CN115948440A

CN115948440A - Construction method of escherichia coli probiotics for secretory expression of human proinsulin

Info

Publication number: CN115948440A
Application number: CN202211319503.6A
Authority: CN
Inventors: 张帮周; 朱钰; 徐炜; 李源涛; 何剑全; 肖传兴
Original assignee: Shanghai Chengge Pharmaceutical Technology Co ltd
Current assignee: Shanghai Chengge Pharmaceutical Technology Co ltd
Priority date: 2022-10-26
Filing date: 2022-10-26
Publication date: 2023-04-11

Abstract

The invention relates to a construction method of escherichia coli probiotics for secreting and expressing human proinsulin, which is characterized in that EcN is used as a chassis for modification, an escherichia coli CRISPR/Cas9 dual-plasmid system pEcas/pEcgRNA is used for integrating an arabinose promoter, an escherichia coli outer membrane protein ompF signal peptide and a human proinsulin gene into an EcN chromosome, a mural lipoprotein lpp gene of EcN is knocked out at the same time, the outward leakage of periplasmic protein is promoted, a genetic engineering strain EcN-delta lpp-INS strain capable of secreting and expressing human proinsulin and having no resistance marker is constructed, the strain is taken probiotics, an inducer L-arabinose is also an edible component, and the strain is constructed for delivering human proinsulin to intestinal mucosa and stimulating immune tolerance.

Description

Construction method of escherichia coli probiotics for secreting and expressing human proinsulin

Technical Field

The invention relates to the field of microorganisms, in particular to a construction method of escherichia coli probiotics for secretory expression of human proinsulin.

Background

Escherichia coli Nissle 1917 (EcN), the only non-pathogenic E.coli, was isolated from Alfred Nissle in 1917 and has been used for the treatment of various gastrointestinal disorders, including inflammatory bowel disease and irritable bowel syndrome. As a probiotic of escherichia coli, ecN has excellent safety performance and is widely applied to the research fields of clinic, vaccine, drug research and development, bioengineering and the like. In the field of bioengineering, ecN has become one of the most widely used underpan bacteria in biomedical therapy, with mature gene manipulation tools and rich knowledge reserves.

Diabetes is a comprehensive metabolic disorder characterized by chronic hyperglycemia caused by multiple causes such as environment and heredity, and the relative or absolute deficiency of insulin secretion in a patient body or insensitivity of target tissues to insulin. Typical clinical manifestations are "more than three and one less": polydipsia, polyphagia, polyuria, weight loss. Diabetes is classified into type i diabetes (insulin-dependent) and type ii diabetes (non-insulin-dependent). Type I diabetes is an abnormal reaction of islet beta cells of a person with autoimmune deficiency or genetic tendency caused by environmental factors such as virus infection, so that the beta cells of pancreas in vivo for generating insulin are completely destroyed, thereby completely losing the function of generating insulin, and leading to absolute deficiency of insulin. Type I diabetes is often developed in teenagers, the incidence rate of the type I diabetes accounts for about 5% -10% of the total number of patients with diabetes, and the patients are often required to be injected with exogenous insulin for life. Insulin is not completely lost in type II diabetes, and the insulin in patients is relatively insufficient, so diet and exercise therapy and oral hypoglycemic drug treatment are generally performed first, and the insulin is needed when the therapy is not enough to control the blood sugar level.

Insulin is a proteinaceous hormone secreted by pancreatic beta cells and consists of 51 amino acid residues. Mature insulin has a relative molecular weight of 5784 and consists of an A chain of 21 amino acid residues and a B chain of 30 amino acid residues. Proinsulin is a precursor of insulin, a single-chain polypeptide, containing 86 amino acids. The biological action of proinsulin is basically the same as that of insulin, but the activity is weak, the capability of the biosynthetic proinsulin for utilizing glucose in the periphery is only 5% -10% of that of the insulin, but the hypoglycemic effect of the proinsulin is longer than that of the insulin, and the capability of inhibiting the liver from releasing glucose is 1.5 times of that of utilizing peripheral glucose.

Immune tolerance refers to the phenomenon that T cells and B cells do not produce specific immune effector cells and specific antibodies and do not generate normal immune response under the stimulation of antigens. Immune tolerance can be divided into innate immune tolerance and adaptive immune tolerance according to the mechanism of development. Innate immune tolerance, also known as natural immune tolerance, is naturally acquired. Adaptive immune tolerance, also known as acquired immune tolerance, can be achieved by a post-day stimulus. The secretion of suppressive cytokines by immunoregulatory cells, such as regulatory T cells (Treg cells), can lead to immune tolerance. Research on NOD mice proves that under the stimulation of proinsulin persistent antigens and the induction of interleukin 10 immunosuppressive cytokines, treg cells can be promoted to generate, and the Treg cells can generate CTLA4 and TGF-beta, so that the proliferation of effector T cells is inhibited, and the proliferation of immunocompetent cells such as NK cells, B cells and the like is inhibited, and the immune tolerance effect combined with an anti-CD 3 antibody can reverse new type I diabetes, retain the function of residual beta cells and stop the development of insulitis.

Disclosure of Invention

In order to solve the problems, the primary object of the invention is to provide a construction method of escherichia coli probiotics for secreting and expressing human proinsulin, which is characterized in that EcN is used as a chassis for reconstruction, an escherichia coli CRISPR/Cas9 dual-plasmid system pEcas/pEcgRNA is used for integrating an arabinose promoter, an escherichia coli outer membrane protein ompF signal peptide and a human proinsulin gene into an EcN chromosome, a mural lipoprotein lpp gene of EcN is knocked out at the same time, the outward leakage of periplasmic protein is promoted, a genetic engineering strain EcN-delta lpp-INS capable of secreting and expressing human proinsulin and having no resistance marker is constructed, the strain is edible probiotics, an inducer L-arabinose is also an edible component, and the strain is constructed for delivering human proinsulin to intestinal mucosa and stimulating immune tolerance.

In order to realize the purpose, the invention adopts the technical scheme that:

a construction method of escherichia coli probiotics for secreting and expressing human proinsulin comprises the following steps:

s1, constructing a pEcgRNA-N20 vector;

the method specifically comprises the following steps: s11, synthesizing a target point N20,

synthesizing a target N20 sequence: N20-F (TAGTGTTACTAAATGTAACTAAA), N20-R (AAACTTTTAGTTACATTTAGTAGAAC). Annealing to prepare the N20 target joint: mixing 10 μ L of N20-F with 10 μ L of N20-R, annealing at 95 deg.C for 5min; at 25 ℃ and an infinite value.

S12, carrying out enzyme digestion on the pEcgRNA vector;

the pEcgRNA vector was digested with Bsa I-HF restriction enzyme as follows: pEcgRNA 2-3. Mu.g, bsa I-HF restriction enzyme 4-5. Mu.L, 10xCutsmart buffer 10. Mu.L, ddH ₂ O to make up 100. Mu.L, and the mixture was digested at 37 ℃ for 4 hours. Carrying out agarose gel electrophoresis on the enzyme digestion product, wherein the electrophoresis conditions are as follows: 150V,15min.

S13, connecting a pEcgRNA Bsa I enzyme digestion vector and an N20 target joint;

the pEcgRNA Bsa I cleavage vector and the N20 target joint were ligated using T4 DNA ligase as follows: pEcgRNA Bsa I restriction enzyme vector recovery fragment 50-100ng, 10xT4 DNA ligase buffer1 uL, N20 target point joint 1 uL, T4 DNA ligase 1 uL, ddH ₂ And supplementing 10 mu L of O, and reacting at room temperature of 22-25 ℃ for 2-4h.

S14, transformation of DH5 alpha competence:

melting DH5 alpha competent cells on 100 mu L of ice, adding T4 connection products, gently mixing, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 50sec, quickly transferring to an ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing and recovering at 37 ℃ for 30min after mixing, coating on a sterile LB + Spec culture medium plate, and standing overnight culture in a constant-temperature incubator at 37 ℃.

S2, overlapping PCR to obtain an INS expression frame;

the method specifically comprises the following steps:

s21, designing a gene sequence of the INS expression frame, wherein the gene sequence of each segment of the INS expression frame is as follows:

up：AAAGGAGTAGGGAACACGCAATAATGCGCCAGAAACGGAGGGTAATGCGGTTAATAAAAAGAGTTGATCGGTAGTGAAATTAAAACCGATTTTATTGAGATTAACAGTAACTGCGCTAAATAGCATCCAGACACAGAAGGCAAAAAGTAGGCAACTGACTGATATCCAGAGGTTTCTTCGTGCAATATGCTTTCCTTTATTTTCCCAGAAGGCCGGATTTTCTGGTTTCCAGTCGCGTAAAAGATAACGACTATTTTTCTCATTTTGCAGTGCCATATTGTTCCTCACATGCACACATTGGTAATGAAAAAAAGACAAAACAGGAGGTAAGGCGCAATAGCCAGTTATTAGAATTAAGGATGAATCAGGTGAAGTGCT；

P _BAD ：AGACATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGCATCGATTTATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGGAATTCGAGCTCTAAGGAGGTTATAAAAA；

ompF：ATGATGAAGCGCAATATTCTGGCAGTGATCGTCCCTGCTCTGTTAGTAGCAGGTACTGCAAACGCT；

INS：TTTGTTAACCAGCATCTGTGCGGGTCGCACTTAGTAGAAGCCCTGTATCTGGTCTGTGGTGAACGCGGTTTTTTCTATACCCCGAAAACTCGTCGCGAAGCGGAAGATCTGCAGGTGGGCCAAGTTGAACTGGGTGGCGGTCCAGGCGCTGGTAGCTTGCAGCCGTTAGCACTGGAAGGGAGTTTGCAAAAACGTGGCATCGTGGAACAGTGCTGTACGTCTATTTGTTCCCTGTACCAGCTGGAAAATTACTGCAACCACCATCATCATCATCATTAA；

T1：CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT；

down：TTAACTGCCGGGCCGTAGACCCGACAATTATTTTAGCCACGACGTGTCGCCAGCCAGCAGAGCAGGGAACCGCCGCAGACCATTAGCGCGCCTTGCCAGAACGAGAACGACAGCGGGGCGCTGAGTAACACGGCTGCAAGTGCTGAGGAAAGGACGGGCGTAAAATACGAACCTACCGCCATTATGGTGACGTTGCCATGTAATATACCGACATTCCATGCAGCATAAGCAAATCCTAAGGTAAATGCCGCAGAGATGAGTTTAATCATGACGGGCGTGCTAAATACCATTTCTGGTTGTGGCGTAAGAAAATAGTAAACCCACAGACTTGCTCCCGTTAGCAGGACAAAAACGGTAATTCCGTTAAATCCACGTGCG。

s22, designing an amplification primer, wherein the gene sequence of the amplification primer is as follows:

up-F：AGAGTCGACCTGCAGAAGCTTAAAGGAGTAGGGAACACGCA；

up-R：CAAATATGTATCCGCTCATGTCTCAGCACTTCACCTGATTCATCC；

BAD-F：GGATGAATCAGGTGAAGTGCTGAGACATGAGCGGATACATATTTG；

BAD-R：AGAATATTGCGCTTCATCATTTTTTATAACCTCCTTAGAG；

ompF-F：CTCTAAGGAGGTTATAAAAA ATGATGAAGCGCAATATTCT；

ompF-R：GCACAGATGCTGGTTAACAAAAGCGTTTGCAGTACCTGCTAC；

INS-F：GTAGCAGGTACTGCAAACGCTTTTGTTAACCAGCATCTGTGC；

INS-R：ACTGAGCCTTTCGTTTTATTTGTTAATGATGATGATGATGGTG；

T1-F：CACCATCATCATCATCATTAACAAATAAAACGAAAGGCTCAGT；

T1-R：GTCTACGGCCCGGCAGTTAAATTTGTCCTACTCAGGAGAGC；

down-F：GCTCTCCTGAGTAGGACAAATTTAACTGCCGGGCCGTAGAC；

down-R：GGAGCTGCACATGAACTCGAGCGCACGTGGATTTAACGGAAT。

and S23, performing amplification.

Firstly, PCR amplification is carried out to obtain up homologous fragment and arabinose promoter P _BAD Signal peptide ompF, human proinsulin gene (with His label), termination sequence T1 and down homologous fragment, and then obtaining INS expression frame up-P by overlap PCR amplification _BAD -ompF-INS-T1-down。

The amplification system is as follows: KOD FX neo mix buffer 18.5. Mu.L, F primer 0.5. Mu.L (10. Mu.M), R primer 0.5. Mu.L (10. Mu.M), template 0.5. Mu.L.

The amplification conditions were:

up: primers are up-F and up-R, a template is an EcN genome, and PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃,30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

P _BAD : primers are BAD-F and BAD-R, a template is pCas plasmid, and PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃ in 1min30s,35cycles; 10min at 68 ℃; infinity at 10 ℃;

ompF: the primers are ompF-F and ompF-R, the template is a synthetic gene sequence ompF, and the PCR conditions are as follows: 94 ℃,2min; 20s at 98 ℃;68 ℃ in20s, 35cycles; 10min at 68 ℃; infinity at 10 ℃;

INS: the primers are INS-F and INS-R, the template is synthetic gene sequence INS, and the PCR condition is as follows: 94 ℃,2min; 20s at 98 ℃;68 ℃ C, 30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

t1: the primers are T1-F and T1-R, the template is a synthetic gene sequence T1, and PCR conditions are as follows: 94 ℃,2min; 20s at 98 ℃;68 ℃ in20s, 35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

down: primers are down-F and down-R, a template is an EcN genome, and PCR conditions are as follows: 94 ℃,2min; at 98 ℃ for 20s;68 ℃,30s,35cycles; 10min at 68 ℃; infinity at 10 ℃;

up-P _BAD : primer is up-F, BAD-R, template is up, P _BAD And PCR conditions are as follows: 94 ℃ for 2min; at 98 ℃ for 20s;68 ℃,2min,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

ompF-INS-T1: the primers are ompF-F and T1-R, the template is ompF, INS and T1, and the PCR conditions are as follows: 94 ℃ for 2min; at 98 ℃ for 20s;68 ℃ C, 30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

up-P _BAD -ompF-INS-T1-down: the primers are up-F and down-R, and the template is up-P _BAD, ompF-INS-T1, down, PCR conditions: 94 ℃ for 2min; 20s at 98 ℃;68 ℃,2min30s,35cycles; at 68 ℃ for 10min; at 10 ℃ and an infinite value.

S3, constructing a pEcgRNA-N20-INS vector;

the method specifically comprises the following steps: s31, carrying out enzyme digestion on the pEcgRNA-N20 vector;

the pEcgRNA-N20 vector was digested with HindIII-HF, xhoI restriction enzymes as follows: pEcgRNA-N20-3. Mu.g, hindIII-HF restriction enzyme 4. Mu.L, xhoI restriction enzyme 4. Mu.L, 10xCutsmart buffer 10. Mu.L, ddH ₂ O to make up 100. Mu.L, and the mixture was digested at 37 ℃ for 4 hours.

S32, connecting pEcgRNA-N20 enzyme digestion vector and up-P _BAD -ompF-INS-T1-down fragment;

the homologous recombination Kit of Tiangen easy Assembly Kit is used for connecting the pEcgRNA-N20 enzyme digestion vector and up-P _BAD -ompF-INS-T1-down fragment, reaction system as follows: pEcgRNA-N20 enzyme digestion vector 4. Mu.L, up-P _BAD -ompF-INS-T1-down purified fragment 1. Mu.L, 2x Mix buffer 5. Mu.L, reacted at 50 ℃ for 20min, placed on ice for subsequent transformation.

S33, transformation of DH5 alpha competence:

melting DH5 alpha competent cells on 100 mu L of ice, adding homologous recombination ligation products, gently mixing, standing on ice for 30min, thermally shocking in water bath at 42 ℃ for 50sec, rapidly transferring to ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing and recovering for 30min at 37 ℃ after mixing, coating on a sterile LB + Spec culture medium plate, and standing in a constant temperature incubator at 37 ℃ for overnight culture.

S4, designing and synthesizing an lpp target;

designing a target lpp-N20 according to the lpp gene sequence of the EcN genome and synthesizing;

wherein: the gene sequence of lpp-N20-F is TAGTAATCCTGGGTTCTACCTGC, and the gene sequence of lpp-N20-R is AAACGCAGAGTAGAACCCAGGATT.

S5, constructing a pEcgRNA-lpp vector;

putting lpp-N20-F10 mu L and lpp-N20-R10 mu L into a PCR instrument for annealing to form double chains, wherein the annealing condition is 95 ℃ and 5min; at 25 ℃ and an infinite value.

The pEcgRNA Bsa I enzyme cutting vector and the lpp-N20 target point are connected by using T4 DNA ligase, and the system is as follows: recovered fragment 50-100ng of pEcgRNA Bsa I enzyme digestion vector, 1 muL of 10xT4 DNA ligase buffer, 1 muL of lpp-N20 target spot, 1 muL of T4 DNA ligase and ddH ₂ And supplementing 10 mu L of O, and reacting at room temperature of 22-25 ℃ for 2-4h.

Transformation of DH5 α competence: melting DH5 alpha competent cells on 100 mu L of ice, adding T4 connection products, gently mixing, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 50sec, quickly transferring to an ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing and recovering at 37 ℃ for 30min after mixing, coating on a sterile LB + Spec culture medium plate, and standing overnight culture in a constant-temperature incubator at 37 ℃.

S6, designing and amplifying primers of homologous sequences of the upstream and downstream of lpp;

designing upstream and downstream homologous sequence primers according to an lpp gene sequence of the EcN genome and synthesizing, wherein the upstream homologous fragment primers are lpp-up-F and lpp-up-R, and the downstream homologous fragment primers are lpp-down-F and lpp-down-R.

Further, the gene sequence of lpp-up-F is TTGTGCCGCAGCTGGTTAAA, the gene sequence of lpp-up-R is GTTGTCCAGACGCTGGTTAGAAGAAGCATCGACGCTGGG, the gene sequence of lpp-down-F is CCCAGCCGTTCGATGCTTCTTCTAACCAGCGTCGACAAC, and the gene sequence of lpp-down-R is CAGCAGGCTTTACGCAATTT.

The upstream homologous fragment lpp-up PCR amplification is carried out by taking the EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-up-F0.5 uL, lpp-up-R0.5 uL, PCR amplification conditions of 95 ℃,5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min; infinity at 10 ℃; downstream homologous fragment lpp-down PCR amplification is carried out by taking the EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-down-F0.5 uL, lpp-down-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min;10 ℃ and infinity.

Performing overlapping PCR amplification by taking the upstream homologous fragment and the downstream homologous fragment as templates to obtain an upstream homologous fragment lpp-up-down, wherein the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, lpp-up 0.5 uL, lpp-down 0.5 uL, lpp-up-F0.5 uL, lpp-down-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,45sec,35cycles;72 ℃ for 10min;10 ℃ and infinity.

S7, gene knockout and effect detection

Preparing the EcN strain into competence by using calcium chloride and glycerol through a chemical method, converting EcN competence by using pEcas plasmids, coating the EcN competence on a sterile LB + Kana culture medium plate, and standing overnight culture in a constant-temperature incubator at 37 ℃ to obtain the EcN strain containing the pEcas plasmids; similarly, the EcN strain containing the pEcas plasmid was made competent by chemical methods, and the pEcgRNA-lpp vector was transformed into the EcN competent strain containing the pEcas plasmid together with the lpp upstream and downstream homologous fragments.

The pEcgRNA-lpp vector and lpp upstream and downstream homologous fragments together transform the EcN competence containing the pEcas plasmid as follows: competence 200. Mu.L, pEcgRNA-lpp 25. Mu.L (1.5. Mu.g), upstream and downstream homologous fragment lpp-up-down 20. Mu.L (5. Mu.g); melting EcN competent cells containing pEcas plasmids on 200 mu L of ice, adding pEcgRNA-lpp vectors and upstream and downstream fragments according to the system, gently mixing uniformly, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 90s, quickly transferring to an ice bath, standing for 2min, adding 300 mu L LB culture medium without antibiotics into a centrifuge tube, standing for 30min after mixing uniformly, recovering after standing at 37 ℃, completely coating on LB + Kana + Spec culture medium, standing overnight culture at 37 ℃, and performing CRISPR gene knockout.

Detecting the gene knockout effect through colony PCR, selecting a monoclonal colony on an LB + Kana + Spec culture medium plate to carry out colony PCR, taking an EcN genome as a template as a control, and adopting the following system: 1x Taq PCR Master Mix 6.5. Mu.L, lpp-up-F0.25. Mu.L, lpp-down-R0.25. Mu.L, 3. Mu.L of monoclonal bacteria (dissolved in 15. Mu.L water). PCR conditions were as follows: 7min at 95 ℃;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,1min20sec,35cycles;72 ℃ for 7min;10 ℃ and infinity.

S8, eliminating plasmids;

plasmid elimination was performed as follows: adding 10 mu L of bacterial liquid into 1.5mL of LB + kana + rhamnose (10 mM) liquid culture medium for eliminating pEcgRNA-lpp plasmids, culturing for 6.5h at 37 ℃ and 200rpm in a shaking incubator, sucking 20uL of bacterial liquid, pumping the gun head into 1.5mL of simple LB culture liquid, culturing for 1.5h at 37 ℃ in a shaking incubator, coating the gun head on an LB + sucrose (10 g/L) plate culture medium for eliminating pEcas plasmids, and standing overnight at 37 ℃.

S9, carrying out shake culture on the EcN-delta lpp monoclonal bacteria with both plasmids eliminated, extracting the genome of the EcN-delta lpp monoclonal bacteria, and amplifying lpp genes by using lpp-up-F and lpp-down-R primers to obtain an EcN-delta lpp strain;

further, taking the monoclonal bacteria with both plasmids eliminated, and carrying out strain genome extraction and PCR amplification, wherein the steps are as follows: taking 100 mu L of overnight culture liquid, centrifuging at 4000rpm for 5min, discarding supernatant, adding 50 mu L of Buffer A, mixing uniformly, reacting at 95 ℃ for 15min, adding 50 mu L of Buffer B, mixing uniformly, centrifuging at 4000rpm for 5min, and obtaining the supernatant as a genome; PCR amplification was performed using the matched T5 enzyme as follows: 15 μ L of mix buffer of T5 enzyme, 2 μ L of genome supernatant, 1.2 μ L of lpp-up-F, 1.2 μ L of lpp-down-R, ddH ₂ O10.6. Mu.L. PCR amplification conditions: 3min at 98 ℃; at 98 ℃ for 10s; at 64 ℃ for 10s;72 ℃ for 15s;35cycles;72 ℃ for 3min;12 ℃ and infinity.

S10, constructing an EcN-delta lpp-INS strain;

the EcN-. DELTA.lpp strain containing the pEcas plasmid was made competent by chemical methods, and the plasmid pEcgRNA-N20-INS containing the human proinsulin expression cassette was used to transform the EcN-. DELTA.lpp competent strain containing the pEcas plasmid: taking EcN-delta lpp (containing pEcas plasmid) competence 200 mu L, adding pEcgRNA-N20-INS-2 mu g, gently mixing uniformly, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 90s, quickly transferring to an ice bath, standing for 2min, adding 300uL LB culture medium without antibiotics into a centrifuge tube, standing for 30min after mixing uniformly at 37 ℃, recovering, completely coating on LB + Kana + Spec culture medium, standing overnight culture at 37 ℃, and performing CRISPR gene knockout and human proinsulin expression cassette insertion.

S11, detecting the insertion effect of the INS expression cassette;

specifically, the insertion of the human proinsulin expression cassette is detected by colony PCR, a monoclonal colony on an LB + Kana + Spec culture medium plate is selected for colony PCR, an EcN-Deltapp genome is used as a template for comparison, an F primer is a primer in an INS expression cassette, and an R primer is a primer on an EcN genome at the downstream of the insertion position, and the system is as follows: 1x Taq PCR Master Mix 6.5. Mu.L, T1-F0.25. Mu.L, INS-EcN-down-R (GACGGTTTTCGCTGTTGACG) 0.25. Mu.L, monoclonal bacteria (dissolved in 15. Mu.L water) 3. Mu.L. PCR conditions were as follows: 95 ℃ for 7min;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,2min,35cycles;72 ℃ for 7min;10 ℃ and infinity.

S12, eliminating plasmids;

plasmid elimination was performed as follows: adding 10 mu L of bacterial liquid into 1.5mL of LB + kana + rhamnose (10 mM) liquid culture medium for eliminating pEcgRNA-N20-INS plasmids, culturing for 6.5h at 37 ℃ and 200rpm in a shaking table incubator, sucking 20uL of bacterial liquid, pumping a gun head into 1.5mL of pure LB culture liquid for shaking table culture at 37 ℃ for 1.5h, coating the gun head on an LB + sucrose (10 g/L) plate culture medium for eliminating pEcas plasmids, and standing overnight at 37 ℃; after the plasmid is eliminated, the EcN-delta lpp-INS strain is prepared.

16-24 (2-3 rows) monoclonal colonies are picked from each culture medium, placed in20 uL water, 3uL is respectively spotted on three plate culture media of LB, LB + Kana and LB + Spec, a constant temperature incubator is used for overnight at 37 ℃, and whether two plasmids are eliminated or not is judged by counting the growth conditions on the three culture media.

Further, monoclonal bacteria (EcN-delta lpp-INS strains) with both plasmids eliminated are taken to carry out strain genome extraction and PCR amplification, and the steps are as follows: taking 100 mu L of overnight culture liquid, centrifuging at 4000rpm for 5min, discarding supernatant, adding 50 mu L of Buffer A, mixing uniformly, reacting at 95 ℃ for 15min, adding 50 mu L of Buffer B, mixing uniformly, centrifuging at 4000rpm for 5min, and obtaining the supernatant as a genome. PCR amplification was performed using the matched T5 enzyme as follows: 15 μ L of mix buffer of T5 enzyme, 2 μ L of genome supernatant, 1.2 μ L of BAD-F, 1.2 μ L of T1-R, ddH ₂ O10.6. Mu.L. PCR amplification conditions: heating at 98 deg.C for 3min; 10s at 98 ℃; at 64 ℃ for 10s;72 ℃ for 30s;35cycles; 3min at 72 ℃;12 ℃ and infinity.

Further, the EcN-Deltapp-INS strain can also be induced to express: taking the EcN-delta lpp-INS strain on an LB plateStreaking on the culture medium, picking single colony from it in 10mL LB liquid medium at 37 deg.C, 200rpm overnight and shaking on the next day, 1:100 into 1000mL LB medium, 37 ℃ culture to OD ₆₀₀ Adding arabinose as inducer to the final concentration of 10mM, inducing at 37 deg.C and 200rpm for 5 hr, centrifuging at 6000rpm/min, centrifuging the culture medium supernatant, and filtering with 0.45 μm filter membrane.

Further, the EcN-Deltapp-INS strain can also be subjected to protein extraction: the centrifuged bacteria are resuspended according to the amount of 1g of bacteria and 10mL of 0.5mg/mL of lysozyme, digested for 1 hour at 4 ℃, centrifuged at 10000rpm/min, and centrifuged supernatant is periplasmic protein which is poured out of a 0.22-micron filter membrane and filtered for later use.

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

according to the invention, edible probiotic EcN is selected as a chassis bacterium, and an Escherichia coli CRISPR/Cas9 dual-plasmid system pEcas/pEcgRNA is utilized to construct a pEcgRNA-lpp vector at first, knock out a murein lpp gene of EcN, and transform the gene into an EcN-delta lpp strain with outwards leaked periplasm protein. Subsequently, the human proinsulin expression cassette, which is an insert containing the arabinose promoter, the escherichia coli outer membrane protein ompF signal peptide and the human proinsulin gene, was obtained by biosynthesis and overlap PCR amplification. A pEcgRNA-N20-INS vector containing a target N20 and a human proinsulin expression frame is constructed through T4 connection and homologous recombination, the human proinsulin expression frame is inserted into an EcN-delta lpp strain by combining with a pEcas plasmid, an EcN-delta lpp-INS strain which is escherichia coli probiotics without resistance markers and capable of secreting and expressing human proinsulin is constructed, human proinsulin is contained in the supernatant of a culture medium which is induced and expressed by the EcN-delta lpp-INS strain, and meanwhile, an inducer L-arabinose is an edible component and has a positive effect on maintaining stable blood sugar.

The escherichia coli probiotic EcN-delta lpp-INS strain secreting and expressing human proinsulin lays a foundation for development of live diabetes mellitus type I drugs, subsequent research focuses on further improvement of the live diabetes mellitus type I drugs, a strain secreting and expressing human interleukin 10 is constructed, and the cytokine has the effect of promoting formation of immune tolerance and is stimulated to cooperate with human proinsulin antigen to induce formation of the immune tolerance.

Drawings

Fig. 1 is a technical route diagram of the present invention.

FIG. 2 shows gel electrophoresis of the digestion product of pEcgRNA vector Bsa I.

FIG. 3 is a PCR schematic of pEcgRNA-N20 colonies.

FIG. 4 shows the pEcgRNA-N20 restriction enzyme.

FIG. 5 is a schematic diagram of overlapping PCR amplified fragments.

FIG. 6 is a schematic diagram of PCR of pEcgRNA-N20-INS colonies.

FIG. 7 shows the restriction enzyme verification of pEcgRNA-N20-INS.

FIG. 8 is a schematic representation of the upstream and downstream homologous sequences of lpp.

FIG. 9 is a diagram of lpp upstream and downstream homology sequences up-down.

FIG. 10 is a PCR schematic of pEcgRNA-lpp colonies.

FIG. 11 shows the restriction of pEcgRNA-lpp.

FIG. 12 shows the confirmation of the deletion of lpp gene by colony PCR.

FIG. 13 shows the elimination of the EcN-. DELTA.lpp plasmid.

FIG. 14 is a schematic of plasmid-deleted EcN-. DELTA.lpp genomic amplification.

FIG. 15 is a schematic diagram of confirmation of insertion of the INS expression cassette by colony PCR.

FIG. 16 shows the plasmid elimination of EcN-. DELTA.lpp-INS.

FIG. 17 is a schematic of plasmid-deleted EcN- Δ lpp-INS genomic amplification.

FIG. 18 shows Western Blot detection of supernatant and periplasmic protein of EcN-. DELTA.lpp-INS.

FIG. 19 is a diagram showing the identification of amino acid sequence matches of peptide fragments.

FIG. 20 is a mass spectrum of the identified peptide fragment sequence.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

FIG. 1 shows a technical route implemented by the present invention, wherein the method for constructing Escherichia coli probiotics secreting and expressing human proinsulin comprises the following steps:

s1, constructing a pEcgRNA-N20 vector;

the method specifically comprises the following steps: s11, synthesizing a target N20 sequence: N20-F (TAGTGTTACTAAATGTAACTAAA), N20-R (AAACTTTTAGTTACATTTAGTAGAAC). Annealing to prepare the N20 target joint: mixing 10 μ L of N20-F with 10 μ L of N20-R, annealing at 95 deg.C for 5min; at 25 ℃ and an infinite value.

S12, carrying out enzyme digestion on the pEcgRNA vector; the pEcgRNA vector was digested with Bsa I-HF restriction enzyme as follows: pEcgRNA 2-3. Mu.g, bsa I-HF restriction enzyme 4-5. Mu.L, 10xCutsmart buffer 10. Mu.L, ddH ₂ The amount of O is 100. Mu.L, and the enzyme is cleaved at 37 ℃ for 4h. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, wherein the electrophoresis conditions are as follows: 150V,15min.

S13, connecting a pEcgRNA Bsa I enzyme digestion vector and an N20 target joint; the pEcgRNA Bsa I cleavage vector and the N20 target joint were ligated using T4 DNA ligase as follows: pEcgRNA Bsa I restriction enzyme vector recovery fragment 50-100ng, 10xT4 DNA ligase buffer1 uL, N20 target point joint 1 uL, T4 DNA ligase 1 uL, ddH ₂ And supplementing 10 mu L of O, and reacting at room temperature of 22-25 ℃ for 2-4h.

S14, transformation of DH5 alpha competence: melting DH5 alpha competent cells on 100 mu L of ice, adding T4 ligation products, gently mixing, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 50sec, rapidly transferring to an ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing and recovering for 30min at 37 ℃ after mixing, coating on a sterile LB + Spec culture medium plate, and standing in a constant temperature incubator at 37 ℃ for overnight culture.

S2, overlapping PCR to obtain an INS expression frame;

the method comprises the following steps: s21, designing a gene sequence of the INS expression frame, wherein the gene sequence of each segment of the INS expression frame is shown in a table 1.

S22, designing an amplification primer, wherein the gene sequence of the amplification primer is shown in Table 2.

And S23, performing amplification.

PCR amplification to obtain up homologous fragment, arabinose promoter P _BAD Signal peptide ompF, human proinsulin gene (with His label), termination sequence T1 and down homologous fragment, and then obtaining INS expression frame up-P by overlap PCR amplification _BAD -ompF-INS-T1-down。

The amplification conditions were:

up: primers are up-F and up-R, a template is an EcN genome, and PCR conditions are as follows: 94 ℃,2min; 20s at 98 ℃;68 ℃ C, 30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

P _BAD : primers are BAD-F and BAD-R, a template is pCas plasmid, and PCR conditions are as follows: 94 ℃,2min; 20s at 98 ℃;68 ℃ in 1min30s,35cycles; 10min at 68 ℃; infinity at 10 ℃;

ompF: the primers are ompF-F and ompF-R, the template is a synthetic gene sequence ompF, and the PCR conditions are as follows: 94 ℃ for 2min; at 98 ℃ for 20s;68 ℃ in20s, 35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

the INS: the primers are INS-F and INS-R, the template is synthetic gene sequence INS, and the PCR condition is as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃ C, 30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

t1: the primer is T1-F and T1-R, the template is a synthetic gene sequence T1, and the PCR condition is as follows: 94 ℃,2min; at 98 ℃ for 20s;68 ℃ in20s, 35cycles; 10min at 68 ℃; infinity at 10 ℃;

down: primers are down-F and down-R, a template is an EcN genome, and PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃ C, 30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

ompF-INS-T1: primers are ompF-F and T1-R, templates are ompF, INS and T1, and PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃ C, 30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

up-P _BAD -ompF-INS-T1-down: the primers are up-F and down-R, and the template is up-P _BAD, ompF-INS-T1, down, PCR conditions: 94 ℃ for 2min; 20s at 98 ℃; 2min30s,35cycles at 68 ℃; at 68 ℃ for 10min;10 ℃ and infinity.

S3, constructing a pEcgRNA-N20-INS vector;

the method comprises the following steps: s31, carrying out enzyme digestion on the pEcgRNA-N20 vector;

s32, pEcgRNA-N20 enzyme digestion vector and up-P are connected _BAD -ompF-INS-T1-down fragment;

s33, transforming DH5 alpha competence:

s4, designing and synthesizing an lpp target; designing a target lpp-N20 according to the lpp gene sequence of the EcN genome and synthesizing;

s5, constructing a pEcgRNA-lpp vector;

Upstream homologous fragment lpp-up PCR amplification is carried out by taking the EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-up-F0.5 uL, lpp-up-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min; infinity at 10 ℃; downstream homologous fragment lpp-down PCR amplification is carried out by taking the EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-down-F0.5 uL, lpp-down-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min; at 10 ℃ and an infinite value.

S7, gene knockout and effect detection;

preparing the EcN strain into competence by a chemical method by using calcium chloride and glycerol, converting EcN competence by using pEcas plasmid, coating the EcN competence on a sterile LB + Kana culture medium plate, and standing overnight in a constant-temperature incubator at 37 ℃ for culture to obtain the EcN strain containing the pEcas plasmid; similarly, the EcN strain containing the pEcas plasmid was made competent by chemical methods, and the pEcgRNA-lpp vector was transformed into the EcN competent strain containing the pEcas plasmid together with the lpp upstream and downstream homologous fragments.

Melting EcN competent cells containing pEcas plasmids on 200 mu L of ice, adding pEcgRNA-lpp vectors and upstream and downstream fragments, lightly mixing uniformly, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 90s, quickly transferring to an ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing for 30min for resuscitation at 37 ℃ after mixing uniformly, completely coating on LB culture medium, kana culture medium and Spec culture medium, standing overnight at 37 ℃, and performing CRISPR gene knockout.

The gene knockout effect was detected by colony PCR.

S8, eliminating plasmids;

elimination of the pEcgRNA-lpp plasmid and elimination of the pEcas plasmid.

s10, constructing EcN-delta lpp-INS strains;

the EcN-. DELTA.lpp strain containing the pEcas plasmid was made competent by chemical methods, and the plasmid pEcgRNA-N20-INS containing the human proinsulin expression cassette was used to transform the EcN-. DELTA.lpp competent strain containing the pEcas plasmid: taking EcN-delta lpp (containing pEcas plasmid) competence 200 mu L, adding pEcgRNA-N20-INS-2 mu g, lightly mixing evenly, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 90s, quickly transferring to an ice bath, standing for 2min, adding 300uL LB culture medium without antibiotics into a centrifuge tube, standing for 30min for resuscitation at 37 ℃ after mixing evenly, completely coating on LB + Kana + Spec culture medium, standing overnight culture at 37 ℃, and performing CRISPR gene knockout and human proinsulin expression cassette insertion.

S11, detecting the insertion effect of the INS expression frame;

specifically, the insertion condition of the human proinsulin expression cassette is detected through colony PCR, a monoclonal colony on an LB + Kana + Spec culture medium plate is selected to carry out colony PCR, an EcN-delta lpp genome is used as a template as a control, an F primer is a primer in an INS expression cassette, an R primer is a primer on an EcN genome at the downstream of the insertion position, and the system is as follows: 1x Taq PCR Master Mix 6.5. Mu.L, T1-F0.25. Mu.L, INS-EcN-down-R (GACGGTTTTCGCTGTTGACG) 0.25. Mu.L, monoclonal bacteria (dissolved in 15. Mu.L water) 3. Mu.L. PCR conditions were as follows: 95 ℃ for 7min;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,2min,35cycles;72 ℃ for 7min; at 10 ℃ and an infinite value.

S12, eliminating plasmids;

eliminating pEcgRNA-N20-INS plasmid and pEcas plasmid.

S13, eliminating the plasmid to obtain the EcN-delta lpp-INS strain.

The S14 and EcN-delta lpp-INS strains can also be induced to express: taking the EcN-Deltapp-INS strain to streak culture on an LB plate culture medium, picking a monoclonal colony from the EcN-Deltapp-INS strain in 10mL LB liquid culture medium at 37 ℃ and 200rpm on the next day, shaking the strain overnight on the third day 1:100 into 1000mL LB medium, 37 ℃ culture to OD ₆₀₀ Adding arabinose as inducer to the final concentration of 10mM, inducing at 37 deg.C and 200rpm for 5 hr, centrifuging at 6000rpm/min, centrifuging the culture medium supernatant, and filtering with 0.45 μm filter membrane.

The S15 and EcN-delta lpp-INS strains can also be subjected to protein extraction: the centrifuged bacteria are resuspended according to the amount of 1g of bacteria and 10mL of 0.5mg/mL of lysozyme, digested for 1 hour at 4 ℃, centrifuged at 10000rpm/min, and centrifuged supernatant is periplasmic protein which is poured out of a 0.22-micron filter membrane and filtered for later use.

S16, western-blot detection and mass spectrum detection.

And (3) carrying out membrane conversion after SDS-PAGE electrophoresis, washing the PVDF membrane in TBST after membrane conversion, washing the membrane in TBST, developing after membrane washing, and taking a picture by an imager, namely Western-blot detection.

SDS-PAGE electrophoresis was performed, and SDS-PAGE gel was removed, stained, and then a blue band of 1cm. Times.2cm in the 5-15kD region of the induced supernatant and the induced periplasmic protein lane was excised, and subjected to tandem mass spectrometry.

The practice of the present invention will be described below with reference to experiments.

Experiment one: pEcgRNA-N20-INS vector construction

1 materials of the experiment

1.1 Experimental apparatus: a Wix electrophoresis apparatus, a PCR apparatus, a water bath, a centrifuge, an Azure300 imager, a shaking incubator and a constant temperature incubator.

1.2 Experimental reagents: bsa I-HF restriction enzyme, T4 DNA ligase, bamHI-HF restriction enzyme, hindIII-HF restriction enzyme, xhoI restriction enzyme, DL2000 DNA Ladder, agarose, all-gold DH5 alpha competence, LB broth, spectinomycin hydrochloride pentahydrate, 2 XTaq PCR Master Mix (Blue), TOYOBO KOD FX Neo high fidelity DNA polymerase, tiangen easygo Assembly Cloning Kit homologous recombination Kit.

1.3 Experimental materials: a full-gold gel rapid purification kit, a plabenaceae biological plasmid miniprep kit, a full-gold 5-minute DNA rapid purification kit and a sterile culture dish.

2 method of experiment

2.1pEcgRNA-N20 vector construction

The pEcgRNA vector was digested with Bsa I-HF restriction enzyme as follows: pEcgRNA 2-3. Mu.g, bsa I-HF restriction enzyme 4-5. Mu.L, 10xCutsmart buffer 10. Mu.L, ddH ₂ O to make up 100. Mu.L, and the mixture was digested at 37 ℃ for 4 hours. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, wherein the electrophoresis conditions are as follows: 150V,15min. And cutting and recovering the gel according to the instruction of the full-type gold gel rapid purification kit.

Use of T4 DNAAnd (3) connecting the pEcgRNA Bsa I enzyme digestion vector and the N20 target joint by using the ligase, wherein the system is as follows: recovered fragment 50-100ng of pEcgRNA Bsa I enzyme digestion vector, 1 uL of 10xT4 DNA ligase buffer, 1 uL of N20 target joint, 1 uL of T4 DNA ligase and ddH ₂ And supplementing 10 mu L of O, and reacting at room temperature of 22-25 ℃ for 2-4h.

Transformation of DH5 α competence: melting DH5 alpha competent cells on 100 mu L of ice, adding T4 ligation products, gently mixing, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 50sec, rapidly transferring to an ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing and recovering for 30min at 37 ℃ after mixing, coating on a sterile LB + Spec culture medium plate, and standing in a constant temperature incubator at 37 ℃ for overnight culture.

Selecting a monoclonal colony for colony PCR verification, wherein colony PCR primers are N20-F (TAGTGTTACTAAATGTAACTAAAA) and pEcgRNA-con-R (GACGCTCAGTGGAACGAAAA), and the reaction system is as follows: 1xTaq PCR Master Mix 6.5. Mu.L, N20-F0.25. Mu.L, pEcgRNA-con-R0.25. Mu.L, 3. Mu.L of monoclonal bacteria (dissolved in 15. Mu.L water). PCR conditions were as follows: 95 ℃ for 7min;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,1min,35cycles;72 ℃ for 7min;10 ℃ and infinity. And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the electrophoresis conditions are as follows: 150V,15min.

The positive clone verified by colony PCR is picked up in LB + Spec liquid culture medium of 4.3mL, shaken overnight at 37 ℃ and 200rpm, the plasmid is extracted the next day according to the instruction of the plasmid miniprep kit of the Oncodinaceae organism, and restriction enzyme digestion verification is carried out by using BamHI-HF and HindIII-HF restriction enzymes. The enzyme digestion system is as follows: plasmid 400ng, bamHI-HF restriction enzyme 0.5. Mu.L, hindIII-HF restriction enzyme 0.5. Mu.L, 10XCutsmart buffer 2. Mu.L, ddH ₂ O complement 20. Mu.L, and the mixture is digested at 37 ℃ for 4h. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, wherein the electrophoresis conditions are as follows: 150V,15min. The correct positive clone is verified by enzyme digestion and sent to the organism of the department of Onchillaceae for sequencing.

2.2 obtaining INS expression cassette by overlapping PCR

The human proinsulin gene is subjected to codon optimization, and is synthesized by the Scopheraceae biosynthesis to be used as a template for PCR amplification, and the signal peptide ompF gene sequence and the T1 gene sequence are synthesized to be used as a template for PCR amplification. The gene sequences of the INS expression cassette fragments are shown in Table 1.

Amplification primers were designed as shown in table 2. Firstly, PCR amplification is carried out to obtain up homologous fragment and arabinose promoter P _BAD Signal peptide ompF, human proinsulin gene (with His label), termination sequence T1 and down homologous fragment, and then obtaining INS expression frame up-P by overlap PCR amplification _BAD -ompF-INS-T1-down. The high-fidelity DNA polymerases used in the PCR experiments are TOYOBO KOD FXneo, and the amplification system is as follows: KOD FX neo mix buffer 18.5. Mu.L, F primer 0.5. Mu.L (10. Mu.M), R primer 0.5. Mu.L (10. Mu.M), template 0.5. Mu.L, and the specific experimental parameters are shown in Table 3.

TABLE 1 INS expression cassette fragment Gene sequences

TABLE 2 amplification primers

Primer name	Primer sequence (5 '-3')
		up-F	AGAGTCGACCTGCAGAAGCTTAAAGGAGTAGGGAACACGCA
up-R	CAAATATGTATCCGCTCATGTCTCAGCACTTCACCTGATTCATCC
		BAD-F	GGATGAATCAGGTGAAGTGCTGAGACATGAGCGGATACATATTTG
BAD-R	AGAATATTGCGCTTCATCATTTTTTATAACCTCCTTAGAG
		ompF-F	CTCTAAGGAGGTTATAAAAA ATGATGAAGCGCAATATTCT
ompF-R	GCACAGATGCTGGTTAACAAAAGCGTTTGCAGTACCTGCTAC
		INS-F	GTAGCAGGTACTGCAAACGCTTTTGTTAACCAGCATCTGTGC
INS-R	ACTGAGCCTTTCGTTTTATTTGTTAATGATGATGATGATGGTG
		T1-F	CACCATCATCATCATCATTAACAAATAAAACGAAAGGCTCAGT
T1-R	GTCTACGGCCCGGCAGTTAAATTTGTCCTACTCAGGAGAGC
		down-F	GCTCTCCTGAGTAGGACAAATTTAACTGCCGGGCCGTAGAC
down-R	GGAGCTGCACATGAACTCGAGCGCACGTGGATTTAACGGAAT

TABLE 3 amplification conditions

2.3 pEcgRNA-N20-INS vector construction

The pEcgRNA-N20 vector was digested with HindIII-HF, xhoI restriction enzymes as follows: pEcgRNA-N20-3. Mu.g, hindIII-HF restriction enzyme 4. Mu.L, xhoI restriction enzyme 4. Mu.L, 10xCutsmart buffer 10. Mu.L, ddH ₂ The amount of O is 100. Mu.L, and the enzyme is cleaved at 37 ℃ for 4h. The enzyme products were recovered from the column according to the instructions of the full-format gold 5-minute DNA rapid purification kit.

up-P _BAD Column recovery of the amplification product of the-ompF-INS-T1-down fragment according to the instructions of the full-format gold 5min DNA Rapid purification kit.

Transformation of DH5 α competence: melting DH5 alpha competent cells on 100 mu L of ice, adding homologous recombination ligation products, gently mixing, standing on ice for 30min, thermally shocking in water bath at 42 ℃ for 50sec, rapidly transferring to ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing and recovering for 30min at 37 ℃ after mixing, coating on a sterile LB + Spec culture medium plate, and standing in a constant temperature incubator at 37 ℃ for overnight culture.

Selecting a monoclonal colony for colony PCR verification, wherein colony PCR primers are BAD-F and BAD-R, and the reaction system is as follows: 1xTaq PCR Master Mix 6.5. Mu.L, BAD-F0.25. Mu.L, BAD-R0.25. Mu.L, monoclonal bacteria (dissolved in 15. Mu.L water) 3. Mu.L. PCR conditions were as follows: 95 ℃ for 7min;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,1min30sec,35cycles;72 ℃ for 7min;10 ℃ and infinity. And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the electrophoresis conditions are as follows: 150V,15min.

The positive clone verified by colony PCR is picked up and put in LB + Spec liquid culture medium of 4.3mL, shaken overnight at the temperature of 37 ℃ and the rpm of 200, the plasmid is extracted on the next day according to the instruction of the plasmid miniprep kit of the department of engine biology, and restriction enzyme digestion verification is carried out by using HindIII-HF and XhoI restriction enzyme. The enzyme digestion system is as follows: plasmid 400ng, xhoI restriction enzyme 0.5. Mu.L, hindIII-HF restriction enzyme 0.5. Mu.L, 10xCutsmart buffer 2. Mu.L, ddH ₂ O complement 20. Mu.L, and the mixture is digested at 37 ℃ for 4h. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, wherein the electrophoresis conditions are as follows: 150V,15min. The correct positive clone is verified by enzyme digestion and sent to the organism of the department of Ongjingkistry for sequencing.

3. Analysis of results

3.1 pEcgRNA-N20 vector construction

Gel electrophoresis results of the enzyme digestion product of the pEcgRNA vector Bsa I are shown in figure 2, and the gel electrophoresis results comprise three bands of 2093bp, 571bp and 441bp, and the fragment with the size of 2093bp is subjected to gel cutting and recovery and is used for connecting a subsequent target point N20.

The PCR gel electrophoresis result of the colony after connection of pEcgRNA-N20T 4 is shown in FIG. 3, the empty load pEcgRNA is used as a control, and the positive clone connected with the target point N20 can amplify a fragment of about 1000 bp. After the positive clone is shaken and plasmids are extracted, the restriction enzyme digestion verification is carried out by using BamHI and HindIII restriction enzymes, the gel electrophoresis result of the restriction enzyme digestion product is shown in figure 4, the positive clone connected with the upper target point N20 can only enzymatically cut fragments of about 2000bp and about 150bp, and the unloaded pEcgRNA can enzymatically cut fragments of about 2000bp, about 1000bp and about 120 bp. And sequencing and verifying the positive clone with correct enzyme digestion to obtain a pEcgRNA-N20 vector with correct sequence.

3.2 obtaining INS expression cassette by overlapping PCR

The result of gel electrophoresis of the amplified fragment is shown in FIG. 5, wherein the up homologous fragment 378bp and the arabinose promoter P _BAD 1329bp, signal peptide ompF66bp, human proinsulin gene (with His label) 279bp, termination sequence T1 87bp, down homologous fragment 378bp, up-P _BAD The 1707bp, ompF-INS-T1 fragment 432bp, INS expression cassette up-P _BAD -ompF-INS-T1-down fragment 2517bp.

3.3 pEcgRNA-N20-INS vector construction

The PCR gel electrophoresis result of the colony after homologous recombination of pEcgRNA-N20-INS is shown in FIG. 6, and the expression frame up-P of INS is connected _BAD Positive cloning of the-ompF-INS-T1-down fragment amplified a fragment of about 1300bp, whereas the blank pEcgRNA-N20 did not. Carrying out enzyme digestion verification by using HindIII and XhoI restriction enzymes after positive cloning and shake bacteria and plasmid extraction, connecting INS expression frame up-P as the gel electrophoresis result of the enzyme digestion product shown in figure 7 _BAD The positive clone of the-ompF-INS-T1-down fragment was enzymatically cleaved to yield two fragments of 2000bp and 2500bp, whereas the blank pEcgRNA-N20 was enzymatically cleaved to yield only a 2000bp fragment. And sequencing and verifying the positive clone with the correct enzyme digestion to obtain a pEcgRNA-N20-INS vector with the correct sequence.

Experiment two: construction of EcN-. DELTA.lpp Strain

1 materials of the experiment

1.2 Experimental reagents: t4 DNA ligase, bamHI-HF restriction endonuclease, hindIII-HF restriction endonuclease, DL2000 DNA Ladder, agarose, all-formula gold DH5 alpha competence, LB broth, spectinomycin hydrochloride pentahydrate, kanamycin sulfate, 2 xTaq PCR Master Mix (Blue), phanta Super-Fidelity DNA Polymerase high Fidelity DNA Polymerase, calcium chloride, glycerol, rhamnose monohydrate, sucrose, and Protillus direct amplification kit.

1.3 Experimental materials: a plasmid mini-extraction kit of the Oncorhynchaceae, a full-expression gold 5-minute DNA rapid purification kit and a sterile culture dish.

2 Experimental methods

2.1 lpp target design and synthesis

Designing a target lpp-N20 according to the lpp gene sequence of the EcN genome and synthesizing: lpp-N20-F (TAGTAATCCTGGGTTCTACCTGC), lpp-N20-R (AAACGCAGAGTAGAACCCAGGATT).

2.2 Lpp upstream and downstream homologous sequence primer design and amplification

And designing and synthesizing upstream and downstream homologous sequence primers according to the lpp gene sequence of the EcN genome, wherein the upstream homologous fragment primers are lpp-up-F and lpp-up-R, and the downstream homologous fragment primers are lpp-down-F and lpp-down-R, and the sequence is shown in Table 4.

The upstream homologous fragment lpp-up PCR amplification is carried out by taking the EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-up-F0.5 uL, lpp-up-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min;10 ℃ and infinity. Downstream homologous fragment lpp-down PCR amplification is carried out by taking the EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-down-F0.5 uL, lpp-down-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min;10 ℃ and infinity.

Performing overlapping PCR amplification by taking the upstream homologous fragment and the downstream homologous fragment as templates to obtain an upstream homologous fragment lpp-up-down, wherein the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, lpp-up 0.5 uL, lpp-down 0.5 uL, lpp-up-F0.5 uL, lpp-down-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,45sec,35cycles;72 ℃ for 10min;10 ℃ and infinity. The amplification product was subjected to agarose gel electrophoresis under 150V,15min. The amplification product was recovered according to the full gold 5min DNA rapid purification kit.

TABLE 4 lpp upstream and downstream homologous fragment amplification primers

Primer name	Primer sequence (5 '-3')
		lpp-up-F	TTGTGCCGCAGCTGGTTAAA
lpp-up-R	GTTGTCCAGACGCTGGTTAGAAGAAGCATCGAACGCTGGG
		lpp-down-F	CCCAGCGTTCGATGCTTCTTCTAACCAGCGTCTGGACAAC
lpp-down-R	CAGCAGGCTTTACGCAATTT

2.3 construction of pEcgRNA-lpp vector

Putting lpp-N20-F10 microliter and lpp-N20-R10 microliter into a PCR instrument for annealing to form double chains, wherein the annealing condition is 95 ℃ and 5min; at 25 ℃ and an infinite value.

The pEcgRNA Bsa I cleavage vector and lpp-N20 target were ligated using T4 DNA ligase as follows: recovered fragment 50-100ng of pEcgRNA Bsa I enzyme digestion vector, 1 uL of 10xT4 DNA ligase buffer, 1 uL of lpp-N20 target spot, 1 uL of T4 DNA ligase and ddH ₂ Adding 10 mu L of O, and reacting at room temperature of 22-25 ℃ for 2-4h.

Selecting a monoclonal colony for colony PCR verification, wherein colony PCR primers are lpp-N20-F and pEcgRNA-con-R, and a reaction system is as follows: 1xTaq PCR Master Mix 6.5. Mu.L, lpp-N20-F0.25. Mu.L, pEcgRNA-con-R0.25. Mu.L, monoclonal bacteria (dissolved in 15. Mu.L water) 3. Mu.L. PCR conditions were as follows: 95 ℃ for 7min;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,1min,35cycles;72 ℃ for 7min; at 10 ℃ and an infinite value. And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the electrophoresis conditions are as follows: 150V,15min.

Positive clones verified by PCR of picked colonies were cloned in 4.3mL LIn B + Spec liquid culture medium, shaking the bacteria overnight at 37 ℃ and 200rpm, extracting the plasmids according to the instruction of the plasmid miniprep kit of the Scirridae organism on the next day, and performing enzyme digestion verification by using BamHI-HF and HindIII-HF restriction enzymes. The enzyme digestion system is as follows: plasmid 400ng, bamHI-HF restriction enzyme 0.5. Mu.L, hindIII-HF restriction enzyme 0.5. Mu.L, 10xCutsmart buffer 2. Mu.L, ddH ₂ O complement 20. Mu.L, and the mixture is digested at 37 ℃ for 4h. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, wherein the electrophoresis conditions are as follows: 150V,15min. The correct positive clone is verified by enzyme digestion and sent to the organism of the department of Ongjingkistry for sequencing.

2.4 Gene knockout and Effect detection

Preparing EcN strain into competence by using calcium chloride and glycerol through a chemical method, converting EcN competence by using pEcas plasmid, coating the EcN competence on a sterile LB + Kana culture medium plate, and standing overnight in a constant-temperature incubator at 37 ℃ for culture to obtain the EcN strain containing the pEcas plasmid. Similarly, ecN strains containing pEcas plasmids were made competent by chemical methods. The pEcgRNA-lpp vector and lpp upstream and downstream homologous fragments together transform the EcN competence containing the pEcas plasmid as follows: competent 200. Mu.L, pEcgRNA-lpp 25. Mu.L (1.5. Mu.g), upstream and downstream homologous fragment lpp-up-down 20. Mu.L (5. Mu.g). Melting EcN competent cells containing pEcas plasmids on 200 mu L of ice, adding pEcgRNA-lpp vectors and upstream and downstream fragments according to the system, lightly mixing the mixture evenly, standing the mixture on ice for 30min, performing water bath heat shock at 42 ℃ for 90s, quickly transferring the mixture to an ice bath, standing the mixture for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing the mixture evenly for 30min at 37 ℃ for resuscitation, completely coating the mixture on LB + Kana + Spec culture medium, standing the mixture at 37 ℃ for overnight culture, and performing CRISPR gene knockout.

Detecting the gene knockout effect through colony PCR, selecting a monoclonal colony on an LB + Kana + Spec culture medium plate to perform colony PCR, taking an EcN genome as a template as a control, and performing the following steps: 1x Taq PCR Master Mix 6.5. Mu.L, lpp-up-F0.25. Mu.L, lpp-down-R0.25. Mu.L, 3. Mu.L of monoclonal bacteria (dissolved in 15. Mu.L water). PCR conditions were as follows: 95 ℃ for 7min;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,1min20sec,35cycles;72 ℃,7min; at 10 ℃ and an infinite value. And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the electrophoresis conditions are as follows: 150V,15min. Positive clones verified by PCR of picked colonies were put in 4.3mL LB + Kana + Spec liquid medium, shaken overnight at 37 ℃ and 200 rpm.

Plasmid elimination was performed as follows: adding 10 mu L of bacterial liquid into 1.5mL of LB + kana + rhamnose (10 mM) liquid culture medium for eliminating pEcgRNA-lpp plasmids, culturing for 6.5h at 37 ℃ and 200rpm in a shaking incubator, sucking 20uL of bacterial liquid, pumping the gun head into 1.5mL of simple LB culture liquid, culturing for 1.5h at 37 ℃ in a shaking incubator, coating the gun head on an LB + sucrose (10 g/L) plate culture medium for eliminating pEcas plasmids, and standing overnight at 37 ℃. 16-24 (2-3 rows) monoclonal colonies are picked from each culture medium, placed in20 mu L of water, 3 mu L of the single colonies are respectively spotted on three plate culture media of LB, LB + Kana and LB + Spec, the temperature of a constant temperature incubator is kept overnight at 37 ℃, and whether two plasmids are eliminated or not is judged by counting the growth conditions on the three culture media.

Taking the monoclonal bacteria with two kinds of plasmids eliminated, and performing strain genome extraction and PCR amplification by using an Optimus sp direct amplification kit, wherein the steps are as follows: taking 100 mu L of overnight culture liquid, centrifuging at 4000rpm for 5min, discarding supernatant, adding 50 mu L of Buffer A, mixing uniformly, reacting at 95 ℃ for 15min, adding 50 mu L of Buffer B, mixing uniformly, centrifuging at 4000rpm for 5min, and obtaining the supernatant as a genome. PCR amplification was performed using the matched T5 enzyme as follows: 15 μ L of mix buffer of T5 enzyme, 2 μ L of genome supernatant, 1.2 μ L of lpp-up-F, 1.2 μ L of lpp-down-R, ddH ₂ O10.6. Mu.L. PCR amplification conditions: 3min at 98 ℃;10 s at 98 ℃; at 64 ℃ for 10s;72 ℃ for 15s;35cycles;72 ℃ for 3min;12 ℃ and infinity. Taking 1 mu L of amplification product for agarose electrophoresis: 150V,15min, and the rest is sent to the engine for sequencing.

3. Analysis of results

3.1 amplification of upstream and downstream homologous sequences

PCR amplification results of the upstream homologous sequence, the downstream homologous sequence and the upstream and downstream homologous sequence are shown in FIGS. 8 and 9, the size of the upstream homologous sequence lpp-up fragment is 343bp, the size of the downstream homologous sequence lpp-down fragment is 280bp, and the size of the upstream homologous sequence lpp-up-down fragment is 623bp.

3.2 construction of pEcgRNA-lpp vector

The PCR gel electrophoresis result of the colony after connection of the pEcgRNA-lpp T4 is shown in FIG. 10, the empty pEcgRNA is used as a control, and a positive clone connected with the target lpp can amplify a fragment of about 1000 bp. After the positive clone is shaken and plasmids are extracted, the restriction enzyme digestion verification is carried out by using BamHI and HindIII restriction enzymes, the gel electrophoresis result of the restriction enzyme digestion product is shown in FIG. 11, the positive clone connected with the upper target point N20 can only enzymatically cut fragments of about 2000bp and about 150bp, and the unloaded pEcgRNA can enzymatically cut fragments of about 2000bp, 1000bp and about 120 bp. And sequencing and verifying the positive clone with correct enzyme digestion to obtain a pEcgRNA-lpp vector with correct sequence.

3.3 Gene knockout and Effect detection

The pEcgRNA-lpp vector and the upstream and downstream homologous fragments lpp-up-down together transform EcN competence containing pEcas plasmids, CRISPR gene knockout is carried out, a single clone colony is picked from an LB + Kana + Spec plate culture medium to carry out colony PCR, the knockout condition of the lpp gene is verified, the gel electrophoresis result of the colony PCR product is shown in figure 12, the positive clone EcN-delta lpp of the successful lpp gene knockout can amplify fragments with the size of about 600bp, and the blank control of the EcN genome amplifies fragments with the size of about 950 bp.

A positive clone with successfully knocked-out lpp gene is selected for plasmid elimination, and clones with both eliminated pEcas and pEcgRNA-lpp can only grow on LB plate culture medium but cannot grow on LB + Kana and LB + Spec plate culture medium, and the plasmid elimination condition is shown in FIG. 13 (only one plate is shown).

The EcN-delta lpp monoclonal bacteria with both plasmids eliminated are shake cultured, the genome is extracted, lpp-up-F and lpp-down-R primers are used for amplifying the lpp gene, and the gel electrophoresis result of the amplified product is shown in figure 14, and the fragment with the size of about 600bp can be amplified. The sequencing result of the amplified product shows that 311bp fragments of the EcN-delta lpp are deleted at the lpp gene, and the EcN-delta lpp strain for knocking out the mural lipoprotein lpp gene is successfully constructed.

Experiment three: constructing EcN-delta lpp-INS strain, inducing expression, extracting protein

1. Experimental Material

1.1, experimental apparatus: the device comprises a Wix electrophoresis apparatus, a PCR (polymerase chain reaction) instrument, a water bath, a centrifugal machine, an Azure300 imager, a shaking incubator, a constant-temperature incubator, an Insses AutoPure protein purification chromatography system and an ultrasonic instrument.

1.2, experimental reagent: DL2000 DNA Ladder, agarose, full-scale gold DH5 alpha competence, LB broth, spectinomycin hydrochloride pentahydrate, kanamycin sulfate, 2 xTaq PCR Master Mix (Blue), calcium chloride, glycerol, rhamnose monohydrate, sucrose, a chromocor direct amplification kit, arabinose, lysozyme, ethanol, sodium phosphate, sodium chloride, imidazole, trichloroacetic acid and loading buffer.

1.3, experimental materials: the kit comprises a platyphenidae biological plasmid minipill kit, a full-scale gold 5-minute DNA rapid purification kit, a sterile culture dish, a GE17524801 HisTrpHigh Performance 5mL chromatographic column, a sand core filtering device 1000mL, a 0.22 mu m filter membrane organic system, a 0.22 mu m filter membrane water system and a 0.45 mu m filter membrane water system.

2. Experimental methods

2.1 construction of EcN-. DELTA.lpp-INS strains

Preparing the EcN-delta lpp strain into competence by a chemical method by using calcium chloride and glycerol, converting the EcN-delta lpp competence by using the pEcas plasmid, coating the competence on a sterile LB + Kana culture medium plate, and standing the culture medium plate in a constant-temperature incubator at 37 ℃ for overnight culture to obtain the EcN-delta lpp strain containing the pEcas plasmid. Similarly, the EcN-. DELTA.lpp strain containing the pEcas plasmid was made competent by a chemical method. EcN-. DELTA.lpp competence of the pEcas-containing plasmid was transformed using the plasmid pEcgRNA-N20-INS containing the human proinsulin expression cassette: taking EcN-delta lpp (containing pEcas plasmid) competence 200 mu L, adding pEcgRNA-N20-INS-2 mu g, lightly mixing evenly, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 90s, quickly transferring to an ice bath, standing for 2min, adding 300uL LB culture medium without antibiotics into a centrifuge tube, standing for 30min for resuscitation at 37 ℃ after mixing evenly, completely coating on LB + Kana + Spec culture medium, standing overnight culture at 37 ℃, and performing CRISPR gene knockout and human proinsulin expression cassette insertion.

Detecting the insertion condition of the human proinsulin expression frame by colony PCR, selecting a monoclonal colony on an LB + Kana + Spec culture medium plate to carry out colony PCR, taking an EcN-delta lpp genome as a template as a control, taking an F primer as a primer in an INS expression frame, taking an R primer as a primer on an EcN genome at the downstream of an insertion position, and adopting the following system: 1xTaq PCR Master Mix 6.5. Mu.L, T1-F0.25. Mu.L, INS-EcN-down-R (GACGGTTTTCGCTGTTGACG) 0.25. Mu.L, monoclonals (dissolved in 15. Mu.L water) 3. Mu.L. PCR conditions were as follows: 7min at 95 ℃;95 ℃ for 30sec;58 ℃ for 30sec;72 ℃,2min,35cycles;72 ℃,7min; at 10 ℃ and an infinite value. And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the electrophoresis conditions are as follows: 150V,15min. Positive clones verified by PCR of picked colonies were put in 4.3mL LB + Kana + Spec liquid medium, shaken overnight at 37 ℃ and 200 rpm.

Plasmid elimination was performed as follows: adding 10 mu L of bacterial liquid into 1.5mL of LB + kana + rhamnose (10 mM) liquid culture medium for eliminating pEcgRNA-N20-INS plasmids, culturing for 6.5h at 37 ℃ and 200rpm in a shaking incubator, sucking 20uL of bacterial liquid, pumping the gun head into 1.5mL of simple LB culture liquid for shaking culture at 37 ℃ for 1.5h, coating the gun head on LB + sucrose (10 g/L) plate culture medium for eliminating pEcas plasmids, and standing overnight at 37 ℃. 16-24 (2-3 rows) monoclonal colonies are picked from each culture medium, placed in20 uL water, 3uL is respectively spotted on three plate culture media of LB, LB + Kana and LB + Spec, a constant temperature incubator is used for overnight at 37 ℃, and whether two plasmids are eliminated or not is judged by counting the growth conditions on the three culture media.

Taking two kinds of monoclone bacteria with eliminated plasmids, and using a prokaryote direct amplification kit to extract the bacterial strain genome and perform PCR amplification, wherein the steps are as follows: taking 100 mu L of overnight culture liquid, centrifuging at 4000rpm for 5min, discarding supernatant, adding 50 mu L of Buffer A, mixing uniformly, reacting at 95 ℃ for 15min, adding 50 mu L of Buffer B, mixing uniformly, centrifuging at 4000rpm for 5min, and obtaining the supernatant as a genome. PCR amplification was performed using the matched T5 enzyme, the system is as follows: 15 μ L of mix buffer of T5 enzyme, 2 μ L of genome supernatant, 1.2 μ L of up-F, 1.2 μ L of down-R, ddH ₂ O10.6. Mu.L. PCR amplification conditions: 3min at 98 ℃;10 s at 98 ℃; at 64 ℃ for 10s;72 ℃ for 30s;35cycles;72 ℃ for 3min;12 ℃ and infinity. 1 μ L of the amplified product was subjected to agarose electrophoresis: 150V,15min, and the rest is sent to the engine for sequencing.

2.2 Induction of expression

Taking the EcN-Deltapp-INS strain to streak culture on an LB plate culture medium, picking a monoclonal colony from the EcN-Deltapp-INS strain in 10mL LB liquid culture medium at 37 ℃ and 200rpm on the next day, shaking the strain overnight on the third day 1:100 into 1000mL LB medium, cultured at 37 ℃To OD ₆₀₀ Adding arabinose as inducer to a final concentration of 10mM, inducing at 37 ℃ and 200rpm for 5 hours, centrifuging at 6000rpm/min, pouring out the supernatant of the centrifugal culture medium, and filtering with a 0.45-micron filter membrane for later use.

2.3 protein extraction

The centrifuged bacteria are re-suspended according to the amount of 1g of bacteria and 10mL of 0.5mg/mL of lysozyme, digested for 1 hour at 4 ℃, centrifuged at 10000rpm/min, and centrifuged supernatant is periplasmic protein which is poured out of a 0.22-micron filter membrane and filtered for later use.

Concentration of His-tagged human proinsulin protein (INS-Histag) was purified using an Ensses AutoPure protein purification chromatography system using a GE17524801HisTraphigh Performance 5mL column, in combination with buffer: 20mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole, pH 7.4, elution buffer: 20mM sodium phosphate, 0.5M sodium chloride, 500mM imidazole, pH 7.4. Elution was performed in 5 column volumes with one step elution. The supernatant protein and periplasmic protein of the induced bacterial liquid were purified by chromatography as above to obtain 25mL of eluents, respectively.

Trichloroacetic acid (TCA) was added to 25mL of the eluate to a final concentration of 10% w/v, the protein was precipitated overnight at 4 ℃ and centrifuged at 11000rpm/min, the supernatant was discarded, and the precipitated protein was dissolved in 50. Mu.L of loading buffer, boiled, cooled on ice and subjected to SDS-PAGE.

After the lysozyme treatment, 1 μ L of the pellet was pipetted into 50 μ L of loading buffer, boiled, and cooled on ice, and used as a protein for inducing the cells for SDS-PAGE electrophoresis.

3. Analysis of results

3.1 Construction of EcN-Deltalpp-INS Strain

pEcgRNA-N20-INS vector transforms EcN-delta lpp competence containing pEcas plasmid, carries out CRISPR gene knockout, picks single clone colony from LB + Kana + Spec plate culture medium to carry out colony PCR, verifies the insertion condition of INS expression frame, the result of colony PCR product gel electrophoresis is shown in figure 15, positive clone EcN-delta lpp-INS inserted successfully by INS expression frame can amplify fragment with size about 1500bp, and the blank control of EcN-delta lpp genome is not existed.

The positive clone with successfully inserted INS expression frame was selected for plasmid elimination, and the clone with both plasmids eliminated pEcas and pEcgRNA-N20-INS could only grow on LB plate culture medium, but could not grow on LB + Kana, LB + Spec plate culture medium, and the plasmid elimination is shown in FIG. 16 (only one plate is shown).

The EcN-delta lpp-INS monoclonal bacteria with both plasmids eliminated are cultured by shaking, the genome of the EcN-delta lpp-INS monoclonal bacteria is extracted, the human proinsulin expression frame is amplified by using up-F and down-R primers, the gel electrophoresis result of the amplified product is shown in figure 17, a fragment with the size of about 2500bp can be amplified, and a blank control of the EcN-delta lpp genome can only amplify a fragment with the size of about 900 bp. Sequencing results of the amplified products show that the human proinsulin expression cassette is successfully inserted into the EcN-Deltapp-INS genome.

Experiment four: western-blot detection

1. Experimental Material

1.1, experimental apparatus: a Wix electrophoresis apparatus, a ThermoScientific Mini Gel Tank electrophoresis Tank, a water bath, a centrifuge, an Azure300 imager and a shaking table.

1.2, experimental reagent: thermoscientific Novex ^TM Tricine SDS loading buffer (2X), thermoscientific NuPAGE Sample Reducing Agent (10X), thermoscientific Novex ^TM Tricine SDS running buffer (10X), TBST, skim milk powder, THE His tag antibody (mAb, mouse), goat Anti-Mouse IgG HRP (secondary antibody), kaiyi biological ECL detection kit, thermoscientific Spectra Multi color Low Range Protein Ladder 26628.

1.3 Experimental materials: 0.22 μm PVDF film, thermoscientific Novex ^TMTM 10-20% tricine 1.0mm small protein pre-gel.

2. Experimental method

2.1 SDS-PAGE

The prepared induced supernatant, induced periplasm, induced thalli and non-induced supernatant, periplasm and thalli protein samples (dissolved in a loading buffer solution) are used for SDS-PAGE electrophoresis, the loading amount is 20 mu L, and the electrophoresis conditions are as follows: 125V,60min.

2.2 transfer of the film

And (2) putting SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) glue into deionized water, washing for 10min, transferring into a pre-cooled membrane transfer liquid, standing for 10min, simultaneously infiltrating filter paper and sponge with the pre-cooled membrane transfer liquid, cutting out PVDF membranes with the same size, putting into methanol, standing for 10min, transferring into the membrane transfer liquid, standing for 10min, placing in the sequence of a blackboard, three layers of filter paper, glue, PVDF membranes, three layers of filter paper and a white board, putting black to black and white to red into an electrophoresis tank, and transferring the membranes at the voltage of 100V for 45 min.

2.3、Western Blot

And (3) after membrane conversion, putting the PVDF membrane in TBST for cleaning, transferring the PVDF membrane into 5% skim milk powder prepared by TBST, and slowly shaking at room temperature for sealing for 3 hours. The 5% skim milk powder was replaced and primary antibody was added at a ratio of 1.

Washing the membrane by TBST: washing 5 times with 10mL each time for 10 min. And (3) replacing new 5% of skim milk powder, adding a secondary antibody according to the proportion of 1. The membranes were washed 5 more times with TBST for the same time and volume as before.

After washing the membrane, developing according to the instruction of a Kaikyi biological ECL detection kit, and taking a picture by an Azure300 imager.

3. Analysis of results

3.1 Western Blot detection

The Western Blot detection results of the EcN- Δ lpp-INS supernatant protein and periplasmic protein are shown in FIG. 18, wherein lane 1 is induced supernatant protein, lane 2 is induced periplasmic protein, lane 3 is induced mycoprotein, lane 4 is non-induced supernatant protein, lane 5 is non-induced periplasmic protein, and lane 6 is non-induced mycoprotein. The non-induced supernatant, periplasm and mycoprotein had no band, the induced supernatant protein had two bands in the range of 10kD to 15kD, the recombinant human proinsulin protein (with 6XHis tag) was expected to be 10.22kD, and the signal peptide ompF was 12.47kD, corresponding to the two bands, and it was presumed that the ompF signal peptide of a part of the recombinant protein was not cleaved or that a part of the cells were cleaved. The weak band of the periplasmic protein at 12.47kD is induced, and the bright band of the mycoprotein at 12.47kD is induced, so that the expression of a large amount of recombinant human proinsulin protein with ompF signal peptide (namely expression of ompF-INS gene) in the bacteria is shown, the ompF signal peptide guides the protein to periplasm space and then secretes the periplasm space into a supernatant culture medium, and the deletion of mural lipoprotein lpp can effectively promote the protein secretion.

Experiment five: mass spectrometric detection

1. Experimental Material

1.2, experimental apparatus: a Wix electrophoresis apparatus, a water bath, a centrifuge, an Azure300 imager and a shaking table.

1.3, experimental reagent: solarbio Protein loading Buffer (containing DTT), kinry Tris-MES-SDS Running Buffer, thermoScientific Spectra Multicolor Low Range Protein Ladder 26628 and Solarbio Coomassie brilliant blue fast staining solution.

1.4, experimental materials: kisry SurePAGE 15% Bio-Tris MES precast gel.

2. Experimental methods

2.1 SDS-PAGE

The prepared protein samples (dissolved in loading buffer) were subjected to SDS-PAGE electrophoresis, with a loading of 20. Mu.L, and electrophoresis conditions: 90V,10min;130V,30min.

2.2, mass Spectrometry detection

Taking out SDS-PAGE gel, dyeing according to the specification of the Solarbio Coomassie brilliant blue fast dyeing solution, cutting out blue bands 1cm x 2cm of the induced supernatant and the induced periplasmic protein lane in a 5-15kD region, and sending to a hundred-funny organism for tandem mass spectrometry.

3. Analysis of results

3.1 Mass spectrometric identification analysis

In order to further determine the protein properties after purification, the protein band in the 5-15kD region was cut off and subjected to mass spectrometry, and the mass spectrometry results show that two peptide fragments consistent with the amino acid sequence of human insulin were identified in both induced supernatant protein and periplasmic protein, namely REAEDLQVGQVELGGGPGAGSLQPLALEGSLQK and EAEDLQVGQVELGGGPGAGSLQPLAGSLQK, respectively, the sequence matching part of the identified peptide fragments is shown in FIG. 19, and the mass spectrometry spectrum of the peptide fragments is shown in FIG. 20. Therefore, the protein induced by the EcN-Deltapp-INS strain to be expressed, separated and purified is recombinant human proinsulin protein.

The expected recombinant human proinsulin amino acid sequence:

MFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCNHHHHHH

4. results and discussion

The invention aims to lay a foundation for the development of live bacteria medicaments for type I diabetes, construct probiotics capable of delivering human proinsulin to intestinal mucosa, and give continuous antigen stimulation, so that the immune tolerance of the type I diabetes is induced, the residual beta cell function is retained, and new onset type I diabetes is improved or even reversed. Therefore, edible probiotic EcN is selected as a chassis bacterium, and a pEcgRNA-lpp vector is constructed by using an Escherichia coli CRISPR/Cas9 dual-plasmid system pEcas/pEcgRNA, so that a murein lpp gene of the EcN is knocked out, and the murein lpp gene is transformed into an EcN-delta lpp strain with periplasmic protein leaking outwards. Then, an insert fragment, namely a human proinsulin expression frame is obtained through biosynthesis and overlapping PCR amplification, and the insert fragment comprises an arabinose promoter, an escherichia coli outer membrane protein ompF signal peptide and a human proinsulin gene; a pEcgRNA-N20-INS vector containing a target N20 and a human proinsulin expression frame is constructed through T4 connection and homologous recombination, the human proinsulin expression frame is inserted into the EcN-Deltapp strain by combining with a pEcas plasmid, and an EcN-Deltapp-INS strain which is escherichia coli probiotics without resistance markers and capable of secreting and expressing human proinsulin is constructed.

Western-blot detection and mass spectrometry prove that human proinsulin is contained in the supernatant of a culture medium induced and expressed by the EcN-delta lpp-INS strain, which fully indicates that the strain can secrete and express human proinsulin to the outside of cells. Meanwhile, the inducer L-arabinose is an edible component and has a positive effect on maintaining the blood sugar stability. The escherichia coli probiotic EcN-Deltpp-INS strain secreting and expressing human proinsulin lays a foundation for development of type I diabetes live bacteria medicaments, subsequent research focuses on further improvement of type I diabetes live bacteria medicaments, a strain secreting and expressing human interleukin 10 is constructed, and the cytokine has the effect of promoting formation of immune tolerance and is stimulated to cooperate with human proinsulin antigen to induce formation of immune tolerance.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A construction method of escherichia coli probiotics for secreting and expressing human proinsulin is characterized by comprising the following steps:

s1, constructing a pEcgRNA-N20 vector;

s2, overlapping PCR to obtain an INS expression frame;

s3, constructing a pEcgRNA-N20-INS vector;

s5, constructing a pEcgRNA-lpp vector;

designing an upstream and downstream homologous sequence primer according to an lpp gene sequence of the EcN genome and synthesizing, wherein the upstream homologous fragment primer is lpp-up-F and lpp-up-R, and the downstream homologous fragment primer is lpp-down-F and lpp-down-R;

s7, gene knockout and effect detection;

preparing the EcN strain into competence by using calcium chloride and glycerol through a chemical method, converting EcN competence by using pEcas plasmids, coating the EcN competence on a sterile LB + Kana culture medium plate, and standing overnight culture in a constant-temperature incubator at 37 ℃ to obtain the EcN strain containing the pEcas plasmids; similarly, preparing the EcN strain containing the pEcas plasmid into competence by a chemical method, converting the pEcgRNA-lpp vector and homologous fragments on the upstream and downstream of lpp into the EcN competence containing the pEcas plasmid, and performing CRISPR gene knockout;

detecting the gene knockout effect by colony PCR;

s8, eliminating plasmids;

eliminating pEcgRNA-lpp plasmid and pEcas plasmid;

s10, constructing EcN-delta lpp-INS strains;

the EcN-. DELTA.lpp strain containing the pEcas plasmid was made competent by chemical methods, and the plasmid pEcgRNA-N20-INS containing the human proinsulin expression cassette was used to transform the EcN-. DELTA.lpp competent strain containing the pEcas plasmid: adding pEcgRNA-N20-INS plasmid about 2 mu g into EcN-delta lpp (containing pEcas plasmid) competence, gently mixing uniformly, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 90s, quickly transferring to an ice bath, standing for 2min, adding an LB culture medium without antibiotics into a centrifuge tube, standing at 37 ℃ for 30min for resuscitation after mixing uniformly, completely coating on the LB culture medium, kana culture medium and Spec culture medium, standing overnight at 37 ℃, and performing CRISPR gene knockout and human proinsulin expression cassette insertion;

s11, detecting the insertion effect of the INS expression frame;

detecting the insertion condition of the human proinsulin expression frame by colony PCR, selecting a monoclonal colony on an LB + Kana + Spec culture medium plate to carry out colony PCR, taking an EcN-delta lpp genome as a template as a control, taking an F primer as a primer in an INS expression frame, taking an R primer as a primer on an EcN genome at the downstream of an insertion position, and adopting the following system: 1x Taq PCR Master Mix 6.5. Mu.L, T1-F0.25. Mu.L, INS-EcN-down-R (GACGGTTTTCGCTGTTGACG) 0.25. Mu.L, monoclonal bacteria (dissolved in 15. Mu.L water) 3. Mu.L;

s12, eliminating plasmids;

eliminating pEcgRNA-N20-INS plasmid and pEcas plasmid;

s13, eliminating the plasmid to obtain the EcN-delta lpp-INS strain.

2. The method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 1, wherein in the step S1, the method comprises the following steps:

s11, synthesizing a target N20 sequence: N20-F (TAGTGTTACTAAATGTAACTAAA), N20-R (AAACTTTTAGTTACATTTAGTAGAAC). Annealing to prepare the N20 target joint: mixing 10 μ L of N20-F with 10 μ L of N20-R, annealing at 95 deg.C for 5min; at 25 ℃ and infinity.

S12, carrying out enzyme digestion on the pEcgRNA vector; the pEcgRNA vector was digested with Bsa I-HF restriction enzyme as follows: pEcgRNA 2-3. Mu.g, bsa I-HF restriction enzyme 4-5. Mu.L, 10xCutsmart buffer 10. Mu.L, ddH ₂ The amount of O is 100. Mu.L, and the enzyme is cleaved at 37 ℃ for 4h.

S13, connecting a pEcgRNA Bsa I enzyme digestion vector and an N20 target joint;the pEcgRNA Bsa I cleavage vector and the N20 target joint were ligated using T4 DNA ligase as follows: pEcgRNA Bsa I restriction enzyme vector recovery fragment 50-100ng, 10xT4 DNA ligase buffer1 uL, N20 target point joint 1 uL, T4 DNA ligase 1 uL, ddH ₂ And supplementing 10 mu L of O, and reacting at room temperature of 22-25 ℃ for 2-4h.

S14, transformation of DH5 alpha competence: melting DH5 alpha competent cells on 100 mu L of ice, adding T4 connection products, gently mixing, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 50sec, quickly transferring to an ice bath, standing for 2min, adding 300 mu L of LB culture medium without antibiotics into a centrifuge tube, standing and recovering at 37 ℃ for 30min after mixing, coating on a sterile LB + Spec culture medium plate, and standing overnight culture in a constant-temperature incubator at 37 ℃.

3. The method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 1, wherein the step S2 comprises:

s21, designing a gene sequence of the INS expression frame,

s22, designing an amplification primer,

s23, amplifying;

PCR amplification to obtain up homologous fragment, arabinose promoter P _BAD Signal peptide ompF, human proinsulin gene (with His label), termination sequence T1 and down homologous fragment, and then obtaining INS expression frame up-P by overlap PCR amplification _BAD -ompF-INS-T1-down；

The amplification system is as follows: 18.5 μ L of KOD FX neo mix buffer, 0.5 μ L (10 μ M) of F primer, 0.5 μ L (10 μ M) of R primer, 0.5 μ L of template;

the amplification conditions were:

P _BAD : primers are BAD-F and BAD-R, a template is pCas plasmid, and PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃ in 1min30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

ompF: the primers are ompF-F and ompF-R, the template is a synthetic gene sequence ompF, and the PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃ in20s, 35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

INS: the primers are INS-F and INS-R, the template is synthetic gene sequence INS, and the PCR condition is as follows: 94 ℃,2min; 20s at 98 ℃;68 ℃ C, 30s,35cycles; 10min at 68 ℃; infinity at 10 ℃;

t1: the primers are T1-F and T1-R, the template is a synthetic gene sequence T1, and PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃ in20s, 35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

down: the primers are down-F and down-R, the template is EcN genome, and PCR conditions are as follows: 94 ℃ for 2min; 20s at 98 ℃;68 ℃,30s,35cycles; at 68 ℃ for 10min; infinity at 10 ℃;

up-P _BAD : primer is up-F, BAD-R, template is up, P _BAD And (3) PCR conditions: 94 ℃,2min; 20s at 98 ℃;68 ℃,2min,35cycles; 10min at 68 ℃; infinity at 10 ℃;

4. The method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 3, wherein the gene sequence of each segment of the INS expression cassette is as follows:

ompF：ATGATGAAGCGCAATATTCTGGCAGTGATCGTCCCTGCTCTGTTAGTAGCAGGTACTGCAAACGCT；

5. the method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 4, wherein the gene sequence of the amplification primer is as follows:

up-F：AGAGTCGACCTGCAGAAGCTTAAAGGAGTAGGGAACACGCA；

up-R：CAAATATGTATCCGCTCATGTCTCAGCACTTCACCTGATTCATCC；

BAD-F：GGATGAATCAGGTGAAGTGCTGAGACATGAGCGGATACATATTTG；

BAD-R：AGAATATTGCGCTTCATCATTTTTTATAACCTCCTTAGAG；

ompF-F：CTCTAAGGAGGTTATAAAAA ATGATGAAGCGCAATATTCT；

ompF-R：GCACAGATGCTGGTTAACAAAAGCGTTTGCAGTACCTGCTAC；

INS-F：GTAGCAGGTACTGCAAACGCTTTTGTTAACCAGCATCTGTGC；

INS-R：ACTGAGCCTTTCGTTTTATTTGTTAATGATGATGATGATGGTG；

T1-F：CACCATCATCATCATCATTAACAAATAAAACGAAAGGCTCAGT；

T1-R：GTCTACGGCCCGGCAGTTAAATTTGTCCTACTCAGGAGAGC；

down-F：GCTCTCCTGAGTAGGACAAATTTAACTGCCGGGCCGTAGAC；

down-R：GGAGCTGCACATGAACTCGAGCGCACGTGGATTTAACGGAAT。

6. the method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 1, wherein the step S3 comprises:

s31, carrying out enzyme digestion on the pEcgRNA-N20 vector;

the pEcgRNA-N20 vector was digested with HindIII-HF, xhoI restriction enzymes as follows: pEcgRNA-N20-3. Mu.g, hindIII-HF restriction enzyme 4. Mu.L, xhoI restriction enzyme 4. Mu.L, 10xCutsmart buffer 10. Mu.L, ddH ₂ Supplementing O to 100 mu L, and carrying out enzyme digestion at 37 ℃ for 4h;

s32, connecting the pEcgRNA-N20 enzyme digestion vector with the up-PBAD-ompF-INS-T1-down fragment;

the homologous recombination Kit of Tiangen easy Assembly Kit is used for connecting the pEcgRNA-N20 enzyme digestion vector and up-P _BAD -ompF-INS-T1-down fragment, reaction system as follows: pEcgRNA-N20 enzyme digestion vector 4. Mu.L, up-P _BAD -ompF-INS-T1-down purified fragment 1. Mu.L, 2x Mix buffer 5. Mu.L, reacted at 50 ℃ for 20min, placed on ice for subsequent transformation;

s33, transformation of DH5 alpha competence:

7. The method for constructing escherichia coli for secretory expression of human proinsulin according to claim 1, wherein in the step S4, the gene sequence of lpp-N20-F is tagtagtaatcctgggttctacctgc, and the gene sequence of lpp-N20-R is aaacgcagtagaacccaggatt.

8. The method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 1, wherein in the step S5, lpp-N20-F10 μ L and lpp-N20-R10 μ L are taken and put into a PCR instrument for annealing to form double chains, wherein the annealing condition is 95 ℃ and 5min; infinity at 25 ℃;

t4 DNA ligase was used to join the pEcgRNA Bsa I cleavage vector and the lpp-N20 target as follows: recovered fragment 50-100ng of pEcgRNA Bsa I enzyme digestion vector, 1 uL of 10xT4 DNA ligase buffer, 1 uL of lpp-N20 target spot, 1 uL of T4 DNA ligase and ddH ₂ Supplementing 10 mu L of O, and reacting at room temperature of 22-25 ℃ for 2-4h;

9. The method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 1, wherein in the step S6, upstream homologous fragment lpp-up PCR amplification is performed by using EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-up-F0.5 uL, lpp-up-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min; infinity at 10 ℃; downstream homologous fragment lpp-down PCR amplification is carried out by taking the EcN genome as a template, and the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, ecN genome template 1 uL, lpp-down-F0.5 uL, lpp-down-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,30sec,35cycles;72 ℃ for 10min; infinity at 10 ℃;

performing overlapping PCR amplification by taking the upstream homologous fragment and the downstream homologous fragment as templates to obtain an upstream homologous fragment lpp-up-down, wherein the system is as follows: phanta Super-Fidelity DNA Polymerase mix buffer 18 uL, lpp-up 0.5 uL, lpp-down 0.5 uL, lpp-up-F0.5 uL, lpp-down-R0.5 uL, PCR amplification conditions of 95 ℃ and 5min; at 95 ℃ for 30s; at 58 ℃ for 30s;72 ℃,45sec,35cycles;72 ℃ for 10min; at 10 ℃ and an infinite value.

10. The method for constructing escherichia coli probiotics for secretory expression of human proinsulin according to claim 1, wherein in the step S7, ecN competent cells containing pEcas plasmids are thawed on 200 μ L of ice, pEcgRNA-lpp vectors and upstream and downstream fragments are added, the mixture is gently mixed, the mixture is stood on ice for 30min, water bath heat shock is performed at 42 ℃ for 90S, the mixture is rapidly transferred to an ice bath for stewing for 2min, 300 μ L of antibiotic-free LB medium is added to a centrifuge tube, the mixture is stood for 30min at 37 ℃ for resuscitation after being blended, all the mixture is coated on LB + Kana + Spec medium, and CRISPR gene knockout is performed after standing overnight culture at 37 ℃.