Disclosure of Invention
The purpose of the application is to overcome the defects of the prior art and provide the pichia pastoris engineering bacteria for secretory expression of the recombinant III type collagen and the application thereof, and the pichia pastoris engineering bacteria can be used for efficiently and safely carrying out extracellular secretory expression of the recombinant III type collagen, and the recombinant III type collagen has obvious promotion effect on cell proliferation and cell migration.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a recombinant type III collagen having an amino acid sequence as set forth in (a) or (b):
(a) The amino acid sequence is shown as SEQ ID NO. 1;
(b) The protein derived from the (a) which has collagen activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of the (a).
The inventor of the application finds that a natural III type collagen integrin signal site or binding site sequence is adopted for splicing, then 12 repeated fragments are connected in series to form a recombinant expression collagen fragment (namely the recombinant III type collagen of the application), the total length of the recombinant expression collagen fragment is 525 amino acids, 1-171, 174-342 and 345-513 are identical human III type collagen fragments, RS connection is arranged between three human III type collagen fragments, and GPPGPCCGGG is adopted for modification after the human III type collagen fragments of 345-513 are subjected to the modification (the stability of the recombinant III type collagen can be improved), so that the recombinant III type collagen obtained by the application has obvious effects on cell proliferation and cell migration.
Due to the impact of the size of the collagen polypeptide on its secretion, too long a collagen polypeptide sequence (greater than 90 kDa) may not be secreted efficiently in pichia pastoris; too short a collagen polypeptide sequence would certainly increase the difficulty of the subsequent purification process. The application selects 12 repeated fragments in series to form a recombinant expression collagen fragment, so that the size of the fragment is controlled to be about 50 kDa.
In a second aspect, the present application provides a nucleic acid molecule encoding said recombinant type III collagen.
In a third aspect, the present application provides a vector carrying said nucleic acid molecule.
Redesign and optimize the nucleotide sequence of the recombinant III type collagen according to the selected amino acid sequence (shown as SEQ ID NO. 2), connect the nucleotide sequence of the recombinant III type collagen after optimization to a vector pGSI to obtain a plasmid pGSI-SEQ ID NO.2 containing the recombinant III type collagen (SEQ ID NO. 2), double-enzyme-cut pGSI-SEQ ID NO.2, and recycle the target fragment of the SEQ ID NO. 2; after single enzyme cutting pGSI-SEQ ID NO.2 to form linearization plasmid, the recovered T4 target fragment is connected in vitro to form two monomer concatemers pGSI- (SEQ ID NO. 2)2 The method comprises the steps of carrying out a first treatment on the surface of the Single enzyme cutting pGSI- (SEQ ID NO. 2)2 After formation of the linearized plasmid, the recovered fragment of SEQ ID NO.2 is ligated into the linearized plasmid pGSI- (SEQ ID NO. 2)2 Becomes a triple tandem pGSI- (SEQ ID NO. 2)3 The method comprises the steps of carrying out a first treatment on the surface of the pGSI- (SEQ ID NO. 2) with double enzyme cutting3 Recovery (SEQ ID NO. 2)3 A triset fragment of interest; after double cleavage of pPIC9K to form linearized plasmid, the plasmid is recovered (SEQ ID NO. 2)3 The target fragment is connected with pPIC9K to form recombinant III collagen expression plasmid pPIC9K-rhCOL3S5.
Fourth, the present application provides genetically engineered bacteria expressing the recombinant type III collagen.
The method converts pichia pastoris GS115 through an electrotransformation method through linearization yeast expression plasmid pPIC9K-rhCOL3S5, directly selects single colony with better growth vigor on an MD culture medium plate to screen on a YPD culture medium plate containing high-concentration G418, and obtains the genetically engineered bacterium (high-copy recombinant yeast) of the method, so that the screening process of the high-copy recombinant yeast is simplified, the time cost of the stage is shortened, and the working efficiency is improved.
As a preferred embodiment of the genetically engineered bacterium, the genetically engineered bacterium is Pichia pastoris @Pichia pastoris) GS115/pPIC9K-rhCOL3S5-248b and Pichia pastorisPichia pastoris) GS115/pPIC9K-rhCOL3S5-523c, pichia pastorisPichia pastoris)GS115/pPIC9K-rhCOL3S5-248b has a preservation number of CGMCC No.27541, and the Pichia pastoris is preparedPichia pastoris) GS115/pPIC9K-rhCOL3S5-523c has a preservation number of CGMCC No.27542, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 5 days of 6 months of 2023.
The genetically engineered bacterium can efficiently and safely carry out extracellular secretion expression of recombinant III type collagen.
As a preferred embodiment of the genetically engineered bacterium, the genetically engineered bacterium uses pPIC9K as an expression vector.
In a fifth aspect, the present application provides an agent for promoting cell proliferation, which comprises the recombinant type III collagen.
In a sixth aspect, the present application provides a cell migration promoting agent comprising the recombinant type III collagen.
The seventh object is to provide the application of the recombinant type III collagen in preparing a reagent for promoting cell proliferation or migration.
Eighth, the application provides an application of the genetically engineered bacterium in producing recombinant type III collagen, wherein the amino acid sequence of the recombinant type III collagen is shown as SEQ ID NO.1, and the nucleotide sequence of the recombinant type III collagen is shown as SEQ ID NO. 2.
The application uses BMM culture medium to induce and express fermentation supernatant of recombinant type III collagen, and the obtained recombinant type III collagen can be directly used for biological function detection through ammonium persulfate precipitation, dialysis and rapid purification of ultrafiltration. A process for precisely screening the functional fragments is established, and the process has short time and is beneficial to rapidly screening the functional fragments.
Compared with the prior art, the application has the following beneficial effects:
the recombinant type III collagen is obtained by splicing natural type III collagen integrin signal sites or binding site sequences, and then carrying out 12 repeated fragments in series to form a recombinant expression collagen fragment. The experimental result of CCK-8 method shows that the cell proliferation rate of ESF cells at the final concentration of 1.3 mg/mL and 0.5 mg/mL is 123.90% and 118.73%, which is obviously higher than that of the negative control group (1X maintenance medium), and the ESF cells are considered to have a certain effect of promoting the proliferation of the ESF cells. The cell scratch experiment result shows that the Wound healing percentage of 24 h of the negative control group (NC) is 59.71%, the Wound healing percentage of 24 h of the recombinant type III collagen sample group (WoundClosure%) of 0.2 mg/mL and 0.1 mg/mL is obviously larger than that of the negative control group (NC), and the Wound healing percentage is about 71.17% and 82.54% respectively, and the recombinant type III collagen sample groups of 0.2 mg/mL and 0.1 mg/mL are considered to have obvious effect of promoting HaCaT Wound healing.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
In the early experiment, the sequence of natural III type collagen integrin signal site or binding site is adopted to splice, and then 12 repeated segments are connected in series to form the design thought of recombinant expression collagen segment, so that more than 4 sequences are designed. Scoring is carried out according to a cytological efficacy detection result and the product purification difficulty (the separation and purification difficulty degree of a main band and a hydrolysis band is taken as judgment), and the scoring of the recombinant III type collagen is higher than that of other sequences.
EXAMPLE 1 construction of recombinant III collagen expression plasmid
In the embodiment, natural III type collagen integrin signal sites or binding site sequences are adopted for splicing, then 12 repeated fragments are connected in series to form a recombinant expression collagen fragment (namely recombinant III type collagen) with the total length of 525 amino acids, wherein 1-171, 174-342 and 345-513 are identical human III type collagen fragments, RS connection is arranged among three human III type collagen fragments, the human III type collagen fragment of 345-513 is modified by adopting GPPGPCCGGG, and the amino acid sequence of the recombinant III type collagen is shown as SEQ ID NO. 1.
According to the preference of pichia pastoris codons, the nucleotide sequence of the selected natural III type collagen integrin signal site or binding site is subjected to codon optimization by adopting the means, and the nucleotide sequence of the recombinant III type collagen is shown as SEQ ID NO. 2.
The nucleotide sequence (SEQ ID NO. 2) of recombinant type III collagen is synthesized by the Guangzhou Ai Ji biotechnology Co-Ltd and cloned to a vector pGSI to obtain a plasmid pGSI-SEQ ID NO.2 containing one recombinant type III collagen (SEQ ID NO. 2), and restriction enzyme sites of isotail enzymes BamHI and Bgl II are arranged at two ends of the SEQ ID NO.2 fragment.
(3) After BamH I and Bgl II double enzyme cutting pGSI-SEQ ID NO.2, recovering the fragment of SEQ ID NO. 2. After Bgl II single enzyme cuts pGSI-SEQ ID NO.2 to form linearization plasmid, the recovered T4 target fragment is connected in vitro to form tandem pGSI- (SEQ ID NO. 2) of two monomers2 。
(4) Bgl II single enzyme cutting pGSI- (SEQ ID NO. 2)2 After formation of the linearized plasmid, the recovered fragment of SEQ ID NO.2 is ligated into the linearized plasmid pGSI- (SEQ ID NO. 2)2 Becomes a triple tandem pGSI- (SEQ ID NO. 2)3 。
(5) EcoR I and Not I double enzyme cutting pGSI- (SEQ ID NO. 2)3 After that, recovery (SEQ ID NO. 2)3 The triset fragment of interest. EcoR I and Not I double enzyme cut pPIC9K to form linearization plasmid, will be recoveredIs (SEQ ID NO. 2)3 The target fragment is connected with pPIC9K to form recombinant III collagen expression plasmid pPIC9K-rhCOL3S5. FIG. 1 is a plasmid map of recombinant type III collagen expression vector pPIC9K-rhCOL3S5.
EXAMPLE 2 construction of high-copy recombinant GS115/pPIC9K-rhCOL3S5 expressing recombinant type III collagen
The present example provides the construction of a high copy recombinant GS115/pPIC9K-rhCOL3S5 expressing recombinant type III collagen, comprising the steps of:
1) 20 mug of pPIC9K-rhCOL3S5 plasmid is extracted, and is subjected to tangential digestion by Sac I enzyme, and is purified and concentrated by an ethanol precipitation method for later use.
2) Electrotransformation pichia pastoris cell GS115: mu.L of electrotransformation competent GS115 and 10. Mu.g of recovered linearized plasmid were added to a sterile, ice-pre-chilled 1.5. 1.5 mL centrifuge tube and mixed well at the bottom of the flick tube to prepare an electrotransformation mixture. The above electrotransport mixture was transferred to a 0.2. 0.2 cm ice pre-chilled electrotransport cup, which was then ice-bathed for 5 min. The electric shock was performed using the sc2 electroporation program on a microplus electroporation machine. After the electric shock was completed, a sorbitol solution of 1M pre-cooled with 12 mL ice was immediately added, and the cells were mixed uniformly. The bacterial suspension was plated onto MD plates, one plate per 500. Mu.L of bacterial suspension, for a total of 10 plates. Plates were placed in an incubator at 29℃and incubated until single colonies appeared. Incubate at 29℃for about 4 days. FIG. 2 is a plate diagram of the electric transformed Pichia pastoris GS 115.
3) Screening of high copy recombinant yeasts: single colonies growing well on MD media plates were picked up and plated on YPD media plates containing 4.0. 4.0 mg/mL G418, and single colonies of Pichia pastoris GS115 (untransformed) were picked up and plated on YPD media plates containing 4.0. 4.0 mg/mL G418 using the same procedure as the control. The incubator was incubated at 29℃for about 5 days. FIG. 3 is a plate diagram of 4.0. 4.0 mg/mL G418 screen.
4) PCR identification of positive clone strains: after the plate developed colonies, single colonies 248b, 523c grown on the plate were picked with an inoculating loop and transferred to a 1.5 mL centrifuge tube containing 10. Mu.L of sterile water. After mixing, 1. Mu.L of 10U/. Mu.L of lysozyme was added and incubated at 30℃for 10 min. The sample was immersed in liquid nitrogen for 1 min and thawed at room temperature for about 5 min. Repeated 1 time. Briefly centrifuging, taking 1 μl of supernatant as a PCR reaction template, and using a vector-borne primer (5' - α -Factor: TACTATTGCCAGCATTGCTGC;
3' ″ AOX: GCAAATGGCATTCTGACATCC) PCR, the results are shown in FIG. 4.
Wherein M: DNA standard, 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100, bp from top to bottom; 1-2: PCR amplified fragments of colonies 248b, 523 c; 3: PCR amplified fragment of plasmid pPIC9K-rhCOL3S5 is used as positive control; 4: PCR amplified fragments of untransformed GS115 strain as negative control; 5: h2 O as a blank.
Example 3 Induction expression, rapid purification and functional detection of recombinant type III collagen
Induction of expression: and selecting 248b and 523c positive bacteria for induction expression.
Positive strains 248b and 523c were inoculated into 250-mL Erlenmeyer flasks containing 25 mL of BMG medium, and incubated at 29℃and 250 rpm for about 24 h. Taking 2.5. 2.5 mL bacterial liquid from the cultured seed liquid, transferring to a 250 mL conical flask containing 22.5 mL BMG culture medium, culturing at 29 deg.C and 250 rpm at 18-22 h to OD600 =6 or so. Subpackaging the bacterial liquid into a 50 mL centrifuge tube, centrifuging at 6000 rpm for 5 min at room temperature, discarding the supernatant, and collecting bacterial cells. The collected bacteria were resuspended to OD with the appropriate amount of BMM medium600 After the resuspension, the bacterial suspension was transferred to 1L conical flasks for cultivation, each flask being 100 mL resuspended. The culture was performed at 29℃and 250 rpm for 72℃ 72 h. After the start of induction, methanol was added to a final concentration of 1% every 24. 24 h. After induction of expression 0, 24, 48, 72 and h, 10000 g was centrifuged for 10 min to collect fermentation supernatant, which was subjected to SDS-PAGE electrophoresis, and the electrophoresis results are shown in FIG. 5.
Wherein M: a protein standard; 1: positive strains induced to express the supernatant for 0 hours; 2: positive strains induced expression for 24 hours of supernatant; 3: positive strains induced expression of the supernatant for 48 hours; 4. positive strains induced expression of the supernatant for 72 hours; the 248b and 523c positive strains are respectively named as Pichia pastoris @, respectivelyPichia pastoris）GS115/pPIC9K-rhCOL3S5-248b and Pichia pastorisPichia pastoris) GS115/pPIC9K-rhCOL3S5-523c, the above strains were deposited in China general microbiological culture Collection center (accession number: beijing, chaoyang, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, postal code: 100101 Pichia pastorisPichia pastoris) GS115/pPIC9K-rhCOL3S5-248b and Pichia pastorisPichia pastoris) The preservation numbers of GS115/pPIC9K-rhCOL3S5-523c are CGMCC No.27541 and CGMCC No.27542 respectively.
And (3) quick purification: to the supernatant obtained above, 500 mL was added 121.5 g ammonium sulfate solids in portions in small amounts to a saturation concentration of 40% ammonium sulfate. And left standing at room temperature (25 ℃) for 1 h. Centrifuging at 25deg.C and 10000 g for 15 min, removing supernatant, and collecting precipitate. Finally, 5 mL of 1 XPBS (pH 7.2) was added, and the mixture was blown and mixed until the precipitate was completely dissolved. After adding the ammonium sulfate precipitation resuspension to a 10 kDa dialysis bag, the dialysis bag is placed in a 1L beaker containing 1000 mL purified water for dialysis, during which time the dialysate is changed 4-5 times for total dialysis of about 24 h. The collected dialysis retention liquid is subjected to ultrafiltration concentration by using a 10 kDa ultrafiltration tube, and the obtained ultrafiltration concentration liquid is used for subsequent biological function detection experiments.
And (3) function detection:
promotion of cell proliferation by recombinant type III collagen of the present application: the experimental result of the CCK-8 method shows that the cell proliferation rate of ESF cells at the final concentration of 1.3 mg/mL and 0.5 mg/mL is 123.90% and 118.73%, which is obviously higher than that of the negative control group (1X maintenance medium), and the ESF cells are considered to have a certain ESF cell proliferation promoting effect.
Promotion of cell migration by recombinant type III collagen of the present application: the cell scratch experiment result shows that the Wound healing percentage of 24 h of the negative control group (NC) is 59.71%, the Wound healing percentage of 24 h of the recombinant type III collagen sample group (WoundClosure%) of 0.2 mg/mL and 0.1 mg/mL is obviously larger than that of the negative control group (NC), and the Wound healing percentage is about 71.17% and 82.54% respectively, and the recombinant type III collagen sample groups of 0.2 mg/mL and 0.1 mg/mL are considered to have obvious effect of promoting HaCaT Wound healing.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present application and not for limiting the scope of protection of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.