WO2022170689A1 - 一种高表达n-糖基化抗vegfr2单体拟抗体的重组渗漏菌株、其构建方法及应用 - Google Patents

一种高表达n-糖基化抗vegfr2单体拟抗体的重组渗漏菌株、其构建方法及应用 Download PDF

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WO2022170689A1
WO2022170689A1 PCT/CN2021/095166 CN2021095166W WO2022170689A1 WO 2022170689 A1 WO2022170689 A1 WO 2022170689A1 CN 2021095166 W CN2021095166 W CN 2021095166W WO 2022170689 A1 WO2022170689 A1 WO 2022170689A1
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vegfr2
recombinant
strain
glycosylated
leaky
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丁宁
胡学军
陆龙臻
晁双英
孙湘皓
林悦
张伟俊
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大连大学
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
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Definitions

  • the invention belongs to the field of biotechnology, and relates to a recombinant leaky strain highly expressing N-glycosylated anti-VEGFR2 monomeric mimetic antibodies and a preparation method thereof.
  • the extracellular production of Escherichia coli can increase N-glycosylated anti-VEGFR2 Application of Monomeric Mimic Antibody Yield.
  • Escherichia coli the most common host bacteria for recombinant protein production is Escherichia coli, which has the characteristics of short growth cycle, low cost and suitable for industrial production.
  • Escherichia coli the lack of protein glycosylation modification mechanism in natural Escherichia coli is the main reason to limit its wide application.
  • prokaryotic expression systems represented by Escherichia coli to produce glycoproteins has become increasingly mature, marking the arrival of the era of customized glycoprotein production using Escherichia coli.
  • Monobody is an antibody analog developed by using phage display technology to transform the 10th repeat region protein (fibronectin type III, FN3 backbone protein) of human fibronectin type III domain. It has weak properties, strong tissue penetration, high affinity and high specificity, and is easy to be prepared in Escherichia coli at low cost, quickly and in large quantities. However, due to its small molecular weight (10kDa), it is easily degraded and has a short serum half-life.
  • the literature shows that the current research usually uses a vector with a strong promoter such as pET28 for intracytoplasmic expression, which is easy to form inclusion bodies, difficult to renature, and poor protein stability, resulting in low protein yield and poor druggability. At present, no Monobody drugs have been certified by the US FDA in clinical practice.
  • Escherichia coli has outer and inner membrane structures, which can be divided into three regions: intracytoplasmic, periplasmic cavity, and extracellular.
  • the target protein is designed to be expressed in the periplasmic cavity, and the products accumulate continuously and rapidly, which increases the metabolic burden of the periplasmic cavity and is not conducive to the stable and continuous expression of the recombinant protein; in addition, the N-glycosylation modification based on pglB must be in the periplasmic cavity.
  • the limited space also affects the total yield of recombinant glycoprotein expression and the efficiency of glycosylation modification.
  • Construction of genetically leaky strains is the preferred method for secreting recombinant proteins into the culture medium.
  • the extracellular secretory expression of recombinant proteins from Escherichia coli provides an effective strategy with many advantages, such as alleviating bacterial metabolic stress, improving product stability, and simplifying downstream processing processes.
  • the previous experiments of our group confirmed that knocking out lpp in Escherichia coli CLM37 ⁇ lpp strain can increase the production of recombinant protein by increasing the permeability of the outer membrane.
  • leaky strains of Escherichia coli for the production of monomeric antibody glycoprotein drugs, and there are no studies on the glycosylation efficiency and biological activity of exocytotic target glycoproteins.
  • the present invention provides a method for increasing the yield of N-glycosylated anti-VEGFR2 monomer mimetic antibody by using leaky strains.
  • the present invention adopts the Escherichia coli CLM37 ⁇ lpp strain constructed in the early stage to knock out the membrane lipoprotein lpp gene, combined with the N-glycosylation mechanism and automatic induction expression method derived from Campylobacter jejuni, and adopts a medium-strength promoter.
  • the periplasmic cavity expression vector was constructed to realize the extracellular production of N-glycosylated anti-VEGFR2 monomimetic antibodies by Escherichia coli. Yield, while simplifying the subsequent separation and purification of low molecular weight glycoprotein steps.
  • the first aspect of the present invention provides a recombinant leaky strain that highly expresses N-glycosylated anti-VEGFR2 monomeric mimetic antibodies;
  • the recombinant leaky strain is a modified strain of Escherichia coli CLM37 ⁇ lpp introduced with a base sequence such as SEQ
  • the recombinant leaky strain can stably and efficiently secrete and produce N-glycosylation modified anti-VEGFR2 (human vascular endothelial growth factor receptor 2) monomer mimetic antibody under auto-induction conditions. And the activity of the anti-VEGFR2 monomer mimetic antibody prepared by the recombinant leaky strain has no effect.
  • N-glycosylation modified anti-VEGFR2 human vascular endothelial growth factor receptor 2
  • the Escherichia coli modified strain CLM37 ⁇ lpp refers to the Escherichia coli strain with the outer membrane lipoprotein lpp gene knocked out, namely the document "Escherichia coli CLM37 strain lpp gene knockout and extracellular expression N-glycosylation recombination" CLM37 ⁇ Lpp strain in "Protein Research”.
  • Described recombinant vector pIG6-FN3 VEGFR2 containing the gene fragment shown in nucleotide sequence as SEQ ID NO.1 refers to the expression of anti-human vascular endothelial growth factor VEGFR2 monomer mimetic antibody in the recombinant vector pIG6-FN3 VEGFR2 with Glycosylation modification site and introduction of pglB-based N-glycosylation modification gene cluster in Campylobacter jejuni.
  • the introduction of the N-glycosylated recombinant protein mechanism increases the yield of N-glycosylated anti-VEGFR2 monomeric mimetic antibodies.
  • the second aspect of the present invention provides a method for constructing the above-mentioned recombinant leaky strain (CLM37 ⁇ lpp/pIG6-FN3 VEGFR2 -Gly) that can highly express glycoproteins, the steps of which include:
  • the step of preparing the recombinant leaky strain CLM37 ⁇ lpp in step (2) includes: 1. PCR amplification containing the lpp gene homology arm and the kan gene fragment, after recovery, the heat shock is transformed into After containing the plasmid pKD46 Escherichia coli CLM37/pKD46 that can induce the expression of Red recombinase, the bacterial solution was coated on a plate containing L-arabinose, kanamycin sulfate and ampicillin for enrichment culture; 2 To remove the plasmid pKD46 , pick positive single clones and culture at 36-43 °C, streak culture on LB plate medium containing ampicillin, and screen out plasmid-free resistant bacteria CLM37 ⁇ lpp:kan strain; 3 In order to eliminate the kan resistance gene, the Plasmid pCP20 heat shock transformed strain CLM37 ⁇ lpp:kan, pick transformants for enrichment culture, take the bacterial solution
  • the third aspect of the present invention provides the application of the above-mentioned recombinant leaky strain in improving the production of N-glycosylated anti-VEGFR2 monomeric mimetic antibodies.
  • the application refers to the use of the recombinant leaky strain described above to induce extracellular production of anti-VEGFR2 monomeric mimetic antibodies in an automatic induction medium; The supernatant was centrifuged and the precipitate was collected, and purified to obtain an N-glycosylated anti-VEGFR2 monomeric mimetic antibody.
  • each liter of medium contains 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of glycerol, Glucose 0.4 ⁇ 0.6g, lactose 1 ⁇ 3g, disodium hydrogen phosphate 6 ⁇ 8g, potassium dihydrogen phosphate 6 ⁇ 8g, ammonium sulfate 3 ⁇ 4g, sodium sulfate 0.5 ⁇ 1.2g, magnesium sulfate heptahydrate 0.2 ⁇ 0.3g; more
  • each liter of medium contains tryptone 10g, yeast extract 5g, glycerol 5g, glucose 0.5g, lactose 2.5g, disodium hydrogen phosphate 7.1g, potassium dihydrogen phosphate 6.8g, ammonium sulfate 3.3g, sodium sulfate 0.9g, magnesium sulfate heptahydrate 0.5g.
  • the induction culture step in the application includes: picking a single clone, inoculating it into an LB liquid medium containing ampicillin and chloramphenicol, and culturing it overnight; Inoculated into an automatic induction medium containing ampicillin and chloramphenicol, and induced expression for 36 hours; more preferably: pick a single clone and inoculate it into a medium containing ampicillin (80-120 ⁇ g/mL) and chloramphenicol (35 ⁇ g/mL) ⁇ 40 ⁇ g/mL) LB liquid medium, cultured overnight for 12 ⁇ 18 hours; then inoculated with ampicillin (80 ⁇ 120 ⁇ g/mL) and chloramphenicol (35 ⁇ 40 ⁇ g/mL at a volume ratio of 1:80 ⁇ 120) ) in LB liquid medium until the OD 600 reaches 0.5-0.8; then, inoculate it into the LB liquid medium containing ampicillin (80-120 ⁇ g/mL
  • the culture condition of the recombinant leaky strain in the application is: inoculate the strain CLM37 ⁇ lpp co-transformed with the pIG6-rFn3-Gly and pACYCpgl plasmids in the cells containing ampicillin, chloramphenicol Supplemented by continuous culture in fresh auto-induction medium.
  • the conditions for centrifugation in the application are: collecting the bacterial liquid induced for at least 40 hours at 8000-14000 r/min, and centrifuging for 18-22 min, more preferably: collecting the induced bacteria The bacterial liquid was centrifuged at 8000 ⁇ 12000r/min for 8 ⁇ 12min.
  • the purification conditions in the application are: 1 Add imidazole with a final concentration of 18-22 mM to the supernatant obtained after the above centrifugation; 2 0.22 ⁇ M filter membrane suction filtration Remove impurities, add Tween-20 with a final concentration of 280-320 mM sodium chloride and 0.8-1.2 wt%, and store in an ice bath; 3 After the His-Trap nickel column is installed, elute with 400-600 mM imidazole to remove residual protein ; 4 Use Binding buffer (18 ⁇ 22mM imidazole+1 ⁇ 2wt% Tween-20) to pass through the column, and wash at a flow rate of 0.8 ⁇ 1.2mL/min.
  • the beneficial effects of the present invention are as follows: using a modified strain of Escherichia coli CLM37 ⁇ lpp to secrete the N-glycosylation monomer mimetic antibody into the extracellular medium, the present invention applies the gene knockout Escherichia coli extracellular
  • the method for producing N-glycosylated recombinant protein can reduce the intracellular degradation of the target protein and increase the expression level of the recombinant mimetic antibody, that is, the average expression level of glycosylated FN3 VEGFR2 -Gly reaches 70 ⁇ 3.4 mg/L;
  • the glycosylation efficiency of the anti-VEGFR2 monomer mimetic antibody prepared by the recombinant leaky strain can reach 100%, and its activity has no effect; at the same time, it reduces the excessive accumulation of recombinant protein in the cell and reduces the formation of inclusion bodies; eliminates the cell wall breaking process, The pyrogen pollution is reduced, the cost of separation and purification is reduced, and it is ultimately
  • FIG. 1 Cell membrane integrity detection diagram (left: CLM37 ⁇ lpp; right: CLM37). Panels A, D: CLM37 ⁇ lpp and CLM37 were stained by Calcein-AM; panels B, E: CLM37 ⁇ lpp and CLM37 were stained by PI; panels C, F: CLM37 ⁇ lpp and CLM37 were simultaneously stained by Calcein-AM and PI.
  • FIG. 1 ELISA detection results of recombinant proteins secreted and expressed by CLM37 and CLM37 ⁇ lpp cells.
  • FIG. 4 CLM37 ⁇ lpp expressing monomeric mimetic antibody FN3 VEGFR2- Gly detection results: Western blot detection: Lane 1 and 2 are CLM37 ⁇ lpp/pIG6-FN3VEGFR2-Gly+pACYC pgl cells before/after purification of supernatant collected after IPTG induction for 48h; Lane 3 Uninduced CLM37 ⁇ lpp/pIG6-FN3VEGFR2-Gly+pACYC pgl supernatant; Lane 4 and 5 are CLM37 ⁇ lpp/pIG6-FN3VEGFR2-Gly+pACYC pgl cells, respectively, before/after purification of the supernatant collected after 48 h of auto-induction; arrows indicate Shown as FN3VEGFR2-Gly protein.
  • Figure 5 Western blot analysis of the stability of N-glycosylated protein FN3 VEGFR2 -Gly serum.
  • Fig. 6 The results of immunofluorescence detection of the antigen-binding activity of sialic acid-modified N-glycosylated protein FN3 VEGFR2 -Sia.
  • A IgG isotype control
  • B sialic acid-modified glycosylated FN3 VEGFR2 -Sia
  • C unglycosylated FN3 VEGFR2 -Gly.
  • Escherichia coli CLM37 ⁇ lpp knocking out the lpp gene constructed by our laboratory, reference "Study on Lpp gene knockout and extracellular expression of N-glycosylated recombinant protein in Escherichia coli CLM37 strain, Ruan Yao, Wang Lifan, Guo Longhua, etc. - "Journal of Shanghai Jiaotong University” (Medical Edition) 2018.11:1332-1337” has a detailed description.
  • CLM37 without lpp gene knockout from BioVector NTCC plasmid vector strain cell gene collection center, in "Proceedings of the National Academy of Sciences of the United States of America (ISSN: 0027-8424), 102(8):3016 -3021" is described in detail.
  • the periplasmic cavity expression plasmid pIG6-rFn3-Gly carrying the model protein FN3-Gly preserved in our laboratory constructed by our laboratory, the document "Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction. Ding N, Yang C, Sun S, etc., Biochem Biophys Res Commun 2017 03 25; 4851" is described in detail.
  • pACYCpgl plasmid carrying the N-glycosylation gene cluster of Campylobacter jejuni a gift from the laboratory of ETH Zurich, reference "N-linked glycosylation in Campylobacter jejuni and its functional transfer into E.coli.Wacker M,Linton D,Hitchen PG,etc.,Science 2002Nov 29;2985599(5599)" has a detailed description.
  • Plasmid pCP20 Shanghai Yubo Biotechnology Co., Ltd., Cat. No. YB1693;
  • Plasmid pKD46 Shanghai Yubo Biotechnology Co., Ltd., product number YB1692.
  • the M1 tag and histidine purification tag were introduced into the end and flexible chain region, and the base sequences encoding different numbers of glycosylation recognition sites were cloned into the downstream of the pIG6 expression vector OmpA signal peptide, and the recombinant protein with the best expression was selected after induction.
  • the gene sequence is shown in SEQ ID NO.1.
  • the steps of preparing the recombinant leaky strain 1 First, PCR amplification containing the homology arm of lpp gene and kan gene fragment, recovery, heat shock transformation to plasmid pKD46 Escherichia coli CLM37/pKD46 containing inducible expression of Red recombinase, The bacterial solution was spread on a plate containing L-arabinose, kanamycin sulfate, and ampicillin for enrichment culture; 2 In order to remove the plasmid pKD46, pick the positive single clone and cultivate it at 36-43 °C, and put it on the LB plate containing ampicillin.
  • CLM37 ⁇ lpp:kan strain 3.
  • the plasmid pCP20 was heat-shocked into the strain CLM37 ⁇ lpp:kan, and the transformants were picked for enrichment and culture, and the bacteria were collected.
  • the solution was spread on the non-resistant LB plate medium and cultivated overnight, and then the monoclonal strains that did not grow on the two antibiotic plates were screened by LB plate culture containing ampicillin and kanamycin respectively, and named CLM37 ⁇ lpp .
  • Escherichia coli CLM37 ⁇ lpp with knockout of lpp gene and CLM37 without knockout of lpp gene were cultured for 12h, diluted 100 times in fresh auto-induction medium, cultured at 200rpm for about 4h, and the OD 600 value was about 0.8.
  • Cells were centrifuged at 3500 rpm for 5 min. Rinse with sterile PBS buffer and resuspend to 1 ⁇ 10 6 -1 ⁇ 10 7 cells/mL. Then, 1000 ⁇ L of the suspension was mixed with 1000 ⁇ staining solution containing 10 mM Calcein-AM and 5 mM PI (propidium iodide) (1 ⁇ L of each was mixed).
  • the periplasmic cavity expression plasmid pIG6-rFn3-Gly carrying the model protein FN3-Gly and the pACYCpgl plasmid carrying the N-glycosylation gene cluster of Campylobacter jejuni were transformed into CLM37 ⁇ lpp; or constructed with sialic acid Plasmid p15 for synthesis of sugar chain (Neu5Ac- ⁇ -2,6-Gal- ⁇ -1,4-GlcNAc- ⁇ -1,3-Gal- ⁇ -1,3-GlcNAc-) and N-glycosylation modification pathway -Ara-lsg (carries E.
  • Centrifuge at 12,000 rpm collect the supernatant as the periplasmic cavity component, and collect the supernatant as the intracellular component. Suspend the precipitate with 2% SDS solution, place for 5 min, centrifuge at 12,000 rpm, and collect the supernatant as the intracellular component (diluted to 1.0 mL with PBS).
  • the system can obtain nearly 100% N-glycosylated monomer mimetic antibodies with high expression and easy separation and purification.
  • the system can be used for the preparation of a large number of N-glycosylated recombinant antibody drug proteins.
  • the harvested supernatant was filtered through a 0.22 [mu]M filter and then added with imidazole in PBS to adjust final concentrations of 20 mM imidazole, 300 mM NaCl and 3% Tween-20.
  • the protein was then loaded onto a 1 mL-Histrap column. 10 column volumes were washed with 20 mM imidazole in PBS, followed by elution with 5 mL of PBS-buffer containing 50 mM sodium phosphate (pH 7.4), 300 mM NaCl and 100 mM imidazole.
  • the expression levels of glycosylated and unglycosylated proteins were detected by Western blot, and the density data of at least three independent experiments were analyzed by image pro plus 6.0 software.
  • the OD value of the purified and desalted unglycosylated protein FN3 VEGFR2 -Gly and N-glycosylated modified FN3 VEGFR2 -Gly protein was measured at 280 nm, and then the concentration coefficient calculated by Vector NTI 11.5.1 software was used to estimate the two. protein concentration. Dilute to 0.1 mol/L with pre-cooled PBS, mix the serum with 0.1 mol/L diluted protein 1:1, let stand at 37°C for 0h, 6h, 18h, 24h, 36h, 48h, 60h, 72h After sampling, Western blot detection and density value analysis were performed.
  • Fluorescent secondary antibody (the secondary antibody is FITC-labeled goat anti-rabbit IgG) binding solution prepared with 0.8% BSA was added, and incubated in a refrigerator at 4°C for 4 hours. After the end, wash 3 times with PBS (5min/time), and remove the residual liquid. DAPI was added for staining and incubated at room temperature for 12 min. Then wash with PBS 3 times (5min/time), and suck off the residual liquid. Take a clean glass slide, add 1 drop of 50% glycerol dropwise, cover glass with cell face down, cover it on the glass slide and fix it. Inverted fluorescence microscope examination.
  • the embodiments of the present invention are not limited to this, but are only preferred specific embodiments of the present invention. According to the above-mentioned content of the present invention, according to common technical knowledge and general methods in the field, and without departing from the above-mentioned basic technical ideas of the present invention, the present invention The invention may also have other embodiments. Any person skilled in the art who is familiar with the technical field of the present invention can make equivalent replacements or changes according to the technical solution of the present invention and its inventive concept within the technical scope disclosed by the present invention, and the present invention can also make other modifications, replacements or changes in various forms.
  • Escherichia coli periplasmic cavity expression vectors and other similar methods can be used to produce N-glycosylated recombinant proteins extracellularly from Escherichia coli, which all fall within the protection scope of the present invention.

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Abstract

提供了一种高表达N-糖基化抗VEGFR2单体拟抗体的重组渗漏菌株、其构建方法及应用，可利用大肠埃希菌胞外高效生产N-糖基化抗VEGFR2单体拟抗体药物蛋白及糖疫苗。主要步骤包括在敲除大肠埃希菌外膜脂蛋白lpp基因的大肠埃希菌菌株中利用基于pglB的N-糖基化机制及自动诱导方法表达N-糖基化修饰的抗VEGFR2单体拟抗体。该方法可实现N-糖基化抗VEGFR2单体拟抗体产量的增加，降低了外源基因表达产物在质周腔过度积累所造成的代谢负荷，无需破碎细菌而从培养基中直接分离纯化N-糖基化蛋白，简化了分离纯化步骤。

Description

一种高表达N-糖基化抗VEGFR2单体拟抗体的重组渗漏菌株、其构建方法及应用 技术领域
本发明属于生物技术领域，涉及一种高表达N-糖基化抗VEGFR2单体拟抗体的重组渗漏菌株、其制备方法，利用大肠埃希菌胞外生产可增加N-糖基化抗VEGFR2单体拟抗体产量的应用。
背景技术
目前生产重组蛋白的最常见宿主菌是大肠埃希菌(Escherichia coli)，它具有生长周期短、成本低和适合工业化生产等特点。但天然的大肠埃希菌缺乏蛋白质糖基化修饰机制，是限制其广泛应用的主要原因。近十年来应用以大肠埃希菌为代表的原核表达系统来生产糖蛋白日益成熟，标志着利用大肠埃希菌定制生产糖蛋白时代的到来。
单体拟抗体(Monobody)是利用噬菌体展示技术等对人纤维连接蛋白III型结构域的第10重复序列区蛋白(fibronectin type III，FN3骨架蛋白)改造发展而来的抗体类似物，具有免疫原性弱，组织穿透力强，具有高亲合力和高特异性，并且易于在大肠埃希菌中低成本、快速、大量制备的特点。然而因为分子量较小(10kDa)，易降解，血清半衰期短。文献显示目前研究通常采用带有较强启动子的载体如pET28等进行胞质内表达，容易形成包涵体，复性困难，蛋白稳定性差，造成蛋白产量低，成药性差的现状。目前临床还没有Monobody类药物通过美国FDA认证。
大肠埃希菌具有细胞外膜和内膜结构，可以将其分为三个区域：胞质内、质周腔、细胞外。通常设计目标蛋白在质周腔表达，产物持续、快速累积，增加了质周腔的代谢负担，不利于重组蛋白的稳定持续表达；另外基于pglB的N-糖基化修饰必须是在质周腔中完成，有限的空间也影响了重组糖蛋白表达的总产量及糖基化修饰的效率。
构建遗传渗漏菌株是将重组蛋白分泌到培养基中的首选方法。大肠埃希菌重组蛋白的胞外分泌表达提供了一种有效的策略，具有许多优点，如减轻菌体代谢压力，提高产物稳定性，简化下游处理工艺等。本课题组前期实验证实大肠埃希菌CLM37Δlpp菌株中敲除lpp可以通过增加外膜的通透性来提高重组蛋白的产量。但目前鲜见利用大肠埃希菌渗漏菌株进行单体拟抗体类糖蛋白药物生产的报导，也未见针对胞外分泌目标糖蛋白的糖基化效率及生物活性的研究。
发明内容
针对现有技术中的不足之处，本发明提供了一种应用渗漏菌株提高N-糖基化抗VEGFR2单体拟抗体产量的方法。本发明通过应用前期构建的敲除胞膜脂蛋白lpp基因的大 肠埃希菌CLM37Δlpp菌株，结合来源于空肠弯曲杆菌(Campylobacter jejuni)N-糖基化机制和自动诱导表达方式，采用中等强度启动子构建质周腔表达载体，实现大肠埃希菌胞外生产N-糖基化抗VEGFR2单体拟抗体，重组N-糖基化蛋白高效分泌到培养基中，可提高抗VEGFR2单体拟抗体的产量，同时简化了后续分离纯化低分子量糖蛋白步骤。
本发明第一方面提供了一种高表达N-糖基化抗VEGFR2单体拟抗体的重组渗漏菌株；所述重组渗漏菌株为大肠埃希菌改造菌株CLM37Δlpp中引入含有碱基序列如SEQ ID NO.1所示基因序列的重组载体pIG6-FN3 VEGFR2及空肠弯曲杆菌(Campylobacter jejuni)来源的N-糖基化基因簇pgl。该重组渗漏菌株在自动诱导条件下能够实现稳定、高效地分泌生产N-糖基化修饰的抗VEGFR2(人血管内皮生长因子受体2)单体拟抗体。且该重组渗漏菌株制备所得的抗VEGFR2单体拟抗体的活性没有影响。
所述的大肠埃希菌改造菌株CLM37Δlpp是指外膜脂蛋白lpp基因被敲除的大肠埃希菌株，即文献“大肠埃希菌CLM37菌株lpp基因敲除及胞外表达N-糖基化重组蛋白研究”中的CLM37ΔLpp菌株。
所述的含有碱基序列如SEQ ID NO.1所示基因片段的重组载体pIG6-FN3 VEGFR2是指在重组载体pIG6-FN3 VEGFR2中表达抗人血管内皮生长因子VEGFR2单体拟抗体、并带有糖基化修饰位点，且引入空肠弯曲杆菌基于pglB的N-糖基化修饰基因簇。引入的N-糖基化重组蛋白机制，使N-糖基化抗VEGFR2单体拟抗体的产量提升。
本发明第二方面提供了上文所述的可高表达糖蛋白的重组渗漏菌株(CLM37Δlpp/pIG6-FN3 VEGFR2-Gly)的构建方法，其步骤包括：
(1)含有碱基序列如SEQ ID NO.1所示基因片段的重组载体pIG6-FN3 VEGFR2的构建步骤：通过PCR方法在抗人VEGFR2单体拟抗体基因5′端、3′端以及柔链区引入M1标签和组氨酸纯化标签及编码不同数量的糖基化识别位点碱基序列，并克隆到pIG6表达载体OmpA信号肽下游，载体命名为pIG6-FN3 VEGFR2；重组蛋白基因片段的序列如SEQ ID NO.1所示。
(2)制备重组渗漏菌株CLM37Δlpp，将pIG6-FN3 VEGFR2-Gly质粒转化到CLM37Δlpp细胞中可高表达重组蛋白的渗漏菌株CLM37Δlpp/pIG6-FN3 VEGFR2-Gly；该重组渗漏菌株进一步转入携带N-糖基化基因簇的pACYCpgl质粒，然后进行自动诱导，可以用于生产糖基化的FN3 VEGFR2-Gly。
上文所述的技术方案中，进一步优选地，步骤(2)中制备重组渗漏菌株CLM37Δlpp的步骤包括：①首先PCR扩增含有lpp基因同源臂和kan基因片段，回收后热激转化至 含有能诱导表达Red重组酶的质粒pKD46大肠埃希菌CLM37/pKD46后，取菌液涂布于含L-阿拉伯糖、硫酸卡那霉素、氨苄青霉素的平板增菌培养；②为去除质粒pKD46，挑取阳性单克隆于36～43℃培养，于含氨苄青霉素的LB平板培养基上划线培养，筛选出不含质粒的抗性菌CLM37Δlpp:kan菌株；③为消除kan抗性基因，将质粒pCP20热激转化菌株CLM37Δlpp:kan，挑取转化子增菌培养，取菌液涂布于无抗性的LB平板培养基上，培养过夜，然后分别用含氨苄青霉素和卡那霉素的LB平板培养筛选出在两种抗生素平板都不生长的单克隆菌株，命名为CLM37Δlpp。本发明第三方面提供了上文所述的重组渗漏菌株在提高N-糖基化抗VEGFR2单体拟抗体产量方面的应用。
上文所述的技术方案中，进一步优选地，所述的应用是指利用前文所述的重组渗漏菌株在自动诱导培养基中诱导胞外生产抗VEGFR2单体拟抗体；收集培养所得的上清液，离心并收集沉淀，纯化后获得N-糖基化抗VEGFR2单体拟抗体。
上文所述的技术方案中，进一步优选地，所述的应用中使用的自动诱导培养基为：每升培养基中含有胰蛋白胨8～12g，酵母提取物4～6g，甘油4～6g，葡萄糖0.4～0.6g，乳糖1～3g，磷酸氢二钠6～8g，磷酸二氢钾6～8g，硫酸铵3～4g，硫酸钠0.5～1.2g，七水硫酸镁0.2～0.3g；更优选为：每升培养基中含有胰蛋白胨10g，酵母提取物5g，甘油5g，葡萄糖0.5g，乳糖2.5g，磷酸氢二钠7.1g，磷酸二氢钾6.8g，硫酸铵3.3g，硫酸钠0.9g，七水硫酸镁0.5g。
上文所述的技术方案中，进一步优选地，所述的应用中的诱导培养步骤包括：挑取单克隆后将其接种到含有氨苄青霉素和氯霉素的LB液体培养基，过夜培养；再接种到含有氨苄青霉素和氯霉素的自动诱导培养基中，诱导表达36小时；更优选为：挑取单克隆后将其接种到含有氨苄青霉素(80～120μg/mL)和氯霉素(35～40μg/mL)的LB液体培养基，过夜12～18小时培养；再以1:80～120的体积比接种到含有氨苄青霉素(80～120μg/mL)和氯霉素(35～40μg/mL)的LB液体培养基中，直至OD 600达到0.5～0.8；然后，以1:80～120接种到含有氨苄青霉素(80～120μg/mL)和氯霉素(35～40μg/mL)自动诱导培养基中诱导表达。优选的，所述诱导表达条件为28～35℃、200rpm。
上文所述的技术方案中，进一步优选地，所述的应用中重组渗漏菌株的培养条件为：将共转化有pIG6-rFn3-Gly和pACYCpgl质粒的菌株CLM37Δlpp接种于含有氨苄青霉素、氯霉素的新鲜自动诱导培养基中，连续培养增菌。
上文所述的技术方案中，进一步优选地，所述的应用中离心的条件为：收取诱导至少40小时的菌液于8000～14000r/min，18～22min离心，更优选为：收取诱导的菌液于8000～12000r/min，8～12min离心。
上文所述的技术方案中，进一步优选地，所述的应用中纯化的条件为：①上文离心后获得的上清液加入终浓度为18～22mM的咪唑；②0.22μM滤膜抽滤除杂质，并加入终浓度为280～320mM氯化钠及0.8～1.2wt％的Tween-20，冰浴保存；③装好His-Trap镍柱后，400～600mM咪唑洗脱，洗去残留蛋白；④用Binding buffer(18～22mM咪唑+1～2wt％的Tween-20)过柱，流速0.8～1.2mL/min清洗。⑤将上清液进行过柱，流速0.8～1.2mL/min；⑥上清液全部过柱后，再用18～22mM的咪唑以1.5～2.5mL/min去除杂蛋白；⑦将镍柱取下，装在干净的注射器上，按40～240mM范围进行梯度洗脱，浓度为400～600mM的咪唑进行蛋白洗脱，收集洗脱液。
本发明的有益效果是：应用一种大肠埃希菌改造菌株CLM37Δlpp使N-糖基化单体拟抗体分泌到胞外的培养基中，本发明所述应用基因敲除大肠埃希菌胞外生产N-糖基化重组蛋白的方法，可降低目标蛋白的胞内降解，提高重组拟抗体的表达水平，即糖基化FN3 VEGFR2-Gly的平均表达水平达70±3.4mg/L；且该重组渗漏菌株制备所得的抗VEGFR2单体拟抗体的糖基化效率可达到100％，其活性没有影响；同时减少重组蛋白的胞内过度积累，而降低包涵体形成；消除细胞破壁工艺，减少热原污染，降低了分离纯化成本，最终有利于N-糖基化药物蛋白或糖疫苗的大规模生产。
附图说明
图1细胞膜完整性检测图(左：CLM37Δlpp；右：CLM37)。图A,D：CLM37Δlpp和CLM37被Calcein-AM染色；图B,E：CLM37Δlpp和CLM37被PI染色；图C,F：CLM37Δlpp和CLM37同时被Calcein-AM和PI染色。
图2CLM37和CLM37Δlpp菌体分泌表达重组蛋白的ELISA检测结果。
图3CLM37Δlpp分泌表达重组蛋白FN3-Gly的Western blot检测结果。
图4CLM37Δlpp表达单体拟抗体FN3 VEGFR2-Gly检测结果：Western blot检测：Lane 1和2分别为CLM37Δlpp/pIG6-FN3VEGFR2-Gly+pACYC pgl细胞于IPTG诱导48h后收集的上清纯化前/后；Lane 3未诱导的CLM37Δlpp/pIG6-FN3VEGFR2-Gly+pACYC pgl上清；Lane 4和5分别为CLM37Δlpp/pIG6-FN3VEGFR2-Gly+pACYC pgl细胞于自动诱导48h后收集的上清纯化前/后；箭头所示为FN3VEGFR2-Gly蛋白。
图5N-糖基化蛋白FN3 VEGFR2-Gly血清稳定性的Western blot检测统计结果。
图6唾液酸修饰的N-糖基化蛋白FN3 VEGFR2-Sia抗原结合活性的免疫荧光检测结果。
A:IgG同型对照；B:唾液酸修饰的糖基化FN3 VEGFR2-Sia；C:未糖基化FN3 VEGFR2-Gly。
具体实施方式
下面结合附图及具体实施方式对本发明作进一步的说明，但并不影响本发明的保护范围。下述实施例中使用的生物材料来源如下：
敲除lpp基因的大肠埃希菌CLM37Δlpp：由本实验室构建，参考文献“大肠埃希菌CLM37菌株Lpp基因敲除及胞外表达N-糖基化重组蛋白研究,阮瑶，王力凡，郭龙华等-《上海交通大学学报》(医学版)2018.11:1332-1337”有详细描述。
未敲除lpp基因的CLM37：来源于BioVector NTCC质粒载体菌种细胞基因保藏中心，在“Proceedings of the National Academy of Sciences of the United States of America(ISSN：0027-8424)，102(8):3016-3021”中有详细描述。
本实验室保存的携带模式蛋白FN3-Gly的质周腔表达质粒pIG6-rFn3-Gly：由本实验室构建，文献“Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction.Ding N,Yang C,Sun S,etc.,Biochem Biophys Res Commun 2017 03 25；4851”有详细描述。
携带空肠弯曲杆菌N-糖基化基因簇的pACYCpgl质粒：由苏黎世联邦理工学院实验室馈赠，参考文献“N-linked glycosylation in Campylobacter jejuni and its functional transfer into E.coli.Wacker M,Linton D,Hitchen PG,etc.,Science 2002Nov 29；2985599(5599)”有详细描述。
质粒pCP20：上海钰博生物科技有限公司，货号YB1693；
质粒pKD46：上海钰博生物科技有限公司，货号YB1692。
实施例1
抗VEGFR2单体拟抗体N-糖基化识别位点设计及表达载体构建
确认抗VEGFR2单体拟抗体FN3 VEGFR2氨基酸序列，通过改变AB Loop、EF Loop、CD Loop区及C端，在相应部分插入寡糖转移酶的糖基化识别位点的氨基酸序列，依据SWISS-MODEL、PyMOL等软件进行合理蛋白模型的优化分析，选出最稳定最合理的重组蛋白的基因序列，根据大肠埃希菌密码子偏好性优化序列，然后分别通过PCR方法在基因5′端、3′端以及柔链区引入M1标签和组氨酸纯化标签及编码不同数量的糖基化识别位点碱基序列，并克隆到pIG6表达载体OmpA信号肽下游，诱导后选出表达最优的重组蛋白基因序列如SEQ ID NO.1所示。
制备重组渗漏菌株的步骤：①首先PCR扩增含有lpp基因同源臂和kan基因片段，回收后热激转化至含有能诱导表达Red重组酶的质粒pKD46大肠埃希菌CLM37/pKD46后，取菌液涂布于含L-阿拉伯糖、硫酸卡那霉素、氨苄青霉素的平板增菌培养；②为去除质粒pKD46，挑取阳性单克隆于36～43℃培养，于含氨苄青霉素的LB平板培养基上划线培养，筛选出不含质粒的抗性菌CLM37Δlpp:kan菌株；③为消除kan抗性基因，将质粒pCP20热激转化菌株CLM37Δlpp:kan，挑取转化子增菌培养，取菌液涂布于无抗性的 LB平板培养基上，培养过夜，然后分别用含氨苄青霉素和卡那霉素的LB平板培养筛选出在两种抗生素平板都不生长的单克隆菌株，命名为CLM37Δlpp。
实施例2 效果验证实验
效果实验一、检验细胞膜的完整性
本效果实验中将敲除lpp基因的大肠埃希菌CLM37Δlpp和未敲除lpp基因的CLM37培养12h，在新鲜自动诱导培养基中稀释100倍后，200rpm培养约4h，OD 600值约为0.8，细胞经3500rpm离心5min。用无菌PBS缓冲液冲洗，再悬浮至1×10 6-1×10 7细胞/mL。然后，将1000μL的悬浮液与含10mM Calcein-AM和5mM PI(碘化丙碇)的1000×染色液混合(各取1μL混匀)。在室温下避光孵育30min后，染色细胞用PBS清洗2次后进行离心。然后用无菌的PBS缓冲液稀释至100μL，在显微镜下观察染色细胞。用400×放大率的荧光显微镜检测，荧光发射光谱用490/545nm激发和515/617nm发射测量。结果表明图A,D分别为CLM37Δlpp和CLM37被Calcein-AM染色，都呈现绿色；图B,E分别为CLM37Δlpp和CLM37被PI染色，仅图B呈现红色；图C,F为CLM37Δlpp和CLM37被Calcein-AM和PI染色后叠加，图F呈现出绿色，只有具有渗透膜的细菌才会同时被Calceim-AM和PI染色叠加出黄色(图1C)。结果表明lpp基因的敲除破坏了细菌胞膜的完整性。
效果实验二、诱导大肠埃希菌表达N-糖基化重组蛋白
本效果实验使用本实验室保存的携带模式蛋白FN3-Gly的质周腔表达质粒pIG6-rFn3-Gly和携带空肠弯曲杆菌N-糖基化基因簇的pACYCpgl质粒转化入CLM37Δlpp；或构建有唾液酸糖链(Neu5Ac-ɑ-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-)合成及N-糖基化修饰途径的质粒p15-Ara-lsg(携带大肠杆菌O-特异多糖合成起始酶基因WecA、空肠弯曲杆菌寡糖转移酶基因pglB、翻转酶基因pglK及流感嗜血杆菌脂寡糖基因簇lsgCDEF)和质粒pIG6-Sia-FN3 VEGFR2-Gly-P2-plst6(主要包括合成CMP-Neu5Ac的neuBCA基因簇，携带N-糖基化识别序列的FN3 VEGFR2-Gly基因，及α-2,6-唾液酸转移酶基因plst6)共转化入CLM37Δlpp进行研究。过夜培养后挑选一个菌落，在3mL LB(培养基中含有34μg/mL氯霉素和100μg/mL氨苄青霉素)中于37℃过夜培养。使用该培养物1/100(v/v)接种到100mL的LB肉汤中，并在37℃下振荡培养(220rpm)至OD 600为0.8，用含有相同抗生素浓度的100mL的ZYP-5052自动诱导培养基再次接种。28℃连续培养48h，每隔12h测量菌液OD 600值，收取2OD菌液并离心，将菌体沉淀与上清分离，菌液上清为胞外成分(用PBS稀释至1.0mL)，沉淀为菌体部分。配制缓冲液[0.2M Tris-HCl(pH＝8.2),0.25mM EDTA，0.25M蔗糖，溶菌酶160μg/mL]。细胞再悬浮于1.0mL上述缓冲液，然后加入1M MgSO 4溶液60μL，细胞在冰上孵育25min。离心12000rpm，收集上清液为质周腔成分，沉淀为胞内成分，用2％的SDS溶液悬浮沉淀，放置5min，离心12000rpm，收集上清为胞内成 分(用PBS稀释至1.0mL)。
收集上述诱导后的上清及菌体，分别经ELISA、Western blot法检测糖基化效率，然后收集上清，用亲和层析柱分离纯化上清中重组蛋白，该蛋白即包含N-糖基修饰的重组糖蛋白。该系统可以获得表达量高、便于分离纯化的近100％N-糖基化单体拟抗体。该系统可以用于大量N-糖基化重组拟抗体药物蛋白的制备。
效果实验三、ELISA方法检测大肠埃希菌各组分中N-糖基化重组蛋白的表达
为检测CLM37Δlpp表达N-糖基化重组蛋白情况，将未敲除lpp基因的CLM37用作对照进行Western blot分析。将各组各成分分别取3复孔，按不同时间点包被12h以上(4℃，过夜，空白孔加标准品和标本稀释液)。用200μL PBS洗板5次，每次5min，100μL 3％BSA封闭2h，洗板5次，100μL His-HRP一抗孵育2h，洗板5次，100μL HRP试剂盒显色20min，100μL 2M浓硫酸终止，酶标仪450nM检测后进行数据分析。
结果显示大肠埃希菌CLM37Δlpp表达重组体的胞内的蛋白量很少，说明lpp基因的敲除不影响ompA信号肽对重组蛋白的分泌转运；上清中大肠埃希菌CLM37Δlpp表达重组蛋白的量远高于CLM37(图2A)，表明CLM37Δlpp表达的重组蛋白大量渗漏到胞外；并且36h之后CLM37Δlpp在质周腔的表达量低于大肠埃希菌CLM37(图2B)，表明大肠埃希菌CLM37Δlpp表达蛋白的峰值在36h左右。
效果实验四、大肠埃希菌各组分中N-糖蛋白糖基化效率的检测
为检测CLM37Δlpp分泌表达糖蛋白的N-糖基化效率，以大肠埃希菌CLM37作为对照菌株，将收集的上清液、细胞质周腔和胞内成分分别用anti-M1-FLAG抗体进行Western blot分析。重组蛋白分泌到质周腔后在信号肽酶作用下脱去信号肽，暴露出DYKD标签，可被anti-M1-FLAG抗体特异性识别。重组蛋白FN3理论分子量约为12800，N-糖基化修饰后分子量增大约为14200，所以可根据迁移速度判断其是否被糖基化修饰，并进一步根据免疫印迹条带的相对密度值判定N-糖基化效率。
如图3所示，经自动诱导36h，由于菌株CLM37部分裂解，上清中检测到少量的重组蛋白，但大部分还存在于质周腔中。相比之下，敲除了lpp的菌株CLM37Δlpp胞膜渗漏能力强，其生产的重组蛋白FN3-Gly大部分则存在于上清液中。但两种菌株生产的重组蛋白的糖基化效率均可达到100％，与前期实验结果一致。
效果实验五、大肠埃希菌分泌表达N-糖基化抗VEGFR2单体拟抗体
为生产糖基化的FN3 VEGFR2-Gly，将pACYCpgl和pIG6-FN3 VEGFR2-Gly质粒转化到CLM37和CLM37Δlpp细胞中，自动诱导方法同本实施例效果实验二。为了生产未糖基化的FN3 VEGFR2，对含pIG6-FN3 VEGFR2-Gly和pACYC184载体的大肠埃希菌CLM37Δlpp进行上述 操作步骤。通过在4℃下8000rpm离心10min获取细胞。然后，根据效果实验二描述的方法制备各组分。离心后将收获的上清液用0.22μM过滤器进行过滤，然后加入含有咪唑的PBS，调整终浓度为20mM咪唑，300mM NaCl和3％Tween-20。然后将蛋白质上样至1mL-Histrap柱。用含20mM咪唑的PBS洗涤10个柱体积，接着用5mL含50mM磷酸钠(pH7.4)，300mM NaCl和100mM咪唑的PBS-buffer缓冲液洗脱。Western blot检测糖基化及未糖基化蛋白的表达量，并通过image pro plus 6.0软件分析至少三个独立实验的密度值数据。
结果显示糖基化FN3 VEGFR2-Gly的条带与预期分子量一致。然后根据Western blot条带密度定量计算出菌体和培养基中糖基化FN3 VEGFR2-Gly的平均表达水平，达70±3.4mg/L。综合上述实验结果表明，lpp基因的单缺失对于实现糖蛋白的渗漏表达是有效的，并且从自动诱导培养基中可以获得大量的N-糖基化单体拟抗体FN3 VEGFR2-Gly。
效果实验六、N-糖基化抗VEGFR2单体拟抗体血清稳定性检测
将纯化脱盐后的未糖基化蛋白FN3 VEGFR2-Gly和N-糖基化修饰的FN3 VEGFR2-Gly蛋白于280nm处测量OD值，然后利用Vector NTI 11.5.1软件计算的浓度系数，估算出两种蛋白的浓度。用预冷的PBS稀释至0.1mol/L，将血清分别与0.1mol/L稀释蛋白1:1混匀，37℃静置，分别在0h，6h，18h，24h，36h，48h，60h，72h取样后进行Western blot检测及密度值分析。
统计发现未糖基化FN3 VEGFR2-Gly在18h后开始明显降解(在36h几乎消失)，而糖基化FN3 VEGFR2-Gly在24h内保持稳定，在36～48h内明显降解，表明糖基化FN3 VEGFR2-Gly比未糖基化FN3 VEGFR2-Gly更稳定。**表示与对照蛋白相比有显著差异(**p<0.01，均与糖基化FN3 VEGFR2-Gly相比较)。
效果实验七、细胞免疫荧光法检测唾液酸修饰的N-糖基化FN3 VEGFR2-Sia蛋白生物活性
取6孔细胞培养板，放入乙醇处理过的盖玻片，接种脐静脉内皮细胞(HUVEC)中，置于含有5％CO 2的恒温培养箱中培养。待细胞汇合率达70-80％时，取出细胞爬片，PBS洗3次，吸去残液。加入含有1mL含有3.7％甲醛的PBS溶液，室温静置固定15min。用PBS洗2次(5min/次)，吸去残液。加入1mL Triton X-100含量为0.1％的PBS溶液，室温静置打孔10min。用PBS洗2次(5min/次)，吸去残液。加入0.8％BSA封闭液，室温静置封闭30min。吸去封闭液，加入0.8％BSA配制的各蛋白稀释液(包括)，4℃冰箱静置孵育过夜。第二日，用PBS清洗3次(5min/次)，吸去残液。加入0.8％BSA配制的His一抗，室温孵育1h；用PBS清洗3次(5min/次)，吸去残液。加入0.8％BSA配制的荧光二抗(二抗为FITC标记的羊抗兔IgG)结合液，4℃冰箱静置孵育4h。结束后用PBS清洗3次(5min/次)，吸去残液。加入DAPI染色，室温孵育12min。然后用PBS清洗3次(5min/次)，吸去残液。 取洁净载玻片，滴加1滴50％的甘油，盖玻片细胞面向下，盖在载玻片上并固定。倒置荧光显微镜镜检。
结果显示末端唾液酸N-糖基化(Neu5Ac-ɑ-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-)修饰的和未糖基化修饰的抗VEGFR2单体拟抗体均见特异染色(图6B,C)，说明可结合到HUVEC细胞表面的VEGFR2，且差别不大，表明唾液酸化N-糖基化修饰对蛋白FN3 VEGFR2-Gly生物活性影响不大。从具体效果实验来看，该方法可以大量、快速、高效获得N-糖基化重组蛋白，为进一步相关N-糖基化药物蛋白及N-糖基化疫苗奠定基础。
本发明实施方式不限于此，仅为本发明较佳的具体实施方式，根据本发明的上述内容，按照本领域的普通技术知识和通用方法，在不脱离本发明上述基本技术思想前提下，本发明还可以有其它的实施方式。任何熟悉本技术领域的技术人员在本发明披露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，本发明还可以做出其它多种形式的修改、替换或变更，如可以用其它的大肠埃希菌质周腔表达载体等类似方法，用大肠埃希菌胞外生产N-糖基化类型重组蛋白，均落在本发明权利保护范围之内。
Figure PCTCN2021095166-appb-000001

Claims (10)

  1. 一种高表达N-糖基化抗VEGFR2单体拟抗体的重组渗漏菌株；其特征在于，所述重组渗漏菌株为大肠埃希菌改造菌株CLM37Δlpp中引入含有碱基序列如SEQ ID NO.1所示基因序列的重组载体pIG6-FN3 VEGFR2及空肠弯曲杆菌(Campylobacter jejuni)来源的N-糖基化基因簇pgl。
  2. 根据权利要求1所述的高表达N-糖基化抗VEGFR2单体拟抗体的重组渗漏菌株；其特征在于，所述的含有碱基序列如SEQ ID NO.1所示基因片段的重组载体pIG6-FN3 VEGFR2是指在重组载体pIG6-FN3 VEGFR2中表达抗人血管内皮生长因子VEGFR2单体拟抗体、并带有糖基化修饰位点，且引入携带空肠弯曲杆菌基于pglB的N-糖基化修饰基因簇的质粒。
  3. 如权利要求1所述的高表达N-糖基化抗VEGFR2单体拟抗体的重组渗漏菌株的构建方法，其步骤包括：
    (1)含有碱基序列如SEQ ID NO.1所示基因片段的重组载体pIG6-FN3 VEGFR2的构建步骤：通过PCR方法在抗人VEGFR2单体拟抗体基因5′端、3′端以及柔链区引入M1标签和组氨酸纯化标签及编码不同数量的糖基化识别位点碱基序列，并克隆到pIG6表达载体OmpA信号肽下游，载体命名为pIG6-FN3 VEGFR2；
    (2)制备重组渗漏菌株CLM37Δlpp，将pIG6-FN3 VEGFR2-Gly质粒转化到CLM37Δlpp细胞中，可高表达抗VEGFR2单体拟抗体-的重组渗漏菌株CLM37Δlpp/pIG6-FN3 VEGFR2-Gly；该重组渗漏菌株进一步转入携带N-糖基化基因簇的pACYCpgl质粒，然后进行自动诱导，用于生产糖基化的FN3 VEGFR2-Gly。
  4. 如权利要求1所述的重组渗漏菌株在提高N-糖基化抗VEGFR2单体拟抗体产量方面的应用。
  5. 根据权利要求4所述的应用，其特征在于：所述应用为利用权利要求1所述的重组渗漏菌株在自动诱导培养基中诱导胞外生产抗VEGFR2单体拟抗体；收集培养所得的上清液，离心并收集沉淀，纯化后获得N-糖基化抗VEGFR2单体拟抗体。
  6. 根据权利要求5所述的应用，其特征在于：所述的应用中使用的自动诱导培养基为：每升培养基中含有胰蛋白胨8～12g，酵母提取物4～6g，甘油4～6g，葡萄糖0.4～0.6g，乳糖1～3g，磷酸氢二钠6～8g，磷酸二氢钾6～8g，硫酸铵3～4g，硫酸钠0.5～1.2g，七水硫酸镁0.2～0.3g。
  7. 根据权利要求5所述的应用，其特征在于：所述的应用中的诱导培养步骤包括：将 其权利要求1所述的重组渗漏菌株接种到含有80～120μg/mL氨苄青霉素和35～40μg/mL氯霉素的LB液体培养基，过夜12～18小时培养；再以1:80～120的体积比接种到含有80～120μg/mL氨苄青霉素和35～40μg/mL氯霉素的LB液体培养基中，直至OD 600达到0.5～0.8；然后，以1:80～120接种到含有80～120μg/mL氨苄青霉素和35～40μg/mL氯霉素的自动诱导培养基中诱导表达。
  8. 根据权利要求5所述的应用，其特征在于：所述的应用中重组渗漏菌株的培养条件为：将共转化有pIG6-Fn3-Gly和pACYCpgl质粒的菌株CLM37Δlpp接种于含有氨苄青霉素、氯霉素的新鲜自动诱导培养基中，连续培养增菌。
  9. 根据权利要求5所述的应用，其特征在于：所述的应用中离心的条件为：收取诱导至少40小时的菌液于8000～14000r/min，18～22min离心。
  10. 根据权利要求5所述的应用，其特征在于：所述的应用中纯化的条件为：①上文离心后获得的上清液加入终浓度为18～22mM的咪唑；②0.22μM滤膜抽滤除杂质，并加入终浓度为280～320mM氯化钠及0.8～1.2wt％的Tween-20，冰浴保存；③装好His-Trap镍柱后，400～600mM咪唑洗脱，洗去残留蛋白；④用由18～22mM咪唑及1～2wt％的Tween-20组成的Bindingbuffer过柱，流速0.8～1.2mL/min清洗；⑤将上清液进行过柱，流速0.8～1.2mL/min；⑥上清液全部过柱后，再用18～22mM的咪唑以1.5～2.5mL/min去除杂蛋白；⑦将镍柱取下，装在干净的注射器上，按40～240mM范围进行梯度洗脱，浓度为400～600mM的咪唑进行蛋白洗脱，收集洗脱液。
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