CN117683700A - Genetically engineered bacterium for high-expression recombinant collagen and application thereof - Google Patents
Genetically engineered bacterium for high-expression recombinant collagen and application thereof Download PDFInfo
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Abstract
The invention discloses a genetically engineered bacterium for high-expression recombinant collagen and application thereof, and relates to the technical field of bioengineering. The genetically engineered bacterium of the high-expression recombinant collagen inserts the gene vhb into the ptsG gene locus and replaces the gene lacZ with the gene P4HTM. The invention provides a CRISPR-Cas 9-based technology, which constructs a genetic engineering bacterium by sequentially knocking in a gene vhb and a gene P4HTM into a genome of escherichia coli, and the engineering bacterium realizes the ultra-efficient expression of recombinant collagen and simultaneously ensures that the prepared recombinant collagen has high thermal stability. The genetic engineering bacteria provided by the invention has the advantages of simple construction method, convenience in use and good application prospect.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a genetically engineered bacterium for high-expression recombinant collagen and application thereof.
Background
The use of gene recombination to express exogenous protein plays an important role in the development and application of modern biological technology. Expression systems can be broadly divided into two classes depending on the host in which the exogenous gene is expressed: prokaryotic expression systems and eukaryotic expression systems. Prokaryotic expression systems include E.coli expression systems and B.subtilis expression systems. Eukaryotic expression systems include mammalian cell expression systems, yeast expression systems, insect expression systems, and plant expression systems. The escherichia coli expression system has the advantages of clear genetic background, simple culture operation, high conversion efficiency, rapid growth and propagation, low cost, capability of rapidly producing target proteins on a large scale and the like, and is widely applied.
Host bacteria commonly used for recombinant protein expression include BL21 (DE 3), BL21 (DE 3) pLysS, BL21Star (DE 3), rosetta (DE 3) pLysS, rosetta-gami 2 (DE 3), origami B (DE 3), over express C43 (DE 3), BLR (DE 3) and the like.
BLR (DE 3) genotype: tn10 (TetR), a RecA (recombinase A-deficient) strain, derived from BL21, F-ompT hsdSB (rB-mB-) gal dcm (DE 3) delta (srl-recA) 306, which contributes to the increased yield of plasmid monomers, and also to the increased stability of plasmids that can lead to loss of DE3 phage. The BLR strain is both lon and ompt protease deficient. DE3 is a lysogenic λDE3 and therefore carries a chromosomal copy of the T7 RNA polymerase. The strain is suitable for pET series vectors and other T7 promoter series vectors. How to improve the yield of the recombinant protein expressed by the escherichia coli is a continuous problem solved by the industry.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a genetically engineered bacterium for high-expression recombinant collagen and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a genetically engineered bacterium for high expression of recombinant collagen is prepared through inserting gene vhb in ptsG locus, and substituting gene lacZ by gene P4HTM.
Based on the problem of low expression level of recombinant proteins of escherichia coli in the prior art, the inventor inserts a UZP69255.1 protein coding gene vhb into a ptsG gene locus of escherichia coli genome by adopting a gene editing technology to obtain a strain BLR (DE 3) pLysS delta ptsG; the AAH11710.3 protein coding gene P4HTM is substituted and inserted into the genome lacZ gene locus of the escherichia coli on the basis of BLR (DE 3) pLysS delta ptsG:: vhb to obtain the strain BLR (DE 3) pLysS delta ptsG::: vhb delta lacZ:: P4HTM, named as LTlank (DE 3). The genetically engineered bacterium LTlank (DE 3) constructed by the invention realizes the ultra-efficient expression of the recombinant collagen, and the proline of the collagen is subjected to hydroxylation modification to form a stable triple-helix structure, so that the stability of the recombinant collagen is improved.
As a preferred embodiment of the genetically engineered bacterium for high-expression of recombinant collagen, the genetically engineered bacterium uses escherichia coli as a host cell.
As a preferred embodiment of the genetically engineered bacterium for high-expression of recombinant collagen, the escherichia coli is escherichia coli BL21 (DE 3) pLysS.
As a preferred implementation mode of the genetically engineered bacterium for high-expression recombinant collagen, the nucleotide sequence of the gene ptsG is shown as SEQ ID NO. 1; the nucleotide sequence of the gene lacZ is shown as SEQ ID NO. 2.
As a preferred embodiment of the genetically engineered bacterium for high-expression of recombinant collagen, the GenBank ID of the gene vhb is UZP69255.1; the GenBank ID of the gene P4HTM is AAH11710.3.
As a preferred implementation mode of the genetically engineered bacterium for high-expression recombinant collagen, the genetically engineered bacterium is constructed based on a CRISPR-Cas9 technology.
The invention also provides a construction method of the genetically engineered bacterium for high-expression recombinant collagen, which comprises the following steps:
(1) Designing and synthesizing sgRNAs of ptsG gene and lacZ gene, and connecting the sgRNAs with a vector to obtain recombinant plasmids;
(2) Constructing a Donor sequence according to the gene vhb and the gene P4 HTM;
(3) Transferring the recombinant plasmid and the Donor sequence into competent cells, and screening to obtain the genetically engineered bacterium of the high-expression recombinant collagen.
The invention also provides application of the genetically engineered bacterium in preparation of recombinant collagen.
The invention also provides a method for producing the recombinant collagen, which is prepared by adopting the genetically engineered bacterium for fermentation.
The invention also provides the recombinant collagen prepared by the method for producing the recombinant collagen.
The invention has the beneficial effects that: the invention provides a CRISPR-Cas 9-based technology, which constructs a genetic engineering bacterium by sequentially knocking in a gene vhb and a gene P4HTM into a genome of escherichia coli, and the engineering bacterium realizes the ultra-efficient expression of recombinant collagen and simultaneously ensures that the prepared recombinant collagen has high thermal stability. The genetic engineering bacteria provided by the invention has the advantages of simple construction method, convenience in use and good application prospect.
Drawings
FIG. 1 shows the partial sequence results of the E.coli BLR (DE 3) pLysS proposed site.
Fig. 2: (a) is a schematic diagram of the expression cassette of pSynbio-sgRNA plasmid sgRNA; (b) sequencing the sgrnas of the ptsG gene; (c) sequencing of the sgRNA of the lacZ gene.
FIG. 3 shows construction of an electrophoresis pattern of a donor fragment of a knock-in gene.
FIG. 4 is a diagram showing PCR amplification electrophoresis of a monoclonal target gene after a knock-in experiment of the ptsG site of a BLR (DE 3) pLysS strain and a diagram showing the result of the monoclonal sequencing after the knock-in.
FIG. 5 shows the PCR amplification electrophoresis pattern of the monoclonal target gene after the knock-in experiment of the lacZ site of the BLR (DE 3) pLysS strain and the result pattern of the monoclonal sequencing after the knock-in.
FIG. 6 shows the growth of E.coli BLR (DE 3) pLysS.DELTA.ptsG: vhb strain on different resistant plates after plasmid removal, where a, b, c represent the non-resistant LB medium, kan and Spec resistant medium, respectively.
FIG. 7 shows the growth of E.coli BLR (DE 3) pLysS.DELTA.ptsG: vhb/. DELTA.lacZY:: P4HTM strain on different resistant plates after plasmid removal, where a, b, c represent the antibiotic-free LB medium, kan and Spec resistant medium, respectively.
FIG. 8 construction of sequencing results of E.coli BLR (DE 3) pLysS.DELTA.ptsG:: vhb strain.
FIG. 9 shows the sequencing results of the construction of the E.coli BLR (DE 3) pLysS.DELTA.ptsG:: vhb/DELTA lacZY:: P4HTM strain.
FIG. 10 shows recombinant human collagen expression of BL21 (DE 3), BLR (DE 3) pLysS, BL21Star (DE 3), LTlank (DE 3).
FIG. 11 shows the results of the thermal stability test of collagen expressed by LTlank (DE 3) and BL21Star (DE 3).
Detailed Description
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
EXAMPLE 1 construction of genetically engineered bacterium highly expressing recombinant collagen
1. Design and synthesis of sgRNA: in this example, the software primer5 was used to design amplification primers for ptsG gene and lacZ gene, and the PCR amplification procedure shown in Table 1 was used to perform PCR amplification recovery and sequencing to determine the exact sequence of the site to be knocked out, and the comparison result is shown in FIG. 1.
TABLE 1 amplification primers for E.coli BLR (DE 3) pLysS sites to be edited
2. design, synthesis and construction verification of the donor: the sgrnas for the ptsG gene and the lacZ gene were designed using software primer5, as shown in table 2. And uniformly mixing the single-stranded DNA primers of the 2 gene sgRNAs with the PCR reaction liquid, and then carrying out annealing reaction, and recovering and purifying after the reaction is finished to obtain a double-stranded DNA product. Constructing a double-stranded DNA product to a vector pSynbio-sgRNA by a Golden Gate method; the following day the monoclonal extracted plasmid was picked and sequenced to verify that the correct clones were named pSynbio-sgRNA-ptsG and pSynbio-sgRNA-LacZ and the sequencing results of the sgRNA sequences are shown in FIG. 2.
TABLE 2 sgRNA sequence of E.coli BLR (DE 3) pLysS pseudoediting site
In this example, the gene vhb encoded by UZP69255.1 and the gene P4HTM encoded by AAH11710.3 were knocked in into the genome of E.coli BLR (DE 3) pLysS: homologous sequences of about 200bp upstream and downstream of target sites of ptsG gene and lacZ gene are respectively added at the head and tail ends of the UZP69255.1 encoding gene vhb and the AAH11710.3 encoding gene P4HTM, and complete donor fragments are obtained through a gene synthesis technology, wherein specific DNA sequences are shown in Table 3, and the results are shown in FIG. 3.
TABLE 3 donor sequence of E.coli BLR (DE 3) pLysS quasi-knock-in
3. The method for knocking in the encoding genes of the escherichia coli BLR (DE 3) pLysS strain UZP69255.1 and AAH11710.3 protein comprises the following specific steps:
(1) Streaking a BLR (DE 3) pLysS strain on a non-resistant LB plate for strain activation;
(2) Selecting a monoclonal and inoculating the monoclonal into LB culture solution, and culturing for 12-14 h at 37 ℃;
(3) Inoculating the bacterial liquid into fresh LB culture solution according to the proportion of 1%, and culturing for 2-3 h at 37 ℃ until OD reaches 0.6; (4) Transferring the bacterial liquid into a centrifuge tube, centrifuging the bacterial liquid at 5000rpm, discarding the supernatant, and collecting cells;
(5) Re-suspending the cells with pre-chilled 10% glycerol, centrifuging the bacterial liquid again at 5000rpm, discarding the supernatant, and collecting the cells;
(6) Repeating the step (5) for 2 times;
(7) Adding appropriate amount of 10% glycerol, and slightly oscillating to make cells uniformly resuspended;
(8) Sub-packaging competent cells according to 100ul/tube specification, directly carrying out plasmid transformation or preserving at-80 ℃;
(9) Transforming pSynbio-Cas9 plasmid (offered by Souzhou Hongxun Biotechnology Co., ltd.) into competent cells in step (8), and plating onto Kan-resistant LB plates;
(10) Selecting a monoclonal and inoculating the monoclonal into LB culture solution, and culturing for 12-14 h at 37 ℃;
(11) Preparing new competent cells according to steps (3) - (8);
(12) Co-transferring pSynbio-sgRNA plasmid and donor fragment into new competent cells, and coating on Kan/Spec resistant LB plate;
(13) The next day, selecting a monoclonal from a Kan/Spec resistance LB plate, inoculating the monoclonal to a deep hole plate, and culturing for 2-3 hours at 37 ℃;
(14) Fragments of the monoclonal target genes ptsG and lacZ are amplified by PCR technology, and primers are ptsG-KI-JJF, ptsG-KI-JJR and LacZ-KI-PCR-F, lacZ-KI-PCR-R in Table 4, and the amplification results are shown in FIG. 4 (a) and FIG. 5 (a);
(15) Selecting positive clones, recovering PCR products, and performing sequencing verification;
(16) Screening gave the correct knock-in strain, whose knock-in site sequencing results are shown in FIG. 4 (b) and FIG. 5 (b).
TABLE 4 construction of PCR amplification primers, amplification procedure and product Length used in knock-in Gene experiments
As can be seen from the sequencing results, the present example successfully obtained BLR (DE 3) pLysSΔptsG:: vhb strain and BLR (DE 3) pLysSΔptsG::: vhb/ΔlacZY:: P4HTM strain.
4. The pSynbio-Cas9 and pSynbio-sgRNA plasmids were removed from the cells of the above strains as follows:
(1) Transferring the knocked-out strain to a fresh non-antibiotic LB culture medium, adding an inducer, and culturing for 12-14 h at 37 ℃;
(2) Diluting and coating the bacterial liquid onto an antibiotic-free LB plate, performing cell separation and screening, and continuing to culture at 37 ℃ until obvious bacterial colonies are observed;
(3) Extracting monoclonal antibodies, respectively inoculating the monoclonal antibodies into antibiotic-free LB, kan-resistant LB and Spec-resistant LB culture solutions, and culturing at 37 ℃ for 4-5 hours;
(4) Comparing the turbidity of LB bacteria solutions with different resistances, and screening to obtain strains sensitive to Kan and Spec antibiotics;
(5) The bacterial solution of the partially resistant sensitive strain was inoculated onto LB plates without resistance, LB with Kan resistance and LB with Spec resistance, and cultured at 37℃for 12-14 hours, and the plasmid removal was judged, and the results are shown in FIG. 6 and FIG. 7.
5. Verification of E.coli BLR (DE 3) pLysS.DELTA.ptsG:: vhb strain and BLR (DE 3) pLysS.DELTA.ptsG::: vhb/DELTA lacZY::: P4HTM strain
(1) Genome sequencing of E.coli BLR (DE 3) pLysS.DELTA.ptsG:: vhb strain: after successfully removing the resistance of the E.coli BLR (DE 3) pLysS.DELTA.ptsG:: vhb strain, the ptsG gene fragment was again amplified with the ptsG-KI-JF/ptsG-KI-JJR primer of Table 4, and after purification and recovery, the sequence was verified, and the sequencing result was shown in FIG. 8, and it was found that the strain was a BLR (DE 3) pLysS.DELTA.ptsG::: vhb strain from which the resistance was successfully removed.
(2) Genomic sequencing of the BLR (DE 3) pLysS.DELTA.ptsG:: vhb/DELTA.lacZY:: P4HTM strain: after successful removal of the resistance by the E.coli BLR (DE 3) pLysS.DELTA.ptsG: vhb/. DELTA.lacZY:: P4HTM strain, the lacZ gene fragment was again amplified with LacZ-KI-PCR-F/LacZ-KI-PCR-R primers in Table 4, and after purification recovery, sequencing was performed, the result of which was shown in FIG. 9, and it was found that the strain was a BLR (DE 3) pLysS.DELTA.ptsG::: vhb/. DELTA lacZY:: P4HTM strain, i.e., LThin (DE 3).
EXAMPLE 2 recombinant human collagen expression level test experiment
1. Competent cell preparation: (1) The strain LTlank (DE 3) was picked from a plate incubated at 37℃for 16-20h as a single colony (diameter 2-3 mm) and transferred to a 1L flask containing 100ml LB or SOB medium. The culture was vigorously shaken at 37℃for 3 hours. 1OD600 about contains E.coli 10 9 And each mL. (2) The bacteria were transferred to a sterile, single-use, 50ml centrifuge tube pre-chilled with ice, and placed on ice for 10min, and the culture was allowed to cool to 0 ℃. (3) centrifuging at 4℃at 4100r/min for 10min to recover the cells. (4) The culture broth was decanted and the tube was inverted for 1min to drain out the last traces of culture broth. (5) 30ml of pre-chilled 0.1mol/LCaCl was used per 50ml of initial broth 2 -MgCl 2 Solution (80 mmol/L MgCl) 2 ，20mmol/L CaCl 2 ) Each cell pellet was resuspended. (6) The cells were recovered by centrifugation at 4100r/min at 4℃for 10 min. (7) The culture broth was decanted and the tube was inverted for 1min to drain out the last traces of culture broth. (8) 2ml of ice-precooled 0.1mol/L CaCl were used per 50ml of initial culture 2 Each cell pellet was resuspended. (9) Transformation experiments can be performed directly with freshly prepared competent cells.
2. Conversion: taking 50 mu L of BL21 (DE 3), BLR (DE 3) pLysS, BL21Star (DE 3) and LTlank (DE 3) competent cells respectively, adding 10pg of COL3-pET-30a plasmid DNA into the mixture for 30min, immediately placing the mixture on ice after heat shock for 90s at 42 ℃, and carrying out ice bath for 2min; adding 400 μL LB culture medium, and shake culturing at 37deg.C for 45-60min; 50-100. Mu.L of the culture solution was spread on LB solid medium containing ampicillin (100. Mu.g/mL), and the culture was inverted at 37℃overnight.
3. Recombinant human collagen expression: (1) The single colonies were picked up and cultured overnight at 37℃and 250rpm in 4mL of LB medium with ampicillin (kan, final concentration 100 g/mL). (2) The overnight culture broth was transferred at 1:100 into 100mL of LB containing the above antibiotics at 37℃and 250rpm for 3.5h (culture to OD600 = 0.6). (3) 1mL of the bacterial liquid is taken out as an uninduced electrophoresis control, and the remaining culture liquid is added with inducer IPTG to a final concentration of 1mmol/L, and the shaking culture is continued for 4h. (4) Comparing the non-induced bacterial liquid with the bacterial liquid after IPTG induction, and detecting by SDS-PAGE.
4. (1) the above-mentioned induced bacterial liquid at 37℃was centrifuged (4℃12000g,5 min), the supernatant (LB medium) was discarded, and the pellet was resuspended in 0.9% NaCl solution for bacterial washing. (2) The cell solution resuspended in 0.9% NaCl was centrifuged (4 ℃,12000g,5 min), and the supernatant (0.9% NaCl wash) was discarded to obtain cells, which were weighed and resuspended in 8mL PBS (pH 7.0) buffer, the buffer amount was according to the final concentration of 50-100 mg/mL. (3) ultrasonic sterilization: under ice bath conditions, the escherichia coli is crushed by ultrasonic waves, and the ultrasonic waves are set as follows: 400W, work: 15s, interval: 45s, whole time: the culture medium is changed from white to transparent and is not sticky after 30min (about 30 times). (4) The sonicated bacterial solutions were centrifuged (4 ℃,12000g,15 min), the supernatant and pellet were collected, 1mL each, 1mL of 2 Xloading buffer was added, the boiling water bath was 10min, and SDS-PAGE was performed.
The experimental results are shown in FIG. 10, in which the expression level of LTlank (DE 3) is about 50% or more, and most suitably used for expression of collagen.
EXAMPLE 3 recombinant collagen thermal stability experiment
The specific experimental steps are as follows: the LTlank (DE 3) and BL21Star (DE 3) expressed collagen were placed in an oven at 45℃for 0h,12h,24h, and 48h, respectively. Cell proliferation assay was performed: 3T3-L1 cells were cultured according to 5X 10 3 Density of individual/well was inoculated into 96-well plates and blank DMEM high sugar medium was added at 5% co 2 And culturing at 37 ℃, adding different collagens to be detected after the cells are attached, adding CCK8 detection reagent according to the instruction after 24 hours, incubating for 2 hours, and detecting by using an enzyme-labeled instrument (wavelength of 450 nm).
As shown in FIG. 11, recombinant collagen expressed by LTlank (de 3) has optimal thermal stability.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention 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 solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. The genetically engineered bacterium for high expression of recombinant collagen is characterized in that the genetically engineered bacterium inserts a gene vhb into a ptsG gene locus and replaces a gene lacZ with a gene P4HTM.
2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium comprises an escherichia coli host cell.
3. The genetically engineered bacterium of claim 2, wherein the escherichia coli is escherichia coli BL21 (DE 3) pLysS.
4. The genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the gene ptsG is shown in SEQ ID No. 1; the nucleotide sequence of the gene lacZ is shown as SEQ ID NO. 2.
5. The genetically engineered bacterium of claim 1, wherein the GenBank ID of the gene vhb is UZP69255.1; the GenBank ID of the gene P4HTM is AAH11710.3.
6. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is constructed based on CRISPR-Cas9 technology.
7. A method for constructing genetically engineered bacteria of the recombinant collagen highly expressed according to any one of claims 1 to 6, comprising the steps of:
(1) Designing and synthesizing sgRNAs of ptsG gene and lacZ gene, and connecting the sgRNAs with a vector to obtain recombinant plasmids;
(2) Constructing a Donor sequence according to the gene vhb and the gene P4 HTM;
(3) Transferring the recombinant plasmid and the Donor sequence into competent cells, and screening to obtain the genetically engineered bacterium of the high-expression recombinant collagen.
8. The use of the genetically engineered bacterium of any one of claims 1 to 6 in the preparation of recombinant collagen.
9. A method for producing recombinant collagen, characterized in that the recombinant collagen is prepared by fermenting the genetically engineered bacterium according to any one of claims 1 to 6.
10. The recombinant collagen produced by the method of claim 9.
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