CN116355825A - Method for enhancing production efficiency of human epidermal growth factor secretion by strengthening cellulose synthesis - Google Patents
Method for enhancing production efficiency of human epidermal growth factor secretion by strengthening cellulose synthesis Download PDFInfo
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- CN116355825A CN116355825A CN202310382114.6A CN202310382114A CN116355825A CN 116355825 A CN116355825 A CN 116355825A CN 202310382114 A CN202310382114 A CN 202310382114A CN 116355825 A CN116355825 A CN 116355825A
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Abstract
The invention discloses a method for enhancing cellulose synthesis to improve the secretion production efficiency of human epidermal growth factor, fermenting the reformed recombinant escherichia coli in a culture medium; the transformation method is to transform in the following way: enhancing the expression of bcsB gene. The method provided by the invention improves the capability of the escherichia coli for secreting and producing the human epidermal growth factor, so that the efficiency of the escherichia coli for producing the human epidermal growth factor in single batch free fermentation and immobilized continuous fermentation is improved by 1-2 times, and the continuous production of the human epidermal growth factor is realized.
Description
The invention relates to a 202210176910X divisional application of a method for improving the secretion production efficiency of human epidermal growth factor, which is filed in 2022, 02 and 25 days.
Technical Field
The invention belongs to the fields of genetic engineering technology and fermentation engineering, and in particular relates to a method for improving secretion production efficiency of human epidermal growth factor by using escherichia coli.
Background
Biosynthesis of pharmaceutical proteins is of increasing interest because of the higher safety of biosynthesis compared to chemical synthesis. Coli has many advantages such as short growth cycle, easy realization of high-density culture, clear genetic background, and rapid transformation of exogenous genes, and is therefore commonly used for expression of recombinant proteins. However, achieving commercial production of recombinant proteins remains a challenge, and one of the most important problems is the poor ability of E.coli to secrete proteins. The problems of inclusion body formation, protein degradation, cellular metabolic burden, or inefficiency in the transport/delivery system of expressed proteins during production all result in inefficient secretion of proteins. In order to improve the protein secretion efficiency of E.coli, various methods have been attempted, such as selection of highly potent signal peptides (e.g., ompA and PelB signal sequences) for directing proteins into the E.coli protein secretion pathway; modification of key transport proteins or accessory proteins involved in transmembrane transport has also been applied to increase the secretory efficiency of proteins; furthermore, co-expression of fusion proteins and chaperones has also been shown to increase protein expression and extracellular secretion; other methods of improving protein secretion also include optimizing fermentation process parameters and media formulation.
Recombinant human epidermal growth factor (rhEGF) is a potential therapeutic protein widely used as a healing agent for various chronic wounds, and has been successfully expressed extracellularly in E.coli BL21 (DE 3) using the PelB signal peptide (Chinese patent No. CN 111471636A). Secretion of hEGF into the culture medium greatly simplifies recovery of the product and reduces the cost of separation in downstream fermentation processes. However, hEGF secretion is not efficient and is typically produced in a traditional free fermentation mode. The cell activity of the human epidermal growth factor is reduced under the conditions of shearing force and the like, and the continuous use can not be realized. At present, some researches for improving the secretion of human epidermal growth factor protein mainly realize the yield improvement through modes of optimizing codons (Chinese patent No. CN 1360022A), optimizing signal peptides (Chinese patent No. CN 1854294A), temperature induction (Chinese patent No. CN 102952817A) and the like.
In order to improve the secretion efficiency of hEGF protein, the research adopts different ideas to develop new strategies, such as trying to examine the influence of the expression of the biofilm formation related genes or some other unknown genes on the secretion efficiency of human epidermal growth factor from the perspective of physiological process such as escherichia coli biofilm and the like, and further creating a continuous fermentation process based on the biofilm. Coli biofilm is a complex population of cells consisting of colonies embedded in an Extracellular Polymeric (EPS) matrix. Cells encapsulated in the biofilm are adsorbed on the surface of the solid medium, can continuously self-renew and withstand adverse conditions by absorbing nutrients. The immobilized continuous fermentation system based on the biofilm is applied to the production of small molecular products such as L-threonine (China patent No. CN 201910392765.7), but no report on human epidermal production factors is available at present, and the influence of related genes (such as bcsB, fimH, csgAB) of the biofilm of escherichia coli and other physiological genes (such as moaE, gshB, yceA, ychJ) on hEGF secretion and production is not reported.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a recombinant escherichia coli and a construction method thereof aiming at the defects of the prior art.
The invention also solves the technical problem of providing a method for improving the secretion production efficiency of human epidermal growth factor.
In order to solve the first technical problem, the invention discloses a recombinant escherichia coli, which is modified by adopting any one or more of the following modes;
A. weakening the expression of one or more genes selected from moaE gene, gshB gene, yceA gene and ychJ gene;
B. enhancing the expression of one or more genes selected from bcsB gene, csgA gene, csgB gene and fimH gene.
Wherein the moaE Gene (NCBI Gene ID: 945399) encodes a protein involved in the biosynthesis of molybdenum pterin; the gshB Gene (NCBI Gene ID: 947445) encodes a glutathione synthetase; the yceA (NCBI GeneID: 945601) gene encodes a tRNA U34 hydroxylase; the ychJ Gene (NCBI Gene ID: 945828) encodes an NTF 2-like domain protein.
Wherein the bcsB Gene (Gene ID: 948045) is involved in cellulose synthesis, the csgA Gene (Gene ID: 949055) and the csgB Gene (Gene ID: 947391) are involved in frizzled pili synthesis, and the fimH Gene (Gene ID: 948847) is involved in type I pili synthesis.
Wherein the expression mode of the weakening gene is that gene knockout is carried out in the escherichia coli genome; the gene knockout mode is CRISPR/Cas9 editing technology (Jiang, Y, et al, multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.appl environmental Microbiol.2015058.81 (7), 2506-14), or lambda-red homologous recombination technology (Madyagol, M.et al, 2011.Gene replacement techniques for Escherichia coli genome modification.Folia Microbiol (Praha), 56 (3), 253-63).
Wherein, the mode of strengthening gene expression is to integrate expression in the escherichia coli genome, and the integrated mode adopts the CRISPR/Cas9 editing technology or the homologous recombination technology; or connecting the target gene to a gene expression plasmid, such as common gene expression plasmids of pBbE1a, pET28, pETDuet and the like, for expression.
The escherichia coli recombinant strain expressing bcsB genes, csgcacsgB genes and fimH genes in a genome integration mode is named as BL21-bcsB, BL 21-csgcacsgB and BL 21-fimH; coli recombinant strain expressing bcsB gene, csgcsgB gene and fimH gene by plasmid was designated BL21-bcsB + ,BL21-csgAcsgB + ,BL21-fimH + 。
The gene integration sites of BL21-bcsB, BL 21-csgAcsB and BL21-fimH can be any one of the gene integration sites of escherichia coli reported in the prior literature, such as yjiP_yjiR, thrW_ykfN, ykgH_betA and ileY_ygaQ (Goormans, A.R. et al.,2020.Comprehensive study on Escherichia coli genomic expression:Does position really matterMetab Eng,62,10-19), and can also be any one of the knockout sites of moaE gene, gshB gene, yceA gene, ychJ gene and the like, and the integration fragment is an expression cassette consisting of a promoter+target gene fragment+terminator; preferably, the integration site is a moaE knockout site, the promoter in the expression cassette is a trc promoter, and the genome integration mode is CRISPR/Cas9 editing technology or lambda-red homologous recombination technology
The construction method of the recombinant escherichia coli is also within the protection scope of the invention.
In order to solve the second technical problem, the invention discloses a method for improving the secretion production efficiency of human epidermal growth factor, which comprises the steps of fermenting the recombinant escherichia coli in a culture medium until the Optical Density (OD) of the bacterial growth is reached 600 ) The numerical value reaches 0.6-0.8, and the production of the human epidermal growth factor is carried out after IPTG induction, so as to obtain the fermentation liquor containing the human epidermal growth factor; by using the recombinant escherichia coli, the human epidermal growth factor can be more effectively secreted into a liquid culture medium for production by introducing genes producing the human epidermal growth factor and fermenting the human epidermal growth factor in the culture medium, and meanwhile, a high-efficiency continuous fermentation system can be established.
Wherein the culture medium contains 5-15 g/L of tryptone, 1-30 g/L of yeast powder extract, 0-12 g/L of sodium chloride, 0-50 g/L of glucose, 0-10 mL/L of glycerol, 0-14.4 g/L of dipotassium hydrogen phosphate and 0-5 g/L of potassium dihydrogen phosphate.
Wherein, when the content of sodium chloride is not 0g/L, the content of glycerin, dipotassium hydrogen phosphate and potassium dihydrogen phosphate is 0g/L, namely, the culture medium contains 5-15 g/L of tryptone, 1-30 g/L of yeast powder extract, 0.001-12 g/L of sodium chloride, 0-50 g/L of glucose, preferably 8-12 g/L of tryptone, 2-7 g/L of yeast powder extract, 8-12 g/L of sodium chloride, 0-6 g/L of glucose, more preferably 10g/L of tryptone, 5g/L of yeast powder extract, 10g/L of sodium chloride, and 0 or 5g/L of glucose.
Wherein, when the content of sodium chloride is 0g/L, the content of glucose is 0g/L, and the content of glycerin, dipotassium hydrogen phosphate and monopotassium phosphate is not 0g/L, namely, the culture medium contains 5-15 g/L tryptone, 1-30 g/L yeast powder extract, 0.001-10 mL/L glycerin, 0.001-14.4 g/L dipotassium hydrogen phosphate and 0.001-5 g/L monopotassium phosphate.
Wherein isopropyl-beta-D-thiogalactosyl IPTG is added in the fermentation process; preferably, 0.01-1.5 mM isopropyl-beta-D-thiogalactoside is added in the fermentation process; preferably, 1mM isopropyl-beta-D-thiogalactoside is added during the fermentation.
Wherein the temperature of the fermentation is 25-37 ℃; preferably, the temperature of the fermentation is 25 ℃.
Wherein the fermentation is free fermentation or immobilized fermentation; the fermentation is single-batch fermentation, continuous fermentation or repeated batch fermentation; the fermentation is immobilized repeated batch fermentation.
Wherein the immobilized fermentation carrier is any one or a combination of more of cotton fiber, polyester fiber, activated carbon, non-woven fabric, polylactic acid, nylon fiber, wood pulp cotton, activated carbon, polyethylene, polyvinyl alcohol, silk, polyurethane, clay and metal. Wherein the carrier exists in the form of the material itself or any one of cloth strips, resin, sponge analogues, plastic sheets, plastic springs and glass slides which are processed by the carrier.
Wherein the dosage of the carrier in the immobilized fermentation is 5-100 g/L; preferably, the dosage of the carrier in the immobilized fermentation is 5-60 g/L; further preferably, the amount of carrier used in the immobilized fermentation is 10 to 50g/L.
In the present invention, the free fermentation means that the cells exist in a free suspension state in a medium without adding a solid carrier to the medium for fermentation.
In the invention, the immobilized fermentation refers to adding a solid carrier into a culture medium, and carrying out fermentation by adsorbing and growing cells on the solid carrier.
In the invention, the single-batch fermentation refers to that no nutrient substances are added or removed to the fermentation liquid after inoculation in a culture medium until the fermentation is finished (18-72 h).
In the invention, the continuous fermentation means that a certain amount of fermentation liquid is discharged in the fermentation process, and fresh culture medium is added into a reactor at the same time, so that the continuity of the fermentation process is realized. The fermentation broth can be discharged intermittently, such as 4/5, 2/3, 1/2, 1/3 or 1/4 of the reaction volume can be discharged each time, and then the same volume of fresh culture medium is supplemented for fermentation; the fermentation broth may be discharged continuously, that is, a part of the fermentation broth may be continuously discharged, and the same volume of fermentation broth may be continuously replenished.
In the invention, the immobilized repeated batch fermentation is one of the special modes of continuous fermentation, namely, a solid carrier is added into a culture medium, after one period in the fermentation process is finished, all fermentation liquid is discharged, the solid carrier is reserved, and then a new fermentation culture medium is added to continue the fermentation of the next batch, so that the process is repeated.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. the efficiency of secreting human epidermal growth factor of the escherichia coli cells under various fermentation conditions can be effectively improved through weakening moaE genes, gshB genes, yceA genes and ychJ genes or strengthening bcsB genes, csgcsgB genes and fimH genes, and the cells can be favorably adsorbed and grown on various solid surfaces to form a biological film.
2. The invention discloses a method for producing human epidermal growth factor by immobilized fermentation of escherichia coli, which uses a solid material as an immobilized carrier for the adsorption growth of escherichia coli, wherein the adsorbed and immobilized thalli can be repeatedly utilized, so that the thallus density is improved, the seed is not required to be prepared in the fermentation process, and long-term continuous fermentation can be performed, thereby improving the fermentation speed, reducing the downtime, improving the production intensity and saving the culture resources and the fermentation cost.
3. The invention provides a method for improving the production efficiency of human epidermal growth factor by a biological film, which improves the capability of the escherichia coli for secreting and producing the human epidermal growth factor, improves the efficiency of the escherichia coli for producing the human epidermal growth factor in single batch free fermentation and immobilized continuous fermentation by 1-2 times, and realizes the continuous production of the human epidermal growth factor.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a plasmid map of overexpression bcsB, csgAcsgB, fimH.
FIG. 2 shows the results of the enzyme digestion test of the plasmid extracted from the positive transformant after the thermal transfer of the target gene to the plasmid pBbE1a to BL21, wherein the enzyme digestion results of pBbE1a-bcsB, the enzyme digestion results of pBbE1a-csgAcsgB, the enzyme digestion results of pBbE1a-fimH and the enzyme digestion results of pBbE1a are shown in the sequence from left to right.
FIG. 3 shows a nucleic acid electrophoresis chart of colony PCR verification of a positive transformant selected from the moaE, gshB, yceA, ychJ gene knocked out in BL21 (with the colony PCR result of wild fungus BL21 as a control).
FIG. 4 shows a sequence of PCR-verified nucleic acid electrophoresis of colonies of positive transformants selected for incorporation of the bcsB, csgAcsgB, fimH gene at the moaE knockout site in BL21 (with the result of PCR of colonies of wild-type BL21 as a control).
FIG. 5 is a graph of the relative biomass of crystal violet staining of 96-well plates, the control in A being plasmid pET30a-hEGF and empty plasmid pBbE1a, into which human epidermal growth factor is introduced in wild BL 21; the control in panel B contained only one plasmid pET30a-hEGF.
FIG. 6 shows the results of single-batch fermentation SDS-PAGE gel electrophoresis using LB medium at an IPTG concentration of 1mM and the results of relative quantitative analysis of protein gel using Image Lab.
FIG. 7 shows the result of hEGF SDS-PAGE gel electrophoresis in immobilized continuous fermentation broth using LB medium and cotton fiber as immobilization carrier; FIG. 7A shows lanes with BSA, control 1, BL21-bcsB + 1,BL21-csgAcsgB + 1,BL21-fimH + 1, control 2, BL21-bcsB + 2,BL21-csgAcsgB + 2,BL21-fimH + 2 (control 1 and control 2 are parallel experiments, both plasmids of the empty plasmid pBbE1a and the human epidermal growth factor producer were introduced into BL21 (DE 3); FIG. 7B shows the sequence of BSA lanes, control (plasmid for human EGF in B21 (DE 3) was introduced)),BL21ΔmoaE,BL21ΔgshB,BL21ΔyceA,BL21ΔmoaE,BL21-bcsB*,BL21-csgAcsgB*,BL21-fimH*。
FIG. 8 shows the results of relative quantitative analysis of hEGF using Image Lab software for immobilized continuous fermentation using LB medium and cotton fiber as the immobilization carrier.
FIG. 9 shows the optical density OD of cells in a fermentation broth of immobilized continuous fermentation using a LB medium and cotton fibers as an immobilization carrier 600 。
Detailed Description
The present invention will be more readily understood by those skilled in the art from the following examples. The examples are described for the purpose of illustrating the invention only and should not be construed as limiting the invention as detailed in the claims, but the use of strains for the immobilized expression of recombinant proteins is also intended to be included within the scope of the invention.
The technical scheme of the invention is implemented on the premise, and the embodiment gives a detailed implementation mode and a specific operation process. The reagents used in the following examples are all commercially available. The experimental methods described in the following examples are conventional methods unless otherwise specified.
The following examples illustrate the process of the present invention in detail by genetic modification and surface immobilized continuous fermentation using human epidermal growth factor as the target product.
The E.coli used in the following examples was BL21 (DE 3).
The yeast powder extract used in the following examples was OXOID.
Recombinant bacterium BL21-bcsB related to the following examples + ,BL21-csgAcsgB + ,BL21-fimH + BL21-bcsB, BL21-csgAcsgB, BL21-fimH, BL2 ΔmoaE, BL21 ΔgshB, BL21 ΔyceA and BL21 ΔychJ are respectively the starting bacteria of Escherichia coli BL21 (DE 3).
The control bacterium E.coli BL21 (DE 3) described in the following examples, wherein the control bacterium E.coli BL21 (DE 3) is introduced into the wild BL21 (DE 3) with the plasmid pET30a-hEGF producing human epidermal growth factor and the empty plasmid pBbE1a, if not specified, as compared with the recombinant E.coli expressing the target gene by the plasmid pBbE1a; in comparison with recombinant E.coli modified in two other ways (integrated expression and gene knockout), E.coli BL21 (DE 3) of the control strain was introduced with only human EGF-producing plasmid pET30a-hEGF.
The plasmid for producing human epidermal growth factor is human epidermal growth factor gene with SEQ ID NO.1, and plasmid pET30a is used according to the Chinese invention application: CN111471636A was constructed and designated pET30a-hEGF.
The plasmid producing human epidermal growth factor is introduced into chassis strain with successful construction to obtain monad strain with kana resistance and double plasmid strain with kana+ampicillin resistance, which expresses target gene via plasmid pBbE1 a.
In the process of constructing the strain, software Snap Gene is used for designing primers, and the synthesis and sequencing of related primer sequences SEQ ID NO.2-65 and the like are provided by synthesis of general companies.
The molecular biology experiments in the examples include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc. according to the molecular cloning guidelines.
Example 1Construction of recombinant bacterium BL21-bcsB by over-expressing bcsB, csgAcsgB, fimH Gene by plasmid + ,BL21-csgAcsgB + ,BL21-fimH +
1.1 plasmid pBbE1a was extracted according to the procedure of plasmid extraction kit (Axygen AxyPrep Plasmid Miniprep Kit), and the vector was subjected to double cleavage by selecting BglII site and AvrII site.
1.2 PCR of the target fragment was performed using the E.coli BL21 genome as template, and the terminator portion was PCR-performed using plasmid pBbE1a as template, and then the two fragments were ligated together by overlap PCR for subsequent plasmid construction. The primer of the target gene fragment was designed using Snapge software, the nucleotide sequence was shown in SEQ ID No.2-11 (BglII site followed by trc promoter and terminator followed by AvrII site in two selected cleavage sites on plasmid pBbE1a, SEQ ID No.4, 5 were used as upstream and downstream primers to PCR terminator with this plasmid as template, and ligated to plasmid pBbE1a by one-step cloning after ligation with bcsB gene overlap; SEQ ID No.5 could be used as downstream primer for three gene overlap PCR simultaneously; SEQ ID No.8 and SEQ ID No.11 were the upstream primers for csgAcsgB and fimH gene terminator, respectively)
PCR system (100. Mu.L split into 5 tubes): 5 XPS Buffer 20. Mu.L, dNTP Mix 10. Mu.L, 1. Mu.L each for the upstream and downstream primers, 1. Mu.L for the template, 1. Mu.L for Prime STAR enzyme, dd H 2 O 67μL
PCR conditions: 95 ℃ for 10min;95 ℃ for 10s;55 ℃,15s;72 ℃,2min (extension time varies depending on fragment length), 38 cycles.
1.3 construction of recombinant plasmids
The purified linearization vector obtained in step 1.1 is connected with the target gene fragment obtained in step 1.2 according to the steps of the instruction book of the ClonExpress II One Step Cloning Kit kit, so as to obtain recombinant plasmids pBbE1a-bcsB, pBbE1a-csgAcsgB, pBbE1a-fimH, and a plasmid map of the over-expression bcsB, csgAcsgB, fimH is shown in FIG. 1.
1.4 the constructed recombinant plasmid was transferred hot into BL21 competence and positive transformants were selected on LB plates containing 100. Mu.g/mL ampicillin resistance.
1.5 picking up positive transformant, liquid culturing, enzyme cutting, and running nucleic acid electrophoresis to verify, as shown in figure 2.
Example 2Construction of recombinant E.coli BL 21. Delta. MoaE, BL 21. Delta. GshB, BL 21. Delta. YceA and BL 21. Delta. YchJ genes from which moaE, gshB, yceA and ychJ genes were knocked out
The methods disclosed in the references (Jiang, Y, et al, multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.) use CRISPR-Cas9 to knock out the moaE, gshB, yceA and ychJ genes on the E.coli genome, and the modified strains were named BL 21. Delta. MoaE, BL 21. Delta. GshB, BL 21. Delta. YceA and BL 21. Delta. YchJ, respectively.
The method comprises the following specific steps:
2.1 obtaining repair templates
Taking the escherichia coli BL21 genome as a template, designing primers of 500bp on the upstream and downstream of a target gene respectively, carrying out PCR, recovering product glue, connecting the products through overlap PCR, and carrying out PCR under the conditions similar to the step 1.2 in the example 1, wherein the nucleotide sequence of the primers is shown in SEQ ID NO. 12-27.
2.2 construction of pTarget-moaE plasmid, pTarget-gshB plasmid, pTarget-yceA plasmid, pTarget-ychJ plasmid
a. Inverse PCR is carried out by taking pTargetF plasmid as a template and adopting primer pairs moaE-N20, gshB-N20, yceA-N20 and ychJ-N20, wherein the nucleotide sequence of the primer is shown in SEQ ID NO.28-35, and a DNA fragment with the length of about 2.1kb is obtained.
moaE-N20:CATCCCCGGATAGTGTTCGA
gshB-N20:CGTTAAGTCAGAGAACCAGG
yceA-N20:TCGCCCAAAAACATTCAGCG
ychJ-N20:GCATCCCTCTTGTGGAGCAG
PCR system (100. Mu.L split into 5 tubes): 2 XPS Buffer 50. Mu.L, dNTP 20. Mu.L, 3. Mu.L each for the upstream and downstream primers, 1. Mu.L for the template, 1. Mu.L for KOD enzyme, dd H 2 O 21μL
PCR conditions: 95 ℃ for 10min;95 ℃ for 10s;55 ℃,15s;72 ℃,2min 30s (extension time varies depending on fragment length), 38 cycles.
b. PCR products were purified using Dpn I enzyme according to the system: PCR product 7. Mu.L, dpn I enzyme 1. Mu.L, 10 XBuffer. Mu.L, ddH 2 O1 muL is digested by the original template, then digested by the original template is transferred into Trans 1T 1 competence, a streptavidin LB plate is coated, the culture is carried out at 37 ℃ overnight, and the colony is picked the next day and inoculated into a test tube for bacteria preservation.
2.3 obtaining of recombinant E.coli BL 2. Delta. MoaE, BL 21. Delta. GshB, BL 21. Delta. YceA and BL 21. Delta. YchJ
a. Plasmid pCas was transformed into host BL21 (DE 3) by heat shock and plated on plates containing 50. Mu.g/mL kanamycin resistance for selection at 30 ℃.
b. Positive clones were picked, reference (Jiang, Y, et al Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.) were prepared for electrocompetent cells BL21-Cas9, and 10mM arabinose was added to induce expression of RED during the preparation.
c. The plasmid obtained in step 2.2 and the repair template fragment obtained in step 2.1 were electrotransferred to BL21-Cas9 competent cells, and after resuscitating for 2 hours at 30℃were screened with plates of kanamycin sulfate (50. Mu.g/mL) +streptomycin sulfate (40. Mu.g/mL).
And d, after culturing for 20 hours at 30 ℃, the clone is verified by colony PCR, wherein the colony PCR verification result is shown in figure 3, and the nucleotide sequence of the verification primer is SEQ ID NO.66-73.
e. Positive clones were inoculated with LB liquid (Canada resistance), incubated at 30℃for 8-20 hours with 0.5mM IPTG, single colonies were streaked on Canada resistant plates, and elimination of plasmid pTarget was confirmed. Positive bacteria eliminated by the plasmid pTarget can be subjected to the next round of gene editing again.
f, liquid culture at 37 ℃, streaking single colony on a non-resistant plate, and verifying elimination of plasmid pCas by dot plate or liquid culture to obtain recombinant escherichia coli (non-resistant) after gene knockout, and carrying out expression production of human epidermal growth factor by taking the escherichia coli as a host.
Example 3Construction of recombinant bacteria (exemplified by integration into the moaE knockout site) that integrate and express the bcsB, csgAcsB, fimH genes BL21-bcsB, BL 21-csgAcsB, BL21-fimH
3.1 construction of repair template integration fragments
a. The target gene fragment PCR is carried out by taking the escherichia coli genome as a template, the trc promoter PCR is carried out by taking the plasmid pBbE1a as a template, the target gene fragment PCR is carried out by taking the escherichia coli MG1655 genome as a template, the recovered PCR products are connected by the overlap PCR to form an expression cassette, and the nucleotide sequence of the primer is shown in SEQ ID NO.36-65.
b. Taking the escherichia coli genome as a template, selecting homologous arms on the upstream and downstream of the knocked-out locus gene, and carrying out PCR and then carrying out glue recovery on the product.
c. Ligation was performed using the gel recovered products (upper and lower homology arms and expression cassette) as template overlap PCR.
3.2 construction of mutant sgRNA plasmids: i.e., knockout of the pTarget plasmid used by moaE.
3.3 transformation of integration fragments and selection of transformants: as in example 2, the colony PCR verification result is shown in FIG. 4, and the nucleotide sequence of the verification primer is SEQ ID NO.66-67.
TABLE 1 primer list
Example 496-well plate crystal violet staining method for representing biofilm formation effect
4.1 specific Experimental procedure
a. The recombinant strains constructed in examples 1 to 3 and the corresponding control E.coli BL21 (DE 3) were cultured in LB medium (100. Mu.g/mL ampicillin resistance was added to the strain expressing the target gene via plasmid) at 37℃at 200rpm to logarithmic phase, and the bacterial solution was diluted with sterile water to OD 600 =0.1。
200 mu L of LB liquid culture medium (the culture medium of the over-expression strain is required to be added with IPTG for induction) is added into a 96-well plate, 20 mu L of diluted bacterial liquid is inoculated into the culture medium, and the bacterial liquid is subjected to static culture at 37 ℃ to form a film at the bottom of the 96-well plate.
c. Pouring out LB liquid culture medium, washing with PBS for 2-3 times, fixing with methanol at 4deg.C for 15min, pouring out, air drying, and adding 1% crystal violet for dyeing for 15min; pouring out crystal violet dye solution, washing with PBS, adding 200 μL of 33% glacial acetic acid, slightly shaking for 30min to decolorize and dissolve crystal violet, and measuring OD with enzyme-labeled instrument 570 Lower reading, bottom filming biomass was compared.
d. The experimental procedure described above was repeated by changing the LB medium to the LBG medium (adding 6g/L glucose), and the difference in film formation of the strain in the medium to which glucose was added was observed.
4.2 experimental results: as shown in FIG. 5, the film forming at the bottom of the 96-well plate can be obviously promoted by expressing the bcsB, csgAcsgB, fimH genes through plasmids, and the film forming effect of integrating and expressing the three genes is not better than that of the plasmid expression, but the formation of a biological film is promoted to a certain extent. The gshB gene, the yceA gene and the ychJ gene are knocked out, and no obvious promoting effect exists in LB culture medium, but the film forming effect is better than that of a control after glucose is added. There was no significant promotion of biofilm formation following knockout of the moaE gene.
Example 5Congo red binding method for characterizing biofilm formation effect
5.1 Experimental procedure
a. The recombinant strains constructed in examples 1 to 3 and the control strain E.coli BL21 (DE 3) were selected and cultured overnight in a single-point to 5mL LB liquid (100. Mu.g/mL ampicillin resistance was added to the strain expressing the target gene via a plasmid), 1% was transferred to 5mL LB/LBG medium (6 g/L glucose was added to the LBG medium as compared with the LB medium) and cultured to the logarithmic phase, and 0.5mM IPTG was added to induce the strain at 25℃for 20 hours (no IPTG induction was required for the gene knockout strain).
b. After dilution to the same OD, 2mL of the bacterial solution was centrifuged, gently suspended 2 times with PBS, and centrifuged.
c. The cells were suspended by adding 1mL of PBS, stained with Congo Red (CR) (1 g in 100mL, 10 μl) and reacted at 25 ℃ for 10min with a 150rpm shaker, after centrifugation the precipitate and supernatant were observed for color and the supernatant was scanned at full wavelength to read the value at od=485 nm.
5.2 experimental results: the recombinant bacteria of the three genes of the plasmid expression bcsB, csgAcsgB, fimH are obviously combined with Congo red, and the combination effect is more obvious after glucose is added into a culture medium. No significant binding to congo red was observed after integrating expression bcsB, csgAcsgB, fimH, knockout moaE, gshB, yceA, ychJ, and binding was slightly better than control after glucose addition.
Congo red binding ratio = 1-OD 485 /OD 485(PBS+CR) The congo red binding rate of the engineered strain is shown in table 2.
TABLE 2
Example 6SDS-PAGE gel electrophoresis detection protein band and relative quantification
6.1 preparation of protein electrophoresis samples: 1mL of the fermentation broth was taken into a 1.5mL centrifuge tube, centrifuged at 8000rpm for 2min after OD600 was measured, and 21. Mu.L of the supernatant was added with 7. Mu.L of 4*SDS Loading buffer and thoroughly mixed, and then subjected to a 95℃water bath for 5min.
6.2, glue preparation: the preparation of 16.5% protein gel was performed according to the raw Tricine-SDS-PAGE Gel Preparation kit kit.
6.3, loading: the protein gel plate was mounted in a vertical electrophoresis tank and electrophoresis buffer was added. mu.L of the sample was pipetted into the sample wells and protein Maker was added at the corresponding well.
Preparing electrophoresis buffer solution: 4 XBuffer 35 mL+pure water 105mL+10%SDS 1.4mL
4 XBuffer: tris 6 g+glycine 28.8 g+pure water 500mL
6.4 electrophoresis: and (3) switching on a power supply, adjusting the voltage to 120V, and adjusting the electrophoresis time according to the size of the protein sample.
6.5 dyeing and decolorizing: and taking out the protein gel after electrophoresis for dyeing for 1h, taking out, adding a decolorizing solution for decolorizing until the background color is completely disappeared, pouring the decolorizing solution, and taking a picture of the protein gel by using a gel imager.
6.6 relative quantitative analysis of the protein gel target strips with Image Lab using 50mg/L hEGF standard or 250mg/L BSA as a control, followed by cell optical density OD 600 The values are converted.
Example 7Single batch free fermentation
7.1 activation: glycerol bacteria (recombinant strains constructed in examples 1 to 3 and control strain E.coli BL21 (DE 3)) were inoculated in 5mL of LB medium (corresponding resistance was added, kanamycin sulfate resistance was added to the single plasmid strain at 50. Mu.g/L, kanamycin sulfate resistance and ampicillin resistance at 100. Mu.g/L were added to the double plasmid strain) at an inoculum size of 10. Mu.L, and cultured in a shaking table at 37℃for 12 hours at a rotation speed of 200rpm.
7.2 inoculation: the culture medium is split-packed in 500mL shake flasks and sterilized, the liquid loading amount is 100mL, then the corresponding resistance is added, the inoculation amount is 1%, and the shaking table rotation speed is 200rpm, and the culture is carried out at 37 ℃.
7.3 induction: waiting for OD 600 =0.6-0.8, adding 1mM IPTG, and inducing at 25 ℃ for 48h to obtain the fermentation broth containing the product hEGF.
7.4 detection of product: the detection mode is shown in example 6, the SDS-PAGE electrophoresis result is shown in FIG. 6, and the detection mode is quantitatively analyzed by software according to OD 600 Fold increases in relative hEGF production were calculated as shown in table 3.
Example 8Single batch free fermentation under induction of different IPTG concentrations
8.1 the procedure is as in example 7, except that three different IPTG concentrations are studied in the fermentation: effect of 0.2mM,0.5mM,1mM on efficiency of human EGF production
8.2 experimental results: quantitative analysis by software followed by OD 600 Fold increases in relative hEGF production were calculated as shown in table 4.
TABLE 4 Table 4
Example 9Single batch free fermentation in different media
9.1 the procedure is as in example 7, except that the effect of three different medium components on the efficiency of human epidermal growth factor is studied in fermentation,
LB medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride;
lbg medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 5g/L of glucose;
TB medium, tryptone 12g/L, yeast extract 24g/L, disodium hydrogen phosphate 9.4g/L, potassium dihydrogen phosphate 2.2g/L, and glycerol 5mL/L.
9.2 experimental results: quantitative analysis by software followed by OD 600 Fold increases in hEGF production relative to control strain in different media were calculated as shown in table 5.
TABLE 5
Example 10Immobilized continuous fermentation
10.1 activation, inoculation and induction were performed in the same manner as in example 7, except that an immobilization carrier (cotton fiber, 40 g/L) was added to the flask during inoculation for immobilization and continuous fermentation.
10.2 all broth was evolved at the end of each batch fermentation and fresh fermentation medium was simultaneously replenished for 8 batches.
10.3 OD in the fermentation broth was measured by sampling every 12h 600 Numerical values, the results are shown in FIG. 9. From the figure, it can be seen that the cell density curves for the bcsB gene and the csgAcsB gene expressed by plasmids were somewhat different from the control, and a significant decrease in cell density in the fermentation broth was observed in the fourth and fifth batches. The cell density of the fermentation broth in the second and third batches was also significantly reduced by the integrated expression of the bcsB gene. This may indicate that expression of bcsB and csgAcsB genes effectively promotes extracellular matrix synthesis, allowing better adsorption of cells on the vector.
10.4 detection of product: the relative quantitative analysis of the product concentration was performed by running protein electrophoresis on the fermentation broth at the final time of each batch of fermentation, in the same manner as in example 6, except that the reaction was not performed by OD 600 The values are converted. H in fermentation brothThe results of EGF SDS-PAGE gel electrophoresis are shown in FIG. 7, the results of relative hEGF quantitative analysis after quantitative analysis by software are shown in FIG. 8, and the fold increase in yield is shown in Table 6.
TABLE 6
Example 11Immobilized continuous fermentation using different materials as immobilized carrier
The procedure is as in example 10, except that the effect of the use of five different carriers on the efficiency of producing human epidermal growth factor by immobilized continuous fermentation is studied in immobilized continuous fermentation.
The five immobilization carriers are cotton fiber, non-woven fabric, activated carbon, polyester fiber and nylon fiber respectively.
The fold increase in relative hEGF production after quantitative analysis by software is shown in table 7.
TABLE 7
Example 12Immobilized continuous fermentation using cotton fibers with different amounts
The procedure is as in example 10, except that the effects of the amounts of five different cotton fibers used in the immobilized continuous fermentation on the efficiency of producing human epidermal growth factor by the immobilized continuous fermentation are 10g/L,20g/L,30g/L,40g/L,50g/L, respectively.
The fold increase in relative hEGF production after quantitative analysis by software is shown in table 8.
TABLE 8
The invention provides a method for improving the secretion production efficiency of human epidermal growth factor, and a method for realizing the technical scheme, wherein the method and the way are a plurality of methods, the method is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by one of ordinary skill in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. The recombinant escherichia coli is characterized in that the escherichia coli is modified in the following way;
enhancing expression of a bcsB Gene whose nucleotide sequence is that shown in Gene ID 948045 on NCBI.
2. A method for improving secretion production efficiency of human epidermal growth factor is characterized in that recombinant escherichia coli according to claim 1 is fermented in a culture medium to obtain fermentation liquor containing the human epidermal growth factor.
3. The method according to claim 2, wherein the medium contains 5 to 15g/L of tryptone, 1 to 30g/L of yeast extract, 0 to 12g/L of sodium chloride, 0 to 50g/L of glucose, 0 to 10mL/L of glycerol, 0 to 14.4g/L of dipotassium hydrogen phosphate, and 0 to 5g/L of potassium dihydrogen phosphate.
4. A method according to claim 3, wherein when the sodium chloride content is not 0g/L, the contents of glycerin, dipotassium hydrogen phosphate and potassium dihydrogen phosphate are all 0g/L.
5. A method according to claim 3, wherein when the sodium chloride content is 0g/L, the glucose content is 0g/L and the glycerol, dipotassium hydrogen phosphate and potassium dihydrogen phosphate are not 0g/L.
6. The method according to claim 2, wherein isopropyl- β -D-thiogalactoside is added during the fermentation; preferably, 0.01-1.5 mM isopropyl-beta-D-thiogalactoside is added during the fermentation process.
7. The method according to claim 2, wherein the fermentation temperature is 25-37 ℃.
8. The method of claim 2, wherein the fermentation is an immobilized fermentation.
9. The method of claim 8, wherein the immobilized fermented carrier is any one or more of cotton fiber, polyester fiber, activated carbon, nonwoven fabric, polylactic acid, nylon fiber, wood pulp cotton, activated carbon, polyethylene, polyvinyl alcohol, silk, polyurethane, clay, and metal.
10. The method according to claim 8, wherein the amount of carrier used in the immobilized fermentation is 5 to 100g/L.
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