CN113151126B

CN113151126B - Recombinant leaky strain for high-expression of N-glycosylation anti-VEGFR 2 monomer pseudoantibody, construction method and application thereof

Info

Publication number: CN113151126B
Application number: CN202110196559.6A
Authority: CN
Inventors: 丁宁; 胡学军; 陆龙臻; 晁双英; 孙湘皓; 张伟俊; 林悦; 王倩; 佟思缇
Original assignee: Dalian University
Current assignee: Dalian University
Priority date: 2021-02-10
Filing date: 2021-02-10
Publication date: 2023-08-08
Anticipated expiration: 2041-02-10
Also published as: WO2022170689A1; CN113151126A

Abstract

The invention discloses a recombinant leaky strain for high expression of N-glycosylation anti-VEGFR 2 monomer quasi-antibodies, a construction method and application thereof, and aims to provide a method for efficiently producing N-glycosylation anti-VEGFR 2 monomer quasi-antibody drug proteins and sugar vaccines by using escherichia coli extracellular, belonging to the technical field of biology. The main steps include the expression of N-glycosylation modified anti-VEGFR 2 monomeric-pseudoantibodies in escherichia coli strains knocked out of escherichia coli outer membrane lipoprotein lpp genes by utilizing a pglB-based N-glycosylation mechanism and an automatic induction method. The method can realize the increase of the yield of the N-glycosylation anti-VEGFR 2 monomer pseudo antibody, reduce the metabolic load caused by excessive accumulation of exogenous gene expression products in the periplasm cavity, directly separate and purify the N-glycosylation protein from the culture medium without crushing bacteria, simplify the separation and purification steps, and facilitate large-scale industrialized production.

Description

Recombinant leaky strain for high-expression of N-glycosylation anti-VEGFR 2 monomer pseudoantibody, construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a recombinant leaky strain for high-expression of N-glycosylation anti-VEGFR 2 monomer pseudoantibodies, a preparation method thereof and application of extracellular production of escherichia coli in increasing yield of the N-glycosylation anti-VEGFR 2 monomer pseudoantibodies.

Background

The most common host bacterium for producing recombinant proteins at present is Escherichia coli (Escherichia coli), which has the characteristics of short growth period, low cost, suitability for industrial production and the like. However, native Escherichia coli lacks a protein glycosylation modification mechanism, and is a major cause of limiting its wide application. The use of prokaryotic expression systems typified by escherichia coli for the production of glycoproteins has been increasingly mature in recent decades, marking the advent of the era of customized production of glycoproteins using escherichia coli.

The monomeric pseudo-antibody (Monobody) is an antibody analogue which is obtained by modifying the 10 th repeated sequence region protein (fibronectin type III, FN3 framework protein) of the human fibronectin type III domain by utilizing phage display technology and the like, has the characteristics of weak immunogenicity, strong tissue penetrating power, high affinity and high specificity, and is easy to prepare in large quantities in escherichia coli at low cost. However, because of the small molecular weight (10 kDa), it is easily degraded and has a short serum half-life. The literature shows that the current research generally adopts vectors with stronger promoters, such as pET28 and the like, for intracytoplasmic expression, inclusion bodies are easy to form, renaturation is difficult, protein stability is poor, and the current situations of low protein yield and poor patentability are caused. At present, no Monobody medicine passes the FDA certification in the United states.

The escherichia coli has a cell outer membrane and an inner membrane structure, and can be divided into three regions: intracytoplasmic, pericytoplasmic, extracellular. The target protein is usually designed to be expressed in the periplasmal cavity, and the product is continuously and rapidly accumulated, so that the metabolic burden of the periplasmal cavity is increased, and the stable and continuous expression of the recombinant protein is not facilitated; in addition, pglB-based N-glycosylation modification must be performed in the periplasmic space, and limited space also affects the overall yield of recombinant glycoprotein expression and the efficiency of glycosylation modification.

Construction of the genetically leaky strain is the preferred method of secreting recombinant proteins into the culture medium. The extracellular secretion expression of the escherichia coli recombinant protein provides an effective strategy and has a plurality of advantages, such as reducing the metabolic pressure of thalli, improving the stability of products, simplifying the downstream treatment process and the like. Earlier experiments in this group demonstrated that knockout of lpp in E.coli CLM 37. Delta. Lpp strain can increase recombinant protein production by increasing outer membrane permeability. However, reports of monomeric quasi-antibody glycoprotein drug production by utilizing escherichia coli leaky strains are fresh, and research on glycosylation efficiency and biological activity of extracellular secretion target glycoprotein is not seen.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for improving the yield of N-glycosylation anti-VEGFR 2 monomer pseudo-antibodies by using leaky strains. According to the invention, through the application of the escherichia coli CLM37 delta lpp strain with the cell membrane lipoprotein lpp gene knocked out in the early stage, and the combination of an N-glycosylation mechanism and an automatic induction expression mode derived from campylobacter jejuni (Campylobacter jejuni), a periplasmic cavity expression vector is constructed by adopting a medium-strength promoter, the extracellular production of the N-glycosylation anti-VEGFR 2 monomer pseudoantibody by escherichia coli is realized, the recombinant N-glycosylation protein is secreted into a culture medium with high efficiency, the yield of the anti-VEGFR 2 monomer pseudoantibody can be improved, and the subsequent steps of separating and purifying low molecular weight glycoprotein are simplified.

The first aspect of the present invention provides a recombinant leaky strain that highly expresses an N-glycosylated anti-VEGFR 2 monomer mimetic antibody; the recombinant leaky strain is escherichia coli modified strain CLM37Delpp, and a recombinant vector pIG6-FN3 containing a gene sequence shown as SEQ ID NO.1 is introduced into the recombinant leaky strain _VEGFR2 And the N-glycosylation gene cluster pgl derived from Campylobacter jejuni (Campylobacter jejuni). The recombinant leaky strain can realize stable and efficient secretory production of N-glycosylation modified anti-VEGFR 2 (human vascular endothelial growth factor receptor 2) monomer pseudo-antibodies under the condition of automatic induction. And the activity of the anti-VEGFR 2 monomer pseudoantibody prepared by the recombinant leaky strain is not influenced.

The escherichia coli modified strain CLM37Δlpp refers to an escherichia coli strain with the outer membrane lipoprotein Lpp gene knocked out, namely a CLM37ΔLpp strain in the literature 'escherichia coli CLM37 strain Lpp gene knocked out and extracellular expression N-glycosylation recombinant protein research'.

The recombinant vector pIG6-FN3 containing the gene fragment with the base sequence shown as SEQ ID NO.1 _VEGFR2 Refers to the recombinant vector pIG6-FN3 _VEGFR2 The monoclonal antibody of the anti-human vascular endothelial growth factor VEGFR2 is expressed in the medium and is provided with glycosylation modification sites, and an N-glycosylation modification gene cluster of campylobacter jejuni based on pglB is introduced. The introduced N-glycosylation recombination protein mechanism improves the yield of the N-glycosylation anti-VEGFR 2 monomer quasi-antibody.

In a second aspect, the present invention provides a recombinant leaky strain (CLM37Δlpp/pIG6-FN 3) capable of expressing a glycoprotein as described above _VEGFR2 -Gly), comprising the steps of:

(1) Recombinant vector pIG6-FN3 containing gene fragment with base sequence shown as SEQ ID NO.1 _VEGFR2 Is constructed by the steps of: introducing M1 tag and histidine purification tag and coding different numbers of glycosylation recognition site base sequences into 5 'end, 3' end and flexible chain region of anti-human VEGFR2 monomer pseudoantibody gene by PCR method, cloning to downstream of pIG6 expression vector OmpA signal peptide, and the vector is named pIG6-FN3 _VEGFR2 The method comprises the steps of carrying out a first treatment on the surface of the The sequence of the recombinant protein gene fragment is shown as SEQ ID NO. 1.

(2) Preparation of recombinant leaky strain CLM37Δlpp, pIG6-FN3 _VEGFR2 Transformation of Gly plasmid into CLM37Δlpp cell leaky strain CLM37Δlpp/pIG6-FN3 capable of high expression of recombinant protein _VEGFR2 -Gly; the recombinant leaky strain is further transferred into pACYCpgl plasmid carrying N-glycosylation gene cluster, and then is automatically induced, and can be used for producing glycosylated FN3 _VEGFR2 -Gly。

In the above-described technical scheme, further preferably, the step of preparing the recombinant leaky strain clm37Δlpp in step (2) includes: (1) firstly, amplifying a homologous arm and kan gene fragment containing lpp genes by PCR, recovering and then performing heat shock conversion to obtain a plasmid pKD46 escherichia coli CLM37/pKD46 containing inducible expression Red recombinase, and then, coating bacterial liquid on a flat plate containing L-arabinose, kanamycin sulfate and ampicillin for enrichment culture; (2) for removing the plasmid pKD46, selecting positive monoclonal, culturing at 36-43 ℃, streaking and culturing on LB plate medium containing ampicillin, and screening out a resistant bacterium CLM37.DELTA.lpp: kan strain without plasmid; (3) to eliminate kan resistance gene, plasmid pCP20 heat shock transformant strain CLM37Δlpp: kan was selected for enrichment culture, the bacterial liquid was spread on a LB plate medium without resistance, cultured overnight, and then a monoclonal strain which did not grow on both antibiotic plates was selected by LB plate culture containing ampicillin and kanamycin, respectively, and designated as CLM37Δlpp. In a third aspect, the invention provides the use of a recombinant leaky strain as described above for increasing the yield of N-glycosylated anti-VEGFR 2 monomeric mimetibodies.

In the above technical solution, it is further preferred that the application refers to the use of the recombinant leaky strain described above to induce extracellular production of anti-VEGFR 2 monomeric mimetibodies in an automatic induction medium; collecting supernatant obtained by culture, centrifuging, collecting precipitate, and purifying to obtain the N-glycosylation anti-VEGFR 2 monomer pseudoantibody.

In the above technical solution, it is further preferred that the automatic induction medium used in the application is: each liter of culture medium contains 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of glycerol, 0.4-0.6 g of glucose, 1-3 g of lactose, 6-8 g of disodium hydrogen phosphate, 6-8 g of monopotassium phosphate, 3-4 g of ammonium sulfate, 0.5-1.2 g of sodium sulfate and 0.2-0.3 g of magnesium sulfate heptahydrate; more preferably: each liter of the culture medium contains 10g of tryptone, 5g of yeast extract, 5g of glycerol, 0.5g of glucose, 2.5g of lactose, 7.1g of disodium hydrogen phosphate, 6.8g of monopotassium phosphate, 3.3g of ammonium sulfate, 0.9g of sodium sulfate and 0.5g of magnesium sulfate heptahydrate.

In the above technical solution, further preferably, the step of inducing culture in the application includes: after picking up the monoclonal, inoculating the monoclonal to LB liquid medium containing ampicillin and chloramphenicol, and culturing overnight; inoculating the strain into an automatic induction culture medium containing ampicillin and chloramphenicol, and performing induction expression for 36 hours; more preferably: after picking up the monoclonal, inoculating the monoclonal to LB liquid medium containing ampicillin (80-120 mu g/mL) and chloramphenicol (35-40 mu g/mL), and culturing overnight for 12-18 hours; and then the ratio of the components is 1:80-120 to an OD, and inoculating the culture medium to LB liquid medium containing ampicillin (80-120. Mu.g/mL) and chloramphenicol (35-40. Mu.g/mL) ₆₀₀ Reaching 0.5 to 0.8; then, the cells are inoculated into an automatic induction medium containing ampicillin (80-120. Mu.g/mL) and chloramphenicol (35-40. Mu.g/mL) at a ratio of 1:80-120 to induce expression. Preferably, the conditions for the induction of expression are 28 to 35℃and 200rpm.

In the above technical solution, further preferably, the culture conditions of the recombinant leaky strain in application are: the strain clm37Δlpp co-transformed with the pIG6-rFn3-Gly and pACYCpgl plasmids was inoculated into fresh auto-induction medium containing ampicillin and chloramphenicol, and cultured continuously for enrichment.

In the above technical solution, further preferably, the centrifugation conditions in the application are: collecting bacterial liquid induced for at least 40 hours, centrifuging at 8000-14000 r/min and 18-22 min, and more preferably: collecting induced bacterial liquid at 8000-12000 r/min, and centrifuging for 8-12 min.

In the above technical solution, further preferably, the conditions for purification in the application are: (1) adding imidazole with the final concentration of 18-22 mM into the supernatant obtained after the centrifugation; (2) filtering out impurities with a 0.22 mu M filter membrane, adding 280-320 mM sodium chloride and 0.8-1.2 wt% Tween-20, and preserving in an ice bath; (3) after the His-Trap nickel column is arranged, 400-600 mM imidazole is eluted, and residual proteins are washed away; (4) passing the mixture through a column by using Binding buffer (18-22 mM imidazole+1-2 wt% Tween-20), and cleaning at the flow rate of 0.8-1.2 mL/min. (5) Passing the supernatant through a column with the flow rate of 0.8-1.2 mL/min; (6) after passing through the column, removing the impurity protein by using 18-22 mM imidazole at 1.5-2.5 mL/min; (7) the nickel column is taken down and is arranged on a clean injector, gradient elution is carried out according to the range of 40mM to 240mM, protein elution is carried out by imidazole with the concentration of 400 mM to 600mM, and the eluent is collected.

The beneficial effects of the invention are as follows: the method for producing the N-glycosylated recombinant protein by using the escherichia coli extracellular by using the gene knockout escherichia coli can reduce the intracellular degradation of the target proteinIncreasing the expression level of recombinant mimetibodies, i.e., glycosylated FN3 _VEGFR2 -average expression level of Gly reaches 70±3.4mg/L; the glycosylation efficiency of the anti-VEGFR 2 monomer quasi-antibody prepared by the recombinant leaky strain can reach 100%, and the activity is not influenced; simultaneously reducing the intracellular excessive accumulation of the recombinant protein and reducing the formation of inclusion bodies; eliminating cell wall breaking process, reducing pyrogen pollution, reducing separation and purification cost, and being beneficial to mass production of N-glycosylated pharmaceutical protein or sugar vaccine.

Drawings

FIG. 1 shows a diagram of cell membrane integrity assay (left: CLM 37. DELTA.lpp; right: CLM 37). Figures a, D: clm37Δlpp and CLM37 were stained with Calcein-AM; graphs B, E: clm37Δlpp and CLM37 were PI stained; drawing C, F: clm37Δlpp and CLM37 were stained with Calcein-AM and PI simultaneously.

FIG. 2 results of ELISA assays for clM37 and clm37Δlpp cells secreting expressed recombinant proteins.

FIG. 3 Western blot detection results of CLM37Δlpp secretory expression of recombinant protein FN 3-Gly.

FIG. 4CLM37Δlpp expression of monomeric mimetic antibody FN3 _VEGFR2 Gly detection results: (A) The Western blot detection method is similar to that described in FIG. 3, wherein lane 1 is the FN3VEGFR2-Gly protein modified by incomplete glycosylation as a negative control, and lane 2 is the FN3VEGFR2-Gly protein modified by complete glycosylation as a positive control. (B) SDS-PAGE detection: lane 1 and 4, uninduced CLM37Δlpp/pIG6-FN3 _VEGFR2 -Gly+pACYC pg and CLM37/pIG6-FN3 _VEGFR2 -culture supernatant of gly+pacyc pgl strain; lane 2 and 3: clm37Δlpp/pIG6-FN3 collected after induction _VEGFR2 -gly+pacyc pgl bacterial supernatant and pellet; lane 5: FN3 purified from supernatant _VEGFR2 -Gly。

FIG. 5N-glycosylated protein FN3 _VEGFR2 Western blot detection of Gly serum stability.

FIG. 6N-glycosylated protein FN3 _VEGFR2 Immunofluorescence assay of Gly biological Activity.

IgG isotype control; D-F glycosylation FN3 _VEGFR 2-Gly; G-I non-glycosylated FN3 _VEGFR2 -Gly。

Detailed Description

The present invention will be further described with reference to the drawings and the detailed description, but the scope of the invention is not affected. The sources of the biomaterials used in the examples below were as follows:

coli clm37Δlpp with lpp gene knocked out: constructed by this laboratory, reference "Escherichia coli CLM37 strain Lpp gene knockout and extracellular expression N-glycosylation recombinant protein research, ruan Yao, wang Lifan, guo Longhua, etc. -Shanghai university of transportation (medical edition) 2018.11:1332-1337" is described in detail.

CLM37 without lpp gene knocked out: the collection of cell genes from the Biovector NTCC plasmid vector strain is described in detail in "Proceedings of the National Academy of Sciences of the United States of America (ISSN: 0027-8424), 102 (8): 3016-3021".

The periplasmic cavity expression plasmid pIG6-rFn3-Gly which is preserved in the laboratory and carries the model protein FN3-Gly: constructed by this laboratory, literature "Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-introduction. Ding N, yang C, sun S, etc., biochem Biophys Res Commun 2017 03 25;4851 "is described in detail.

The pACYCpgl plasmid carrying the campylobacter jejuni N-glycosylation gene cluster: the literature is given away by the labs of the Federal regulatory academy of Zurich, ref "N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. Coll. Wacker M, linton D, hitchen PG, etc., science 2002Nov 29;2985599 (5599) "have detailed description.

Plasmid pCP20: the Shanghai Yubo biotechnology Co., ltd., product number YB1693;

plasmid pKD46: shanghai Yubo Biotechnology Co., ltd., product number YB1692.

Example 1

anti-VEGFR 2 monomer pseudoantibody N-glycosylation recognition site design and expression vector construction

Confirmation of anti-VEGFR 2 monomer mimetibodies FN3 _VEGFR2 Amino acid sequence, by changing AB Loop, EF Loop, CD Loop region and C terminal, inserting glycosyl of oligosaccharase in corresponding partThe amino acid sequence of the recognition site is optimized and analyzed by reasonable protein MODELs according to SWISS-MODEL, pyMOL and other software, the gene sequence of the most stable and reasonable recombinant protein is selected, the optimal sequence is optimized according to the codon preference of escherichia coli, then an M1 tag and a histidine purification tag are respectively introduced into the 5 'end, the 3' end and the flexible chain region of the gene by a PCR method, the nucleotide sequences of the glycosylation recognition sites with different numbers are encoded, the nucleotide sequences are cloned to the downstream of the OmpA signal peptide of the pIG6 expression vector, and the gene sequence of the recombinant protein with optimal expression is selected after induction, wherein the gene sequence is shown as SEQ ID NO. 1.

The preparation of recombinant leaky strains: (1) firstly, amplifying a homologous arm and kan gene fragment containing lpp genes by PCR, recovering and then performing heat shock conversion to obtain a plasmid pKD46 escherichia coli CLM37/pKD46 containing inducible expression Red recombinase, and then, coating bacterial liquid on a flat plate containing L-arabinose, kanamycin sulfate and ampicillin for enrichment culture; (2) for removing the plasmid pKD46, selecting positive monoclonal, culturing at 36-43 ℃, streaking and culturing on LB plate medium containing ampicillin, and screening out a resistant bacterium CLM37.DELTA.lpp: kan strain without plasmid; (3) to eliminate kan resistance gene, plasmid pCP20 heat shock transformant strain CLM37Δlpp: kan was selected for enrichment culture, the bacterial liquid was spread on a LB plate medium without resistance, cultured overnight, and then a monoclonal strain which did not grow on both antibiotic plates was selected by LB plate culture containing ampicillin and kanamycin, respectively, and designated as CLM37Δlpp.

Example 2 Effect verification experiment

Effect experiment one, checking the integrity of cell membranes

In this effect experiment, escherichia coli CLM 37. DELTA. Lpp with the lpp gene knocked out and CLM37 without the lpp gene knocked out were cultured for 12 hours, diluted 100-fold in fresh automatic induction medium, and then cultured at 200rpm for about 4 hours, OD ₆₀₀ The value was about 0.8 and the cells were centrifuged at 3500rpm for 5min. Rinsed with sterile PBS buffer and resuspended to 1X 10 ⁶ -1×10 ⁷ cells/mL. Then, 1000. Mu.L of the suspension was mixed with 1000 Xstaining solution containing 10mM Calcein-AM and 5mM PI (propidium iodide) (1. Mu.L of each was mixed). After incubation at room temperature for 30min in the absence of light, stained cells were cleared with PBSAfter washing 2 times, centrifugation was performed. The stained cells were then observed under a microscope by dilution to 100 μl with sterile PBS buffer. Fluorescence emission spectra were measured with 490/545nm excitation and 515/617nm emission, as measured with a 400 Xmagnification fluorescence microscope. The results show that graphs A, D, CLM 37. DELTA.lpp and CLM37 stained with Calcein-AM, respectively, both appear green; panels B, E are clm37Δlpp and CLM37 stained with PI, respectively, only panel B appears red; panels C, F are superimposed on clm37Δlpp and CLM37 stained with Calcein-AM and PI, panel F appears green, and only bacteria with permeable membranes will be superimposed on yellow by Calcein-AM and PI staining simultaneously (figure 1C). The results indicate that the knockout of the lpp gene disrupts the integrity of the bacterial envelope.

Effect experiment II, inducing Escherichia coli to express N-glycosylated recombinant protein

The effect experiments were studied using the periplasmic cavity expression plasmid pIG6-rFn3-Gly carrying the model protein FN3-Gly stored in the laboratory and the pACYCpgl plasmid carrying the Campylobacter jejuni N-glycosylation gene cluster co-transformed into CLM37 Deltatpp. One colony was selected and cultured in 3mL of LB (medium containing 34. Mu.g/mL of chloramphenicol and 100. Mu.g/mL of ampicillin) overnight at 37 ℃. The culture was used to inoculate 1/100 (v/v) of LB broth into 100mL and shake cultured (220 rpm) to OD at 37 ℃ ₆₀₀ 0.8 was re-inoculated with 100mL of ZYP-5052 auto-induction medium containing the same antibiotic concentration. Culturing at 28deg.C for 48 hr continuously, and measuring bacterial liquid OD every 12 hr ₆₀₀ The bacterial liquid was collected at 2OD and centrifuged, and the bacterial pellet was separated from the supernatant, which was extracellular component (diluted to 1.0mL with PBS) and was the bacterial fraction. Buffer [0.2M Tris-HCl (pH=8.2), 0.25mM EDTA,0.25M sucrose, lysozyme 160. Mu.g/mL was prepared]. Cells were resuspended in 1.0mL of the above buffer and then 1M MgSO was added ₄ 60. Mu.L of solution and cells were incubated on ice for 25min. Centrifuging at 12000rpm, collecting supernatant as periplasmoid component, precipitating as intracellular component, suspending the precipitate with 2% SDS solution, standing for 5min, centrifuging at 12000rpm, and collecting supernatant as intracellular component (diluted to 1.0mL with PBS).

Collecting the induced supernatant and thalli, detecting glycosylation efficiency by ELISA and Western blot respectively, collecting the supernatant, and separating and purifying recombinant protein in the supernatant by using an affinity chromatographic column, wherein the protein comprises N-glycosyl modified recombinant glycoprotein. The system can obtain near 100% N-glycosylation monomer quasi-antibody with high expression level and convenient separation and purification. The system can be used for preparing a large number of N-glycosylation recombination quasi-antibody drug proteins.

Effect experiment III and ELISA method for detecting expression of N-glycosylated recombinant protein in Escherichia coli components

To examine the case where clm37Δlpp expressed N-glycosylated recombinant protein, western blot analysis was performed using CLM37, in which lpp gene was not knocked out, as a control. 3 wells were individually coated with each group of components at different time points for 12h (4 ℃ C., overnight, blank Kong Jiabiao standard and sample diluent). The plates were washed 5 times with 200. Mu.L PBS, each time for 5min, blocked with 100. Mu.L 3% BSA for 2h, washed 5 times, incubated with 100. Mu.L His-HRP primary antibody for 2h, washed 5 times, developed for 20min with 100. Mu.L HRP kit, and terminated with 100. Mu.L 2M concentrated sulfuric acid, and data analysis was performed after detection with an ELISA reader at 450 nM.

The results show that the escherichia coli clm37Δlpp expresses a small amount of intracellular protein of the recombinant, indicating that the knockout of lpp gene does not affect the secretory transport of ompA signal peptide to recombinant protein; the amount of escherichia coli clm37Δlpp expressed recombinant protein in the supernatant was much higher than CLM37 (fig. 2A), indicating that clm37Δlpp expressed recombinant protein leaked out of the cell in large amounts; and after 36 hours, the expression level of clm37Δlpp in the periplasmic space was lower than that of escherichia coli CLM37 (fig. 2B), indicating that the peak value of the escherichia coli clm37Δlpp expression protein was about 36 hours.

Effect experiment four, detection of N-glycoprotein glycosylation efficiency in E.coli Components

To examine the N-glycosylation efficiency of the expressed glycoprotein secreted by CLM37Δlpp, western blot analysis was performed on the collected supernatant, periplasmic space and intracellular components using anti-M1-FLAG antibody, respectively, using E.coli CLM37 as a control strain. The recombinant protein is secreted into the periplasm cavity, and then the signal peptide is removed under the action of signal peptidase, so that DYKD label is exposed, and can be specifically identified by anti-M1-FLAG antibody. The recombinant protein FN3 has a theoretical molecular weight of about 12800 and an N-glycosylation modified molecular weight of about 14200, so that whether the recombinant protein FN3 is glycosylated or not can be judged according to the migration speed, and the N-glycosylation efficiency can be further judged according to the relative density value of the immunoblotting band.

As shown in FIG. 3, by auto-induction for 36h, a small amount of recombinant protein was detected in the supernatant due to partial lysis of strain CLM37, but most was also present in the periplasmal cavity. In contrast, the lpp-knocked-out strain clm37Δlpp was strong in membrane leakage, and most of the recombinant protein FN3-Gly produced was present in the supernatant. However, the glycosylation efficiency of the recombinant protein produced by the two strains can reach 100%, which is consistent with the previous experimental result.

Effect experiment five, escherichia coli secretion expression N-glycosylation anti-VEGFR 2 monomer quasi-antibody

To produce glycosylated FN3 _VEGFR2 Gly, pACYCpgl and pIG6-FN3 _VEGFR2 Gly plasmid was transformed into CLM37 and CLM37Δlpp cells and the automatic induction method was the same as in effect experiment two of this example. To produce non-glycosylated FN3 _VEGFR2 For pIG6-FN3 containing _VEGFR2 Coli clm37Δlpp of the Gly and pACYC184 vectors were subjected to the above procedure. Cells were obtained by centrifugation at 8000rpm for 10min at 4 ℃. The components were then prepared according to the procedure described in effect experiment two. After centrifugation, the harvested supernatant was filtered through a 0.22. Mu.M filter, and then PBS containing imidazole was added to adjust the final concentration to 20mM imidazole, 300mM NaCl and 3% Tween-20. The protein was then loaded onto a 1mL-Histrap column. 10 column volumes were washed with 20mM imidazole in PBS followed by elution with 5mL of PBS-buffer containing 50mM sodium phosphate (pH 7.4), 300mM NaCl and 100mM imidazole. Western blot detects the expression levels of glycosylated and non-glycosylated proteins and analyzes the density value data of at least three independent experiments by image pro plus 6.0 software.

The results show that glycosylated FN3 _VEGFR2 The band of Gly is consistent with the expected molecular weight. Then, the bacterial cells and glycosylated FN3 in the culture medium are quantitatively calculated according to the Western blot band density _VEGFR2 Average Gly expression level up to 70+ -3.4 mg/L. Taken together with the above experimental results, it was shown that single deletion of lpp gene was effective for achieving leaky expression of glycoprotein, and that a large amount of N-sugar could be obtained from the auto-induction mediumGlycosylated monomeric mimetic antibody FN3 _VEGFR2 -Gly。

Effect experiment six, N-glycosylation anti-VEGFR 2 monomer quasi-antibody serum stability detection

Purifying desalted non-glycosylated protein FN3 _VEGFR2 -Gly and N-glycosylation modified FN3 _VEGFR2 The OD values of the Gly proteins were measured at 280nm and then the concentration of both proteins was estimated using the concentration coefficient calculated by Vector NTI 11.5.1 software. Diluting the serum to 0.1mol/L by using precooled PBS, uniformly mixing the serum with 0.1mol/L diluted protein 1:1 respectively, standing at 37 ℃, and respectively sampling at 0h,6h,18h,24h,36h,48h,60h and 72h for Western blot detection and density value analysis.

Statistical finding of unglycosylated FN3 _VEGFR2 Gly starts to degrade significantly after 18h (almost disappears at 36 h), whereas glycosylated FN3 _VEGFR2 Gly remains stable within 24h and is obviously degraded within 36-48 h, indicating glycosylation of FN3 _VEGFR2 Gly vs. unglycosylated FN3 _VEGFR2 -Gly is more stable. * Represents a significant difference (.p) from the control protein<0.01, all with glycosylated FN3 _VEGFR2 -Gly comparison).

Seventh effect experiment and cell immunofluorescence method for detecting N-glycosylation FN3 _VEGFR2 -Gly biological Activity

Taking 6-well cell culture plate, placing into an ethanol-treated cover glass, inoculating umbilical vein endothelial cells (HUVEC), and placing into a culture medium containing 5% CO ₂ Is cultured in a constant temperature incubator. When the cell confluence rate reaches 70-80%, taking out the cell climbing sheet, washing with PBS for 3 times, and sucking residual liquid. 1mL of PBS solution containing 3.7% formaldehyde was added thereto, and the mixture was allowed to stand at room temperature for 15 minutes. The residue was removed by washing with PBS 2 times (5 min/time). 1mL of PBS solution with Triton X-100 content of 0.1% is added, and the mixture is stood still and punched for 10min at room temperature. The residue was removed by washing with PBS 2 times (5 min/time). A blocking solution of 0.8% BSA was added thereto, and the mixture was allowed to stand at room temperature for 30 minutes. The blocking solution was removed by pipetting, and a protein dilution prepared by adding 0.8% BSA was incubated overnight at 4℃in a refrigerator. The next day, the residue was removed by washing 3 times with PBS (5 min/time). His primary antibody prepared by 0.8% BSA is added, and incubated for 1h at room temperature; the mixture was washed 3 times (5 min/time) with PBS and the residue was taken up. Adding 0.8% BSA to prepare a fluorescent secondary antibody (the secondary antibody is FITC standard)Goat anti-rabbit IgG) conjugate, and incubated at 4 ℃ for 4 hours in a refrigerator. After the completion, the mixture was washed 3 times (5 min/time) with PBS, and the residual liquid was sucked off. DAPI staining was added and incubated for 12min at room temperature. The residue was then removed by washing 3 times (5 min/time) with PBS. Clean slides were taken, 1 drop of 50% glycerol was added dropwise, the coverslip cells were facing down, covered on the slides and fixed. And (5) performing inverted fluorescence microscopy.

The results showed that both N-glycosylation-modified and non-glycosylation-modified anti-VEGFR 2 monomeric mimetibodies were specifically stained (FIGS. 6B, C), demonstrating that VEGFR2 can bind to the HUVEC cell surface with little difference, indicating that N-glycosylation modification was specific for protein FN3 _VEGFR2 Gly biological activity has little effect. From the specific effect experiment, the method can obtain a large amount of N-glycosylated recombinant proteins rapidly and efficiently, and lays a foundation for further related N-glycosylated pharmaceutical proteins and N-glycosylated vaccines.

The embodiments of the present invention are not limited to the preferred embodiments of the present invention, and other embodiments of the present invention are possible according to the general knowledge and general methods of the art from the above description of the present invention without departing from the basic technical ideas of the present invention. Any person skilled in the art, within the scope of the present disclosure, may make various other modifications, substitutions or alterations according to the technical scheme of the present invention and the inventive concept thereof, and such that the recombinant protein of the N-glycosylation type may be produced extracellularly by other escherichia coli using other escherichia coli periplasmic cavity expression vectors and the like, which fall within the scope of the present disclosure.

Sequence listing

<110> university of Dalian

<120> recombinant leaky strain for high expression of N-glycosylation anti-VEGFR 2 monomer pseudoantibody, construction method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 354

<212> DNA

<213> artificial sequence

<400> 1

gatatcgaag ttgtggcagc aacaccgaca agcctgctga taagctggag acatccgcat 60

tttccgacac ggtattatag aataacatat ggagaaacag gcggaaatag cccggtacag 120

gaatttaccg ttccgctcca gcctccaacc gcaaccattt caggcctgaa acctggtgtc 180

gattatacca tcaccggtta cgcagttaca gatggtcgta atggccggct tctgtctata 240

ccgattagta ttaattaccg taccgaaatt gacaaaccca gtcaaggcgg tgggggtagt 300

gatcaaaacg caacaggcgg tggtggcagc ctcgagcacc accaccacca ccac 354

Claims

1. A recombinant leaky strain highly expressing an N-glycosylated anti-VEGFR 2 monomer mimetic antibody, characterized in that: the recombinant leaky strain is escherichia coli modified strain CLM37Delpp, and a recombinant vector pIG6-FN3 containing a gene sequence shown as SEQ ID NO.1 is introduced into the recombinant leaky strain _VEGFR2 And campylobacter jejuni (campylobacter jejuni) derived N-glycosylation gene cluster pgl.

2. The recombinant leaky strain highly expressing an N-glycosylated anti-VEGFR 2 monomer mimetic antibody according to claim 1, wherein: the recombinant vector pIG6-FN3 containing the gene fragment with the base sequence shown as SEQ ID NO.1 _VEGFR2 Refers to the recombinant vector pIG6-FN3 _VEGFR2 The anti-VEGFR 2 monomer pseudoantibody is expressed in the medium and provided with glycosylation modification sites, and a plasmid carrying a pglB-based N-glycosylation modification gene cluster of campylobacter jejuni is introduced.

3. The method for constructing recombinant leaky strains highly expressing N-glycosylated anti-VEGFR 2 monomer mimetic antibody as claimed in claim 1, comprising the steps of:

(1) Recombinant vector pIG6-FN3 containing gene fragment with base sequence shown as SEQ ID NO.1 _VEGFR2 Is constructed by the steps of: introducing M1 tag into 5 'end, 3' end and flexible chain region of anti-VEGFR 2 monomer pseudoantibody gene by PCR methodAnd histidine purification tag and nucleotide sequences encoding different numbers of glycosylation recognition sites, cloned downstream of OmpA signal peptide of pIG6 expression vector designated pIG6-FN3 _VEGFR2 ；

(2) Preparation of recombinant leaky strain CLM37Δlpp, pIG6-FN3 _VEGFR2 Transforming Gly plasmid into CLM37 delta lpp cell to obtain recombinant leaky strain capable of highly expressing anti-VEGFR 2 monomer pseudoantibody

CLM37Δlpp/pIG6-FN3 _VEGFR2 -Gly; the recombinant leaky strain is further transferred into pACYCpgl plasmid carrying N-glycosylation gene cluster, and then is automatically induced for producing glycosylated FN3 _VEGFR2 -Gly。

4. The use of the recombinant leaky strain of claim 1 for increasing the yield of N-glycosylated anti-VEGFR 2 monomeric mimetibodies.

5. The use according to claim 4, characterized in that: inducing extracellular production of anti-VEGFR 2 monomeric mimetibodies in an auto-induction medium using the recombinant leaky strain of claim 1; collecting supernatant obtained by culture, centrifuging, collecting precipitate, and purifying to obtain the N-glycosylation anti-VEGFR 2 monomer pseudoantibody.

6. The use according to claim 5, characterized in that: the automatic induction culture medium is as follows: each liter of culture medium contains 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of glycerol, 0.4-0.6 g of glucose, 1-3 g of lactose, 6-8 g of disodium hydrogen phosphate, 6-8 g of monopotassium phosphate, 3-4 g of ammonium sulfate, 0.5-1.2 g of sodium sulfate and 0.2-0.3 g of magnesium sulfate heptahydrate.

7. The use according to claim 5, characterized in that: the induction culture step comprises the following steps: inoculating the recombinant leaky strain of claim 1 to LB liquid medium containing 80-120 mug/mL ampicillin and 35-40 mug/mL chloramphenicol, and culturing overnight for 12-18 hours; inoculating to LB liquid containing 80-120 mug/mL ampicillin and 35-40 mug/mL chloramphenicol in a volume ratio of 1:80-120In bulk medium until OD ₆₀₀ Reaching 0.5 to 0.8; then, the strain is inoculated into an automatic induction culture medium containing 80-120 mu g/mL of ampicillin and 35-40 mu g/mL of chloramphenicol at a ratio of 1:80-120 for induction of expression.

8. The use according to claim 5, characterized in that: the culture conditions of the recombinant leaky strain are as follows: the strain clm37Δlpp co-transformed with the pIG6-rFn3-Gly and pACYCpgl plasmids was inoculated into fresh auto-induction medium containing ampicillin and chloramphenicol, and cultured continuously for enrichment.

9. The use according to claim 5, characterized in that: the centrifugation conditions are as follows: collecting bacterial liquid induced for at least 40 hours, centrifuging at 8000-14000 r/min and 18-22 min.

10. The use according to claim 5, characterized in that: the conditions of the purification are as follows: (1) adding imidazole with the final concentration of 18-22 mM into the supernatant obtained after the centrifugation; (2) filtering out impurities with a 0.22 mu M filter membrane, adding 280-320 mM sodium chloride and 0.8-1.2 wt% Tween-20, and preserving in an ice bath; (3) after the His-Trap nickel column is arranged, 400-600 mM imidazole is eluted, and residual proteins are washed away; (4) passing through a Binding buffer consisting of 18-22 mM imidazole and 1-2 wt% Tween-20, and cleaning at a flow rate of 0.8-1.2 mL/min; (5) passing the supernatant through a column with the flow rate of 0.8-1.2 mL/min; (6) after passing through the column, removing the impurity protein by using 18-22 mM imidazole at 1.5-2.5 mL/min; (7) the nickel column is taken down and is arranged on a clean injector, gradient elution is carried out according to the range of 40mM to 240mM, protein elution is carried out by imidazole with the concentration of 400 mM to 600mM, and the eluent is collected.