CN114907467B

CN114907467B - Recombinant spider silk protein fused with carbon ends, preparation method thereof and drug-loaded microsphere based on recombinant spider silk protein

Info

Publication number: CN114907467B
Application number: CN202210521487.2A
Authority: CN
Inventors: 刘义; 黄维; 孟渂偲; 周科
Original assignee: Sichuan University of Science and Engineering
Current assignee: Sichuan University of Science and Engineering
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2023-09-15
Anticipated expiration: 2042-05-13
Also published as: CN114907467A

Abstract

The invention discloses a recombinant spider silk protein fused with a carbon end, a preparation method thereof and a drug-loaded microsphere based on the recombinant spider silk protein, and belongs to the field of protein preparation. The recombinant spider silk protein fused with the carbon end consists of 8 repeated core sequences and a carbon end sequence, and the amino acid sequence of the recombinant spider silk protein is shown as SEQ ID NO. 4. In order to realize simple expression of the recombinant spider silk protein, the recombinant spider silk protein MaSp2 (8R) +CT is cloned on a vector pCold I, so that the recombinant spider silk protein can be successfully induced and expressed without cloning a coding sequence of glycine-tRNA, and a method for purifying the recombinant spider silk protein on the vector pCold I is provided. Compared with the existing drug-loaded microsphere, the drug-loaded microsphere based on the recombinant spider silk protein has smaller toxicity, higher drug-loaded rate and release rate and good application prospect.

Description

Recombinant spider silk protein fused with carbon ends, preparation method thereof and drug-loaded microsphere based on recombinant spider silk protein

Technical Field

The invention belongs to the technical field of protein preparation, and particularly relates to a recombinant spider silk protein, in particular to a recombinant spider silk protein fused with a carbon end, a preparation method thereof and a drug-loaded microsphere based on the recombinant spider silk protein.

Background

Spider species are numerous, up to 4 tens of thousands, and are widely distributed around the world. Different kinds of spiders secrete different amounts and kinds of proteins, and some spiders can produce a variety of proteins, such as major ampullate spidroin protein (major ampullate spidroin, maSp) and minor ampullate spidroin protein (minor ampullate spidroin, miSp), etc. The major ampullate spidroin MaSp consists of two parts: maSp1 and MaSp2. The peptide chains of spider silk proteins have common characteristics in composition and structure, namely, the peptide chains all have repeated GGX (glycine region), poly (A)/poly (GA) (polyalanine) region, a spacer region and a non-repeated carbon end and nitrogen end, the nitrogen end has the function of inhibiting the aggregation of spider silk protein solution before spinning, the carbon end has the function of affecting the switching of the storage state and the assembly state of spider silk, controlling the solubility and the fiber forming property of the protein in an ion solution, promoting the formation of beta-folding, further affecting the fiber structure of the protein due to the secondary structure of the protein, and the carbon end has a larger influence on the performance of the protein. Current studies report that up to 4 repeats of MaSp1 can be expressed with carbon-terminal fusion proteins, and that no more multiple re-core sequences can be expressed.

The expression of the multiple recombinant spider silk protein is very dependent on the quantity of host synthesized transfer RNA because the spider silk protein contains multiple GGX (glycine region) and GPGXX (proline region) in the sequence, so that the requirement for amino acid G (glycine) and corresponding tRNA (transfer RNA) is very large during expression, but the resources for synthesizing glycine and tRNA by the host are limited, so that the expression of the target protein in the host is greatly limited, and the expression quantity is low or even not. The literature shows that common means for successfully expressing multiple rechecked sequences in pET-series vectors such as pET28, pET32, etc. are: the sequence capable of translating glycine-tRNA is introduced in cloning stage, and the induction temperature and rotation speed are reduced, so that the protein is expressed slowly. However, it is complicated to construct and requires multiple operations in multiple vectors. Meanwhile, the translation transfer tRNA consumes nutrition in a culture medium, so that the induction expression is large in volume and difficult to purify. In addition, the inducible expression of multiple recombinant spidroin proteins is dependent on glycine-tRNA, and thus, there is a need to find a recombinant spidroin protein that can be expressed easily.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a recombinant spider silk protein fused with a carbon end, a preparation method thereof and a drug-loaded microsphere based on the recombinant spider silk protein, and solve the problem of complex induction expression of the existing recombinant spider silk protein.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses a carbon-end-fused recombinant spider silk protein, which consists of a carbon-end sequence and one or more core sequences, wherein the core sequences are shown in SEQ ID NO.1, and the carbon-end sequences are shown in SEQ ID NO. 2.

Preferably, the amino acid sequence of the recombinant spider silk protein at the fusion carbon end is shown as SEQ ID NO.4, consists of 8 repeated core sequences and 1 carbon end sequence, and the nucleotide sequence of the recombinant spider silk protein at the fusion carbon end is shown as SEQ ID NO. 3.

The invention also discloses a preparation method of the recombinant spider silk protein fused with the carbon end, which comprises the following specific steps: the method comprises the steps of (1) connecting a fragment containing a carbon end sequence and a fragment containing one or more core sequences on a pET vector through enzyme digestion and connection, transferring the fragment to pColdI, incubating, transforming, inducing expression and purifying to obtain recombinant spider silk protein fused with the carbon end;

when there are a plurality of core sequences, the core sequence multiplication process is required for the core sequences.

Preferably, the transfer is performed at the cleavage site of the common restriction enzyme of pET vector and pColdI.

Preferably, the purification comprises a crude separation comprising steps of sonication, centrifugation and thermal denaturation and a column purification.

Further preferably, the temperature of the thermal denaturation is 30-60℃for 30min.

Further preferably, the temperature of the thermal denaturation is 40℃or 50℃and the centrifugal force after the thermal denaturation is 20000g.

The invention also discloses application of the recombinant spider silk protein fused with the carbon end in preparation of biological materials.

The invention also discloses a drug-loaded microsphere which is prepared by dissolving the recombinant spider silk protein fused with the carbon end in potassium phosphate buffer solution.

Preferably, the pH of the potassium phosphate buffer is 7.

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

the recombinant spider silk protein fused with the carbon end provided by the invention consists of the repetitive core sequence and the carbon end sequence, the storage state and the assembly state of the spider silk can be influenced by the action of the carbon end sequence, the dissolubility and the fiber forming property of the protein in an ion solution are controlled, the formation of beta-sheet is promoted, the secondary structure of the protein is influenced, the fiber structure of the protein is further influenced, and compared with the existing recombinant spider silk protein, a plurality of repetitive core sequences express more repetitive core sequences, so that the expression is simpler. The recombinant spider silk protein can be applied to the field of biological material preparation and has good application prospect.

The preparation method of the recombinant spider silk protein with the fused carbon end is different from the complex method that in the prior art, a sequence capable of translating glycine-tRNA is introduced on pET series vectors, and simultaneously, the induction temperature and the rotation speed are reduced, so that the protein is expressed slowly.

Compared with the existing drug-loaded microsphere, the drug-loaded microsphere provided by the invention has the advantages that the toxicity is smaller, the drug-loaded rate can reach 75%, the release rate is higher than 40% -60% of the release rate in the prior art, the release rate is higher than 60% of the release rate in the prior art, and meanwhile, the preparation method of the drug-loaded microsphere is simple and the application prospect is wide.

Drawings

FIG. 1 is an electrophoretogram of recombinant spider silk protein with fused carbon ends constructed by the vector pColdI of the present invention; wherein, FIG. A is a double-digestion electrophoresis chart of pCold I (1 kb plus (marker), pCold I and pCold I-NdI+HindIII in sequence from left to right), FIG. B is a double-digestion and single-digestion product comparison chart of pCold I (1 kbplus (marker), pCold I-HindIII and pCold I-NdI+HindIII in sequence from left to right), FIG. C is a MaSp2 (8R) +CT double-digestion electrophoresis chart (marker, maSp2 (8R) -NdI+HindIII and MaSp2 (8R) +CT-NdI in sequence from left to right), and FIG. D is a MaSp2 (8R) +CT double-digestion recovery electrophoresis chart (1 kbplus (marker), maSp2 (8R) +CT-NdI+III (1343/5304) and MaSp2 (8R) +CT-NdI+HindIII in sequence from left to right;

FIG. 2 is a diagram showing the identification of NdeI single enzyme digestion of recombinant spidroin fused to the carbon end on pColdI of the present invention; wherein, D15000+2000 (marker), maSp2 (8R) +CT and MaSp2 (8R) +CT-NdeI are sequentially arranged from left to right;

FIG. 3 is an electrophoretogram of recombinant spider silk protein expression on vector pColdI of the present invention; wherein, the left side is the strip of marker, and the unit is: kDa, Y represents induction;

FIG. 4 shows denaturation of protein solutions at different heat denaturation temperatures according to the present invention; wherein, the left side is the strip of marker, and the unit is: kDa, the upper side is the name of the protein sample of each lane, and Y represents induction, A is at 30℃and 40℃temperature, B is at 50℃and 60℃temperature, and C is at 70℃and 80℃temperature, corresponding to the upper side label;

FIG. 5 shows recovery of the fusion carbon-terminal recombinant spider silk protein MaSp2 (8R) +CT-pColdI of the present invention; wherein, the left side is the strip of marker, and the unit is: kDa, Y represents induction, A is the case of the target proteins of the eluents E1-E4, and B is the case of the target proteins of the washings W1-W3;

FIG. 6 is a fluorescent image of blank microspheres and drug-loaded microspheres of the present invention;

FIG. 7 is a scanning electron microscope image of blank microspheres and drug-loaded microspheres of the present invention;

FIG. 8 is a standard graph of doxorubicin according to the present invention;

FIG. 9 is a graph showing a comparison of drug loading of microspheres at two different pH values according to the present invention; wherein, the gray bar graph is group a, ph=4, the white bar graph is group B, ph=7;

fig. 10 is a graph showing the release rate and release profile of microspheres of group B (ph=7) according to the invention; wherein, graph a shows the release rate of PBS at ph=4.5 for 48h at 37 ℃, and graph B shows the release curves of group B microspheres at ph=4.5, 6.0 and 7.2, respectively;

fig. 11 is a microsphere cytotoxicity test of group B (ph=7) according to the present invention.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the attached drawing figures:

in the present invention, maSp2 (nR) represents MaSp2 having n core sequences, and MaSp2 (CT) represents MaSp2 having a carbon-terminal sequence.

As shown in Table 1, the recombinant spider silk protein (named MaSp2 (8R) +CT) fused with the carbon end provided by the invention consists of 8 repeated core sequences and a carbon end sequence, wherein the core sequence of the recombinant spider silk protein is shown as SEQ ID NO.1, the carbon end sequence is shown as SEQ ID NO.2, the nucleotide sequence for encoding the recombinant spider silk protein is shown as SEQ ID NO.3, and the amino acid sequence of the recombinant spider silk protein is shown as SEQ ID NO. 4.

Table 1 sequence listing

TABLE 2 amino acid sequence listing

The preparation method of the recombinant spider silk protein fused with the carbon end provided by the invention comprises the following specific steps:

experimental materials:

vector pET28-a (+) is purchased from Beijing Saint gene technology Co., ltd; vector pCold I was purchased from TakaRa; the core sequence of the recombinant spider silk protein (MaSp 2 (1R)) and the carbon end sequence of the recombinant spider silk protein (MaSp 2 (CT)) are purchased from Beijing Sebai Gene technologies Co., ltd; the reagent formula for recovering the magnetic beads comprises: lysate 1:50mM Tris-HCl, 500mM NaCl, 5mM midazole, pH 8.0; washing liquid 2:50mM Tris-HCl, 500mM NaCl, 20mM midazole, pH 8.0; eluent 3:50mM Tris-HCl, 500mM NaCl, 250mM midazole, pH 8.0.

1. Construction

1) Preparation of MaSp2 (8R)

Carrying out enzyme digestion on a vector pET28-a (+) by taking a plasmid of MaSp2 (1R) as a starting point and using NdeI and BstEII, speI and BstEII, recovering a large fragment I obtained by enzyme digestion of NdeI and BstEII, and a small fragment I obtained by enzyme digestion of SpeI and BstEII, and connecting the large fragment I and the small fragment I to realize core sequence multiplication to obtain MaSp2 (2R); taking a plasmid of MaSp2 (2R) as a starting point, and realizing multiplication of a core sequence by enzyme digestion, recovery and connection to obtain the MaSp2 (4R); taking a plasmid of MaSp2 (4R) as a starting point, and realizing multiplication of a core sequence by enzyme digestion, recovery and connection to obtain the MaSp2 (8R);

wherein, the conditions for multiplying the MaSp2 (2R), maSp2 (4R) and MaSp2 (8R) core sequences are the same as those of MaSp2 (1R).

Note that: maSp2 (nR) was subjected to the same conditions as MaSp2 (1R) for core sequence multiplication.

2) Preparation of MaSp2 (8R) +CT

And (2) performing enzyme digestion on the MaSp2 (8R) obtained in the step (1) by using NdeI and BstEII, speI and BstEII, recovering NdeI and BstEII, performing enzyme digestion on the MaSp2 (CT) by using NdeI and BstEII, speI and BstEII, recovering BstEII and SpeI, performing enzyme digestion on the obtained product to obtain a small fragment II, and connecting the obtained large fragment II and the obtained small fragment II to obtain the MaSp2 (8R) +CT.

3) Enzyme cutting and connection

The MaSp2 (8R) +CT fragment on the carrier pET28-a (+) is subjected to enzyme digestion treatment at enzyme digestion sites of restriction enzymes NdeI and HindIII (the same enzyme digestion sites on the carrier pET28-a (+) and the carrier pCold-I), the small fragment III is recovered, the carrier pCold I is subjected to enzyme digestion treatment with NdeI and HindIII to obtain the large fragment III, gel electrophoresis is used for judging whether enzyme digestion is complete and whether corresponding bands accord with theoretical values after enzyme digestion, and the fragments are separated from gel or enzyme digestion system solution in a gel recovery or column recovery mode; the large fragment III and the small fragment III were then treated with T4 ligase at 1:10, and incubating for 16h at 16℃to give a ligation product, i.e.MaSp 2 (8R) +CT fragment on pCold I.

In FIGS. 1A and 1B, the bands of pColdI after single and double cleavage are substantially indistinguishable, because pColdI after double cleavage of NdeI and HindIII can be calculated from the cleavage sites on the plasmid map and only cut by 35bp, so that small changes are not easily recognized in the electrophoresis map, and because the vast majority of the vector pColdI should be recovered, recovery experiments on pColdI-NdeI+HindIII do not use gel electrophoresis for recovery, and recovery using an adsorption column. FIG. 1C shows the double cleavage of MaSp2 (8R) +CT, and FIG. 1D shows the gel recovery of FIG. 1C. In FIG. D, maSp2 (8R) +CT, after double cleavage with NdeI and HindIII, resulted in two fragments of 1343bp and 5304bp, respectively, with smaller fragments being recovered, so that bands at approximately 1000bp of marker were recovered in gel electrophoresis, respectively.

4) Transformation

Chemical conversion method: taking 100 mu L of competent cells BL21, adding 1 mu L of the ligation product obtained in the step 2), and uniformly mixing; ice bath for 20-30min, water bath for 90S at 42 deg.c, and further placing in ice for 2min; then 650-800 mu LLB liquid culture medium is added, and the mixture is incubated for 1h at 37 ℃ with shaking at 220 rpm; and taking a solid LB culture plate containing antibiotics, respectively adding different bacterial liquid amounts into the solid LB culture plate, and culturing overnight at 37 ℃ to obtain the transformed bacterial strain.

5) Authentication

Vector pColdI has no T7 promoter sequence and does not have the basis for PCR using T7 primers, thus using restriction enzyme identification.

The method comprises the following specific steps: for the transformed strain, the strain was cultured overnight at 37℃to extract the plasmid, and the plasmid was digested with restriction enzymes, as shown in FIG. 2, in which the single digestion of MaSp2 (8R) +CT on pColdI was observed, and the theoretical value of the size of the single digestion product of MaSp2 (8R) +CT on vector pCold I was 5685bp, which was compared with marker and was between 7500 and 5000bp, conforming to the theoretical value.

2. Expression of

According to the property of the vector pColdI, the transformed strain was placed in LB medium containing 100. Mu.g/mL ampicillinCulturing overnight in a shaking table at 37 ℃. The bacterial liquid after overnight transformation is prepared according to the following ratio of 1:100 mass concentration is re-inoculated into LB medium of 100 mug/mL ampicillin, and cultured until OD ₆₀₀ The culture medium is subjected to ice water bath for at least 30min, IPTG with final concentration of 0.2-1mM, preferably 0.2mM, and culturing at 20deg.C in shaking table at 180rpm for 24 hr. Whether expressed or not was identified by SDS-PAGE. As shown in FIG. 3, the number and distribution of bands of the core sequence MaSp2 (8R) +CT on the pColdI vector before and after induction are different. By calculation of the theoretical value, maSp2 (8R) +CT is 37kDa, so that compared with a marker, the theoretical value is basically met, and a conclusion can be drawn that: maSp2 (8R) +CT was successfully expressed on pCold I vector.

3. Preparation and purification

1) Crude separation

Ultrasonic: centrifuging the induced bacterial liquid, collecting bacterial cells, suspending the bacterial cells in the lysate 1, adding 800mL of bacterial liquid into the lysate, and adding 30mL of lysate 1. And (3) uniformly mixing, placing the mixture into an ice-water bath for ultrasonic treatment for 8s, and suspending for 8s, wherein clear and transparent protein ultrasonic solution is obtained after about 30min.

And (3) centrifuging: the protein sonicate solution was centrifuged in a 4000rpm centrifuge for 30min, the supernatant after centrifugation was designated protein solution 1 and the pellet was designated protein pellet 1.

And (3) heat denaturation: 1.5mL of the protein solution 1 obtained after centrifugation was heated in water baths of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 30min, and the heated protein solution 1 was centrifuged in an ultracentrifuge of 20000g and 4 ℃ for 30min to obtain a heat-denatured supernatant and a heat-denatured precipitate heated at different water bath temperatures, respectively, the heat-denatured precipitate at 30 ℃ and 40 ℃ was resuspended with 200 μLPBS, the heat-denatured precipitate at 50 ℃ and 60 ℃ was resuspended with 1000 μLPBS, the heat-denatured precipitate at 70 ℃ and 80 ℃ was resuspended with 2000 μLPBS, and each sample was loaded with 6 μL during SDS-PAGE electrophoresis. As shown in fig. 4, the protein solution was denatured to various degrees after heating. First, the presence of the protein of interest in the ultrasound solution was demonstrated by comparison of the recombinant spidroin MaSp2 (8R) +CT before and after induction of panel A, with the presence of the band of interest at 40-35 kDa. After heating and centrifuging it, it was found that: the non-induced recombinant spider silk protein MaSp2 (8R) +CT does not change much when heated at 30 ℃ and 40 ℃, and the induced recombinant spider silk protein MaSp2 (8R) +CT has target bands in the sediment and the supernatant when heated at 30 ℃ and the content is equivalent, and the induced recombinant spider silk protein MaSp2 (8R) +CT has target bands in the sediment when heated at 40 ℃, but the target bands are shallower than the supernatant, which means that most target proteins exist in the supernatant under the heating condition of 40 ℃. Comparing 30℃with 40℃it was found that the amount of target protein present in the supernatant heated at 30℃was lower than that at 40℃but the denatured target protein bands in the pellet were not significantly different. As can be seen in FIG. B, the extent of protein denaturation increases with increasing temperature at 50℃and 60℃and about half of the target protein denaturation already occurs in the precipitate at 60℃for the induced recombinant spider silk protein MaSp2 (8R) +CT. However, the induced recombinant spider silk protein MaSp2 (8R) +CT shows different performances at 50 ℃: in the figure, the target protein was almost entirely present in the supernatant, and the band of the target protein was hardly visible in the precipitation. Whereas in FIG. C it can be seen that the target protein is substantially completely denatured and present in the precipitate upon heating at 70℃and 80 ℃. Therefore, it can be seen in the thermal denaturation experiments that the heating condition above 50℃can cause denaturation of proteins, the degree of denaturation increases with the increase of temperature, and the method for removing the foreign proteins by thermal denaturation at 40℃and 50℃is more feasible. Therefore, the heat denaturation is preferably carried out at 40℃and 50℃for 30min.

2) Column purification

Combining: uniformly mixing the magnetic beads with the buffer solution, adding the mixed magnetic beads into a 15mL plastic tube, balancing the mixed magnetic beads for 2 times by using 10 times of the volume of 2mL of the lysis solution 1, adsorbing the magnetic beads by using a magnet, and removing the liquid. The denatured and centrifuged protein solution was gently inverted with equilibrated magnetic beads at room temperature for 30min. The bound flow-through was removed and 20. Mu.L was taken as a sample.

Wash (Wash): 1mL of washing liquid 2 is added into the magnetic beads after the combination, the beads are gently turned over for 3-5min at room temperature, washing is repeated for 3 times, 20 mu L of washing liquid is taken as a reserved sample, and the washing liquid is marked as W1 to W3.

Elution (elision): the washed beads were washed 3 times with 1 column volume of eluent 3, 20 μl was taken as a sample for each wash, labeled E1-E4. Removing salt ions from the protein solution in the eluent by dialysis, and freeze-drying the dialyzed protein solution. As shown in FIG. 5, the precipitation after thermal denaturation in FIG. B shows that the target protein is not denatured in large amount by heating during heating, and the impurity protein is denatured by not having resistance to heat. Very little target protein band was still visible in the flow-through, indicating that the target protein was well bound. The bands of the hetero proteins were seen in washes W1, W2 and W3, and a small amount of the target protein was seen in W1, and the amount of the target protein in the eluents E2 to E4 of FIG. A was higher, indicating that the purification was successful.

The drug-loaded microsphere provided by the invention is prepared by dissolving the recombinant spider silk protein in potassium phosphate buffer solution.

1. Preparation of drug-loaded microspheres

And adding 50 mu L of the recombinant spider silk protein solution with the concentration of 5mg/mL into 1000 mu L of 2M potassium phosphate buffer solution with the pH value of 7 (group B) or 4 (group A), and mixing overnight at room temperature to obtain microspheres with different pH values. Microspheres with pH value of 7 (group B) or pH value of 4 (group A) were respectively added with 2mg/mL of drug (doxorubicin; purchased from Shanghai Co., ltd.) 50. Mu.L, 100. Mu.L, and mixed overnight at room temperature, and then the microspheres were dialyzed in ultrapure water overnight to prepare drug-loaded microspheres. And then drying the obtained liquid sample, dripping 10 mu L of the liquid sample on a glass slide, naturally airing at room temperature, repeating for 2-3 times, and then performing metal spraying treatment to obtain a sample of the scanning electron microscope of the drug-carrying microsphere.

2. Performance evaluation of drug-loaded microspheres

1) Fluorescence detection

Since doxorubicin can emit fluorescence after excitation, a fluorescence microscope was used to observe whether the protein microsphere was coated with doxorubicin, and an image of the microsphere was taken under bright field and green light excitation.

The coated microspheres are subjected to green excitation light to respectively shoot images of blank microspheres of the A group and the B group and 50 mu L and 100 mu L microspheres respectively carrying medicine (doxorubicin), as shown in figure 6, the microspheres without the doxorubicin are non-fluorescent under a fluorescent microscope, and the microspheres with the doxorubicin can observe red fluorescent under the green excitation light, which indicates that the doxorubicin is already coated on the surfaces of the microspheres.

2) Observation of drug-loaded microsphere morphology

The shape and size of the drug-loaded microspheres were observed by Scanning Electron Microscopy (SEM), and as can be seen from fig. 7, the group a microspheres had a smaller particle size, about 300nm, and were uniformly dispersed, and after the entrapment of doxorubicin, the particle size was not greatly changed, and the dispersibility was not reduced by the entrapment of doxorubicin. In contrast, the group B microspheres have larger particle size, about 500nm, and have a particle size which is not much different from that before encapsulation when the doxorubicin is encapsulated at 50. Mu.L, but the microspheres aggregate or even agglomerate to form a block when the doxorubicin is encapsulated at 100. Mu.L, and the microspheres with visible surfaces have a larger diameter than those of microspheres not encapsulated.

3) Drug loading rate and release rate

Measurement of drug loading rate: the amount of immobilized microspheres was 250. Mu.g, and the amounts of 2mg/mL drug (doxorubicin) added were 50. Mu.L, 60. Mu.L, 70. Mu.L, 80. Mu.L and 100. Mu.L, respectively, and added to 1000. Mu.L of 2M potassium phosphate buffer at pH 8, and mixed overnight at room temperature. The overnight incubated microspheres were dialyzed against ultrapure water overnight. The solution outside the dialysis bag was collected and the volume was recorded and the absorbance measured at a wavelength of 508 nm.

Drawing an doxorubicin standard curve: a10 mL volumetric flask was prepared, and 100. Mu.L, 50. Mu.L, 40. Mu.L, 30. Mu.L, 20. Mu.L, 10. Mu.L, 5. Mu.L and 1. Mu.L of doxorubicin were added to the respective 2mg/mL flasks, and water was added to the graduation marks. By measuring absorbance A ₅₀₈ A standard curve is prepared, as shown in fig. 8, and the amount of non-entrapment is calculated from the standard curve, thereby obtaining the entrapment rate. The calculation formula is as follows:

in the in vitro drug release link, PBS solutions with pH values of 4.5, 6.0 and 7.2 are prepared respectively, drug-loaded microspheres are placed in a dialysis belt and placed in the PBS solution, and the solution is placed in an incubator at 37 ℃ for 48 hours. The solution outside the dialysis bag was collected and the volume was recorded and the absorbance measured at a wavelength of 508 nm. The calculation formula is as follows:

the measurement points of the doxorubicin standard curve are: doxorubicin 100 μl, 50 μl, 40 μl, 30 μl, 20 μl, 10 μl, 5 μl and 1 μl respectively, standard curves as in fig. 8, y=21.787x, r ² = 0.9979. The amounts of 2mg/mL doxorubicin were 50. Mu.L, 60. Mu.L, 70. Mu.L, 80. Mu.L and 100. Mu.L, respectively, corresponding to the masses of 0.10mg, 0.12mg, 0.14mg, 0.16mg and 0.20mg, respectively, and in the drug loading and release experiments, two kinds of microspheres were used, which were prepared under the conditions corresponding to the groups A and B, respectively.

FIG. 9 is a comparison of microsphere drug loading at different pH values. In general, the drug loading of the two groups of microspheres increases with the increase of the drug amount, but the increase rate of the drug loading slows down with the increase of the drug amount, which indicates that the action part of the doxorubicin and the protein microspheres tends to be saturated. The drug loading rate of the group B is more than 55%, the maximum drug loading rate can reach 75% (40% -60% in the prior art), and the maximum drug loading rate of the group A is lower than 50%, so that the whole microsphere of the group B is higher than that of the microsphere of the group A, and the microsphere of the group B is more suitable for carrying doxorubicin, and therefore, the microsphere of the group B is used for a release experiment.

Fig. 10A shows release of drug-loaded microspheres at different concentrations under the same conditions (PBS ph=4.5, 37 ℃ for 48 h). From the graph, the release rate is between 50% and 83%, and the tendency of lowering and then rising occurs. Of these, the 0.14mg group was the lowest, 50%, and the 0.10mg group was the highest in release efficiency, 83% (60% in the prior art). Thus, the release test of the microspheres in group B revealed that the release was best when 2mg/mL of doxorubicin 50. Mu.L, i.e., doxorubicin 0.10mg, was added under the same conditions, i.e., 250. Mu.g of microspheres. In fig. 10B, the doxorubicin release rate was accumulated with increasing release time, and at pH 4.5 (simulating cancer cell microenvironment), release was faster in the first 3 hours, and the maximum release of group B microspheres was substantially reached around 8 hours, at about 80%, at 48h release rate 83%, and at pH 6.0 and 7.2, at 48h accumulated release rates of 36% and 32%.

4) Cytotoxicity test

The mechanism of cytotoxicity by MTT (thiazole blue) method is that dehydrogenase generated by mitochondrial metabolism in living cells can reduce MTT into water-insoluble blue purple Formazan (Formazan) crystals which are soluble in dimethyl sulfoxide (DMSO) and measure absorbance at 492nm by an enzyme-labeled instrument, the value of absorbance is proportional to the number of living cells in a certain range, and the larger the number of living cells is, the smaller the toxicity to cells is. However, dead cells do not have dehydrogenase, and thus interference with dead cells can be eliminated.

The steps in cytotoxicity testing of empty microspheres and drug-loaded microspheres are divided into two. First, the seeding of Hela cells: diluting 100 μL of cells washed with PBS and digested with pancreatin to 1mL, counting under microscope with a blood cell counting plate, inoculating into 96-well plate with about 5000-6000 cells per well, supplementing 100 μL of culture medium, and culturing at 37deg.C under 5% CO ₂ Is cultured in a constant temperature incubator for 24 hours. The second is the addition of microspheres: after 24h of incubation, the medium was removed and the microsphere suspension was added as follows: the microspheres were prepared in medium to give suspensions at concentrations of 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL and 50. Mu.g/mL, and 100. Mu.L of each concentration gradient of suspension was added to 96-well plates, with 6 wells per concentration. The control group is a medium with cells but without microsphere suspension, and the zeroing group is a cell-free and microsphere-free medium. After adding the microsphere suspension, the culture was continued for 48 hours, the medium was removed and 100. Mu.L of fresh medium containing 5mg/mLMTT was added and the culture was continued in an incubator. After 4h, the old medium was discarded, 100. Mu.L of DMSO was added to each well, the 96-well plate was tightly packed with tinfoil, and the plate was placed on a micro-shaker for shaking for 15min. After Formazan is dissolved in DMSO, the 96-well plate is placed in an enzyme-labeled instrument, OD value is detected and recorded under 492nm, and the OD value is brought into a cell viability formula to calculate the cell viability. The calculation formula is as follows:

as can be seen from fig. 11, the cells can grow in different concentrations of the blank microsphere, and the survival rate is close to 100%, which indicates that the blank microsphere has no toxicity to the cells and good biocompatibility. For the drug-loaded microspheres, the survival rate of cells decreases with the increase of the concentration of the drug-loaded microspheres, which indicates that the toxicity to cells increases with the increase of the concentration of the doxorubicin, and indicates that the killing effect of the doxorubicin on the cells increases.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence table information:

DTD version v1_3

File name PCN 2200469. Xml

Software name WIPO Sequence

Software version 2.3.0

Date of formation 2023-08-15

Basic information:

current application/applicant archive name Sichuan University of Light and Chemical Industry

Applicant name or name of university of light chemical industry

Applicant name or name/language zh

Applicant name or name/latin name Sichuan University of Light and Chemical Industry

The invention discloses a recombinant spider silk protein fused with carbon ends, a preparation method thereof and a drug-loaded microsphere (zh) based on the recombinant spider silk protein

Total amount of sequences 4

Sequence:

serial number (ID) 1

Length 117

Molecular type DNA

Feature location/qualifier:

- source, 1..117

> mol_type, other DNA

> organism, synthetic construct

residues:

gctagcggtc caggtggcta tggtcctggc caacaaggtc catctggtcc tggctctgca 60

gctgcagcag ctgctgcagc tggtccaggt ggctatggtc ctggccagca aactagt 117

serial number (ID) 2

Length 342

Molecular type DNA

Feature location/qualifier:

- source, 1..342

> mol_type, other DNA

> organism, synthetic construct

residues:

gctagcgttg gcagcggtgc gagcgccgcg agcgcggcag ccagtcgcct gtcgtctccg 60

caggcatcca gtcgtgtgag cagtgctgtt agcaacctgg tcgcaagtgg tccgaccaat 120

tccgcagctc tctcgtctac gatctctaac gttgtgagcc agattggcgc aagcaatcct 180

ggtctgagcg gctgcgatgt gctcatccaa gcgctgctcg aagtggtcag tgcgctgatt 240

cagatcctcg gtagcagttc catcggtcag gttaactatg gctcggcagg tcaagcgacg 300

cagattgttg gccagagcgt gtaccaggcg cttggcacta gt 342

serial number (ID) 3

Length 1278

Molecular type DNA

Feature location/qualifier:

- source, 1..1278

> mol_type, other DNA

> organism, synthetic construct

residues:

gctagcggtc caggtggcta tggtcctggc caacaaggtc catctggtcc tggctctgca 60

gctgcagcag ctgctgcagc tggtccaggt ggctatggtc ctggccagca aactagtgct 120

agcggtccag gtggctatgg tcctggccaa caaggtccat ctggtcctgg ctctgcagct 180

gcagcagctg ctgcagctgg tccaggtggc tatggtcctg gccagcaaac tagtgctagc 240

ggtccaggtg gctatggtcc tggccaacaa ggtccatctg gtcctggctc tgcagctgca 300

gcagctgctg cagctggtcc aggtggctat ggtcctggcc agcaaactag tgctagcggt 360

ccaggtggct atggtcctgg ccaacaaggt ccatctggtc ctggctctgc agctgcagca 420

gctgctgcag ctggtccagg tggctatggt cctggccagc aaactagtgc tagcggtcca 480

ggtggctatg gtcctggcca acaaggtcca tctggtcctg gctctgcagc tgcagcagct 540

gctgcagctg gtccaggtgg ctatggtcct ggccagcaaa ctagtgctag cggtccaggt 600

ggctatggtc ctggccaaca aggtccatct ggtcctggct ctgcagctgc agcagctgct 660

gcagctggtc caggtggcta tggtcctggc cagcaaacta gtgctagcgg tccaggtggc 720

tatggtcctg gccaacaagg tccatctggt cctggctctg cagctgcagc agctgctgca 780

gctggtccag gtggctatgg tcctggccag caaactagtg ctagcggtcc aggtggctat 840

ggtcctggcc aacaaggtcc atctggtcct ggctctgcag ctgcagcagc tgctgcagct 900

ggtccaggtg gctatggtcc tggccagcaa actagtgcta gcgttggcag cggtgcgagc 960

gccgcgagcg cggcagccag tcgcctgtcg tctccgcagg catccagtcg tgtgagcagt 1020

gctgttagca acctggtcgc aagtggtccg accaattccg cagctctctc gtctacgatc 1080

tctaacgttg tgagccagat tggcgcaagc aatcctggtc tgagcggctg cgatgtgctc 1140

atccaagcgc tgctcgaagt ggtcagtgcg ctgattcaga tcctcggtag cagttccatc 1200

ggtcaggtta actatggctc ggcaggtcaa gcgacgcaga ttgttggcca gagcgtgtac 1260

caggcgcttg gcactagt 1278

serial number (ID) 4

Length 426

Molecular type AA

Feature location/qualifier:

- source, 1..426

> mol_type, protein

> organism, synthetic construct

residues:

ASGPGGYGPG QQGPSGPGSA AAAAAAAGPG GYGPGQQTSA SGPGGYGPGQ QGPSGPGSAA 60

AAAAAAGPGG YGPGQQTSAS GPGGYGPGQQ GPSGPGSAAA AAAAAGPGGY GPGQQTSASG 120

PGGYGPGQQG PSGPGSAAAA AAAAGPGGYG PGQQTSASGP GGYGPGQQGP SGPGSAAAAA 180

AAAGPGGYGP GQQTSASGPG GYGPGQQGPS GPGSAAAAAA AAGPGGYGPG QQTSASGPGG 240

YGPGQQGPSG PGSAAAAAAA AGPGGYGPGQ QTSASGPGGY GPGQQGPSGP GSAAAAAAAA 300

GPGGYGPGQQ TSASVGSGAS AASAAASRLS SPQASSRVSS AVSNLVASGP TNSAALSSTI 360

SNVVSQIGAS NPGLSGCDVL IQALLEVVSA LIQILGSSSI GQVNYGSAGQ ATQIVGQSVY 420

QALGTS 426

END

Claims

1. the recombinant spider silk protein with the fused carbon end is characterized by comprising an amino acid sequence shown in SEQ ID NO.4, 8 repeated core sequences and 1 carbon end sequence, wherein the core sequences are shown in SEQ ID NO.1, the carbon end sequences are shown in SEQ ID NO.2, and the nucleotide sequence of the recombinant spider silk protein with the fused carbon end is shown in SEQ ID NO. 3.

2. The method for preparing the recombinant spider silk protein fused with the carbon end as claimed in claim 1, which is characterized by comprising the following specific steps: and (3) carrying out core sequence multiplication treatment on the core sequence, then carrying out enzyme digestion and connection on the core sequence on the pET vector, connecting a fragment containing one carbon end sequence and a fragment containing 8 core sequences, transferring the fragment to pColdI, incubating, transforming, inducing expression and purifying to obtain the recombinant spider silk protein fused with the carbon end.

3. The method for preparing the recombinant spider silk protein fused with the carbon end according to claim 2, wherein the enzyme digestion is carried out at the enzyme digestion site of the common restriction enzyme of the pET vector and the pColdI during transfer.

4. A method of preparing a carbon-terminated recombinant spidroin protein as claimed in claim 2, wherein the purification comprises crude separation and column purification, and the crude separation comprises steps of sonication, centrifugation and thermal denaturation.

5. The method for preparing a recombinant spider silk protein fused with carbon ends according to claim 4, wherein the temperature of thermal denaturation is 30-60 ℃ and the time is 30min.

6. The method according to claim 5, wherein the temperature of the thermal denaturation is 40℃or 50℃and the centrifugal force after the thermal denaturation is 20000g.

7. Use of a recombinant spider silk protein fused to a carbon end according to claim 1 for the preparation of drug-loaded microspheres.

8. A drug-loaded microsphere, wherein the drug-loaded microsphere is prepared by dissolving the recombinant spider silk protein with fused carbon ends according to claim 1 in potassium phosphate buffer solution.

9. The drug-loaded microsphere of claim 8, wherein the pH of the potassium phosphate buffer is 7.