃and 28℃and 37℃and subsequently amplified at 28℃to identify whether inclusion bodies or supernatants were expressed, followed by purification.

Example 3: large scale expression and isolation of R-Spondin 1.

PET-28a-R-Spondin 1, his, human was transformed into E.coli competent BL21 (DE 3), plated on Kana-resistant LB plates and incubated overnight at 37 ℃. Then, the monoclonal colony was picked up to 10mLLB (Kana 50 ug/mL), transferred to 1L LB (50 ug/mL) at 37℃overnight for 4 hours, cultured until the OD value was between 0.6 and 0.8, and 1mM IPTG was added at 25-28℃and at not more than 220rpm for at least 12 hours. The invention is preferably cultivated at 28℃for 12h.

The cultured bacterial liquid is collected and centrifuged at 8000rpm for 10min. Discarding the supernatant, adding the precipitate into buffer (50mM Tris,500mM NaCl,pH7.4), stirring for dissolving, and performing ultrasonic sterilization under the following conditions: ultrasound for 3s, pause for 5s, ultrasound for 30min. The following 3 samples were left: ① Whole bacteria after crushing: taking 40 mu L of ultrasonic bacterial suspension, and marking the sample as crushed whole bacteria; ② Supernatant after crushing: taking 100 μl of the disrupted bacterial suspension, 12000 rpm, centrifuging 10min, taking 40 μl of supernatant to another EP tube, and labeling the sample as disrupted supernatant; ③ Precipitation after crushing: after the supernatant was removed, the precipitate was blown off with 40. Mu.L Buffer (50mM Tris,500mM NaCl,pH7.4) solution.

SDS-PAGE identification: the above three samples were each added to 10. Mu.L of 5X Loading Buffer and placed in a 95℃cooker for 10min. 10. Mu.L of the sample was loaded, and 3. Mu.L of the sample was added to the Maker, and the sample was subjected to SDS-PAGE under a constant pressure of 180V for 40 min. The untransformed BL21 bacterial liquid was used as a blank for the identification. The results are shown in FIG. 4, lane 1 being a pilot control; lane 2 is whole bacteria after disruption; lane 3 is the supernatant after disruption; lane 4 is sediment after disruption; m represents a protein Marker.

As can be seen from FIG. 4, 1L amplification was successfully performed at 28℃and inclusion bodies were used for the next purification. The sonicated bacterial liquid was collected into a centrifuge tube, centrifuged at 8000rpm for 15min, the supernatant discarded and the pellet frozen at-80 ℃.

Example 4: the embodiment performs the purification and renaturation of inclusion bodies of R-spondin 1, and specifically comprises the following steps:

and step 1, washing inclusion bodies.

Inclusion bodies mainly contain recombinant proteins, but there are also some impurities such as some outer membrane proteins, plasmid DNA, etc., which can adhere to inclusion bodies, and most of the impurities can be removed by washing, but may not be removed cleanly.

In this example, the pellet was resuspended in Buffer (100 mM Tris,1%TritonX-114, pH 8.0) at a rate of 15mL/g and then disrupted by sonication (sonication for 3s, 6s at 60% power, 5 min) and centrifuged at 12000rpm for 30min at 4 ℃. Repeating once. The centrifuged pellet was resuspended in Buffer (50 mM Tris,PH 8.0) at a rate of 20mL/g and then broken with ultrasound (ultrasound for 3s, 6s at rest, 60% power, 5 min), and centrifuged at high speed at 12000rpm for 30min at 4 ℃. The supernatant was centrifuged and the pellet was separated. The centrifuged pellet was resuspended in Buffer (50 mM Tris,6M guanidine hydrochloride, pH 8.0) at a rate of 20mL/g overnight. Then, the mixture was centrifuged at a high speed at 12000rpm for 30 minutes at 4 ℃. The supernatant was filtered through a 0.45 μm membrane and purified.

Step 2, affinity purification of R-Spondin 1.

The invention removes incorrectly folded R-Spondin 1 by NI column affinity purification to obtain correctly folded protein. The purification steps are as follows:

1) Column balance: the column 5CV was rinsed with ddH2O and the column 5CV was equilibrated with a pre-prepared Buffer (50mM Tris,8M Urea,300mM Nacl,PH8.0); the transmittance (T) of the ultraviolet detector is adjusted to 100%, and then a proper measuring range is selected, and the application selects a measuring range of 0.5A and adjusts A to be 0.

2) Loading: the flow rate is 0.5-1CV/min, the peristaltic pump 1 pumps out about 0.5ml volume, the rotational speed of 1ml column is set to be 0.5-1rpm/min, the rotational speed of 5ml column is set to be 5-10rpm/min, and the flow-through liquid is collected.

3) Rebalancing: rebalancing 5CV with Buffer (50mM Tris,8M Urea,300mM Nacl,PH8.0) without balancing to UV absorbance to baseline;

4) Washing: 20/50/250/500mM Imidazole washing impurities, and eluting the target protein step by using Buffer（50mM Tris,8M Urea,20mM Imidazole,300mM Nacl,PH8.0）、Buffer（50mM Tris,8M Urea,50mM Imidazole,300mM Nacl,PH8.0）、Buffer（50mM Tris,8M Urea,250mM Imidazole,300mM Nacl,PH8.0）、Buffer（50mM Tris,8M Urea,500mM Imidazole,300mM Nacl,PH8.0）.

5) And (3) identification: the fractions were collected, E.coli loading and flow-through fractions were sampled at 5ul (15 ulPBS and 5. Mu.L of 5X Loading Buffer were added to the sample), and after mixing, boiled at 95℃for 10min, 25. Mu.L of loading was obtained, and Maker was added at 3. Mu.L. SDS-PAGE identification is performed at a constant pressure of 180V for 40min, and untransfected cells on the band are required to be used as blank controls for identification. As a result, as shown in FIG. 5, the target proteins were all in the eluting solution. In FIG. 5, lane 1 is the supernatant after inclusion body denaturation; lane 2 is fluid flow through; lane 3 is 20mM imidazole elution; lane 4 is 250mM imidazole elution-1; lane 5 is 250mM imidazole elution-2; lane 6 is 500mM elution; m represents a protein Marker.

And 3, renaturation of inclusion bodies.

Renaturation refers to the process of removing the denaturing agent under certain redox conditions so that the inactive protein returns to an active state by folding. The protein obtained above was purified according to 1:100 (50 mM Tris,0.5M L-arginine,0.5M Urea,2mM GSH,0.4 mM GSSG,150 mM NaCl PH8.0); placing into a refrigerator with the temperature of 4 ℃ for stirring, and dialyzing for 16 hours; the protein was slightly settled and centrifuged at 10000rpm for 30min, and the precipitate was removed. Protein samples were prepared according to 1:100 (50 mM Tris,0.2M L-arginine,0.5M Urea,2mM GSH,0.4 mM GSSG,150 mM NaCl PH8.0); placing into a refrigerator with the temperature of 4 ℃ for stirring, and dialyzing for 16 hours; the protein was slightly settled and centrifuged at 10000rpm for 30min, and the precipitate was removed.

The renaturation sample is prepared according to the following formula 1:100, adding into PBS, stirring in a refrigerator at 4deg.C, and dialyzing for 16 hr; the protein was slightly settled and centrifuged at 10000rpm for 30min, and the precipitate was removed. After centrifugation, filtration was performed, concentration was measured after filtration, and electrophoresis was performed according to the loading amounts of 2ug &4 ug. At this time, the morphology of the protein under the two conditions of Non-Reduced & Reduced is required to be identified, so that one sample is respectively put into two EP pipes when the sample is prepared, and is respectively added with reduction and Non-reduction Loading Buffer, SDS-PAGE electrophoresis identification is carried out, and the result is shown in FIG. 6, wherein lane 1 is 2 mug of post-dialysis R-Spondin 1, and the reduction condition is shown in the following; lane 2 is 2 μg post-dialysis R-Spondin 1, non-reducing conditions; m represents a protein Marker. As can be seen from fig. 6: the size of the purified protein band is 37kDa, accords with the theoretical molecular weight of the protein, has the purity of more than 95 percent and the expression quantity of 1280mg/L.

Example 5: human colon organoid cultures after addition of R-Spondin 1.

In this example, the recombinant protein R-Spondin 1 obtained was added to human colonocytes cultures at various concentrations, with the remaining added factors including WNT3A, EGF and Noggin. The method comprises the following steps:

Step 1, biopsy samples are used for separating human colon organoids, and the specific steps are as follows:

1) 100 μl matrigel was thawed on ice.

2) Pre-chilled D-PBS on ice and DMEM medium with 1% bsa added.

3) The 24-well plate was put into a 37℃cell incubator 2 hours in advance for pre-warming.

4) The tissue samples were washed with 10ml of ice PBS in a15 ml centrifuge tube, waiting for the samples to settle naturally, about 5s or so, and the supernatant was discarded.

5) Repeat step 4) leaving 1ml of liquid in the tube.

6) Transfer tissue and supernatant with a 1mL pipette into a sterile 1.5mL centrifuge tube.

7) The surgical shears thoroughly shear the tissue, and the 1mL pipettes transfer the tissue fragments to a new 15mL centrifuge tube, PBS wash.

8) Wait for the sample to settle naturally, discard the supernatant for about 5 s.

9) Adding 10mL GCDR reagent, incubating 30mi with 40rpm on ice,

10 290G centrifugal force, 5min centrifugal force, discard supernatant.

11 The suspension in the tube was aspirated with a 1mL pipette and added to a 70 mesh cell screen and filtered into a new 15mL centrifuge tube. The collection tube was washed with 1mL of DMEM+1% BSA and the wash was passed through a screen. Preparation for initiation of intestinal organoid culture.

Step 2, human colon crypt culturing organoids, comprising the following steps:

1) Counting the number of the crypts obtained in the separation in the step 1:

2) A centrifugal force of 200g was applied, and the mixture was centrifuged for 5min, most of the supernatant was discarded, and about 100. Mu.L of the supernatant was retained.

3) The 24-well plate was removed from the 37℃incubator, and the plate bottom was wetted with DMEM+1% BSA by pipetting with 200. Mu.L.

4) 100 Mu L of matrigel is added into a sample tube, blown up and down for 10 times, and thoroughly mixed and deposited, and no bubbles are generated.

5) Mu.L of matrigel crypt suspension was pipetted with a wet 200. Mu.L gun head and one drop was added to one of the eight wells in the center of the 24 well plate to prepare a culture hill.

6) Repeating step 5) until all hills are cultivated.

7) The plates were carefully transferred to a 37℃cell incubator and incubated for 10min to solidify the hills without disturbing.

8) 3ML intestinal organoid medium was prepared at room temperature at 15-25 ℃. For primary culture, 10. Mu.L of 3mM Y27632 (10. Mu.M final concentration) was added and mixed well.

9) 750 Μl of intestinal organoid medium was gently added to each well, and the gun tip did not touch the culture mound.

10 Sterile PBS was added to the surrounding wells.

11 Cover, 37 ℃,5% co2 incubator.

12 Every two days, the complete intestinal organoid culture medium was changed.

13 Passage(s)

Step 3, passaging human intestinal organoids, wherein the steps are as follows:

1) 2 hours in advance, a 37℃incubator pre-mixes 24 well plates.

2) Intestinal organoid medium was prepared and pre-warmed at room temperature.

3) Thawing on matrigel ice. 25 μl matrigel was required per well.

4) Dmem+1% bsa was precooled on ice.

5) The original medium per well was carefully aspirated away without disturbing the underlying culture mounds.

6) 1ML of GCDR solution at room temperature after equilibration was added to each well and incubated at room temperature for 1min.

7) After a 1mL gun head is moistened by GCDR reagents, the gun head is used for completely peeling off the culture hill from the plate bottom, GCDR reagents are blown up and down for 2-3 times, the culture hill is destroyed to release organoids, and the matrigel is ensured to be completely separated from the plate bottom.

8) The organoid mixture was transferred to a 15ml centrifuge tube without changing the tip.

9) Continuously adding 1mL GCDR reagent into the original hole, blowing and sucking GCDR reagent for 2-3 times by the wet gun head, thoroughly cleaning the bottom of the plate, and transferring into a 15mL centrifuge tube in the step 8.

10 Repeating steps 7) -9).

11 Room temperature, shaker 40rpm,10min incubation centrifuge tube 10min.

12 290G centrifugal force, 2-8 ℃ centrifugal force for 5min; the supernatant was gently discarded.

13 1ML cold dmem+1% bsa was added to each tube. The moist 1mL gun head stretches into the lower part of the liquid surface to gently blow off the organoids up and down, and the gun head does not need to stab against the pipe wall and the pipe bottom.

14 1ML pipette aspirate the mixture through a 70 mesh cell screen and collect in a 15mL centrifuge tube. 1mL cold DMEM+1% BSA washs the original 15mL centrifuge tube, the mixture was passed through a 70 mesh cell screen, and the filters were combined.

Step 4, observing with a microscope

Performing application of R-Spondin 1 with different concentrations to organoid culture by referring to the steps, and setting a test group 1, a test group 2 and a test group 3, wherein each group is provided with 4 parallel tests; wherein the experimental group 1 concentration: 20ng/ml; experimental group 2 was a blank control group, concentration: 0ng/ul; experimental group 3 concentration: 5ng/ul.

The results under a 40x microscope are shown in fig. 7, with 4 parallel runs of the first behavioral experimental group 1, 4 parallel runs of the second behavioral experimental group 2, and 4 parallel runs of the third behavioral experimental group 3. As can be seen from FIG. 7, the number of human colon organoids after 20ng/ml of R-Spondin 1 addition to culture was most significantly increased compared to the number of blanks.

The method for preparing the R-Spondin 1 by using escherichia coli has the advantages that the expression and purification process of the R-Spondin 1 is simple to operate, the repeatability is good, and the obtained R-Spondin 1 has good function and stability.

SEQ ID NO:1

SRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECSSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKESKEAGAGSRRRKGQQQQQQQGTVGPLTSAGPA

SEQ ID NO:2

HHHHHH

SEQ ID NO:3

SRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECSSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKESKEAGAGSRRRKGQQQQQQQGTVGPLTSAGPAHHHHHH

SEQ ID NO:4

atgagccgtggtattaagggtaaacgtcagcgtcgtattagtgccgaaggcagtcaggcctgcgcaaaaggctgtgaactgtgtagtgaagtgaatggttgtctgaaatgcagcccgaaactgtttattctgctggaacgtaatgatattcgccaggttggtgtttgcctgccgagttgtccgccgggttattttgatgcccgtaatccggatatgaataagtgcattaagtgtaaaatcgagcattgcgaagcctgttttagccataatttttgtaccaaatgcaaggaaggtctgtatctgcataaaggccgctgttatccggcctgcccggaaggtagtagcgccgcaaatggcacaatggaatgcagcagtccggcccagtgtgaaatgagcgaatggagcccgtggggcccgtgcagcaaaaaacagcagctgtgtggttttcgtcgcggcagtgaagaacgtacccgtcgtgtgctgcatgcaccggtgggcgatcatgccgcctgcagtgataccaaagaaacccgtcgttgcaccgtgcgtcgtgtgccgtgtccggaaggtcagaaacgtcgcaaaggcggtcagggtcgtcgtgaaaatgccaatcgtaatctggcacgcaaagaaagcaaagaagcaggcgccggtagtcgtcgtcgcaaaggtcagcagcagcagcaacagcagggcaccgtgggcccgctgaccagtgcaggtccggcacatcatcatcatcaccattaactcgagcaccaccaccaccaccac

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.

Claims

1. A method for preparing R-spondin 1, a key factor for organoid culture, comprising:

A nucleic acid for preparing the key factor R-spondin 1 was constructed, and the nucleic acid was optimized. The optimized nucleotide sequence is shown in SEQ ID NO.4;

The optimized nucleic acid and the plasmid are connected by enzyme digestion to obtain a recombinant plasmid;

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain;

The transformed Escherichia coli BL21 (DE3) strain was cultured to induce the expression of the target protein, and the key factor for organoid culture, R-spondin 1, was obtained, and its amino acid sequence is shown in SEQ ID NO.1.

2. The method according to claim 1 is characterized in that the optimized nucleic acid is connected to the plasmid through two restriction sites, NcoI and XhoI, to obtain a recombinant plasmid.

3. The method according to claim 1, characterized in that the plasmid is pET-28a(+) and the promoter used is T7 promoter.

4. The method according to claim 1, characterized in that the nucleic acid is optimized, comprising:

An NcoI restriction site is added to the 5' end of the nucleic acid, and a stop codon and an XhoI restriction site are added to the 3' end.

5. The method according to claim 4, characterized in that the method further comprises: adding a poly His tag between the 3' end of the nucleic acid and the stop codon, wherein the amino acid sequence of the poly His tag is shown in SEQ ID NO: 2.

6. The method according to claim 1 is characterized in that transforming the recombinant plasmid into the Escherichia coli BL21 (DE3) strain comprises: mixing the recombinant plasmid with BL21 (DE3) competent cells and then adding LB culture medium; wherein the ratio of the number of BL21 (DE3) competent cells to the recombinant plasmid is not less than 250:1.

7. The method according to claim 1, characterized in that the transformed Escherichia coli BL21 (DE3) strain is cultured and the conditions for inducing the expression of the target protein are in the range of 25-28°C, no more than 220 rpm, and at least 12 hours of culture.

8. The method according to claim 1, characterized in that after inducing the expression of the target protein, the bacteria are broken and separated and purified to obtain R-spondin 1, a key factor for organoid culture, which is separated and purified using a His-tag affinity chromatography column.

9. A key factor for organoid culture, R-spondin 1, characterized in that it is prepared by the method described in any one of claims 1 to 8.

10. An application of R-spondin 1, a key factor for organoid culture according to claim 9, characterized in that R-spondin 1 is added for human colon organoid culture.