WO2020133610A1 - 一种重组大肠杆菌质周腔内生产唾液酸修饰n-糖基化重组蛋白方法 - Google Patents

一种重组大肠杆菌质周腔内生产唾液酸修饰n-糖基化重组蛋白方法 Download PDF

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WO2020133610A1
WO2020133610A1 PCT/CN2019/072202 CN2019072202W WO2020133610A1 WO 2020133610 A1 WO2020133610 A1 WO 2020133610A1 CN 2019072202 W CN2019072202 W CN 2019072202W WO 2020133610 A1 WO2020133610 A1 WO 2020133610A1
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sialic acid
recombinant protein
gene
coli
glycosylated
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胡学军
阮瑶
丁宁
付鑫
朱静
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大连大学
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  • the invention specifically relates to a method for producing sialic acid modified N-glycosylated recombinant protein in the periplasmic cavity of recombinant E. coli, which belongs to the field of biotechnology and protein engineering.
  • N-glycosylated drugs 70% of the approved protein drugs are N-glycosylated drugs, and sialic acid modification of N-glycosylated drug proteins can improve their physical and chemical properties. Small molecular weight drug proteins are easily filtered by the kidneys during circulation in the body, mediated by non-sialoglycoprotein receptors in the liver, and degraded by proteases in the peripheral blood. Sialic acid modified N-glycosylated drug protein can significantly increase the stability of the drug protein, reduce the immune response, and prolong the drug half-life.
  • the embryonic kidney cell line (HEK) 293S after genetic engineering, can produce sialylated oligosaccharide chain Neu5Ac- ⁇ -2,3-Gal- ⁇ -1,4-GlcNAc modified recombinant protein, which can reduce the The clearance rate of the recombinant protein in the body, and no new immunogenicity is produced.
  • the cultivation of eukaryotic systems requires a long time and high cost.
  • the sialic acid-modified N-glycosylated recombinant protein can also be obtained by chemical methods in vitro, the method has high cost, complicated process, and low yield.
  • the realization of sialic acid modified N-glycosylated recombinant protein in the periplasmic cavity of Escherichia coli can reduce the cost of producing sialic acid modified N-glycosylated drug protein and improve the efficiency of sialic acid modification.
  • the glycosyltransferase expressed by the glycosyltransferase lsg gene cluster derived from Haemophilus influenzae can synthesize the oligosaccharide chain Gal- ⁇ -1,4-GlcNAc- ⁇ -1 in the periplasmic cavity of E. coli.
  • oligosaccharide chain Neu5Ac-Gal- ⁇ -1,4-GlcNAc- ⁇ -1,3-Gal- ⁇ -1,3-GlcNAc modified recombinant protein can be sialylated at the end of the periplasmic cavity of E. coli
  • the method can be used to produce sialic acid modified N-glycosylated drug protein in the periplasmic cavity of E. coli.
  • the present invention provides a method for recombining sialic acid modified N-glycosylated recombinant protein in the periplasmic cavity of recombinant E. coli, which can quickly, efficiently and mass-produce sialic acid modified N-glycosylated recombinant protein without the need for Adding sialic acid to the culture medium can produce sialic acid modified N-glycosylated recombinant protein at low cost, which provides an effective way for the development of sialic acid modified N-glycosylated drug protein.
  • the technical solution of the present invention is to utilize the glycosyltransferase lsgCDEF gene cluster (GenBank: M94855.1) derived from Haemophilus influenzae (Haemophilus influenzae) and the WecA gene (Gene ID: 948789) derived from E.
  • Oligosaccharide turnover enzyme pglK gene (Gene ID: 905421) derived from Campylobacter jejuni, oligosaccharide transferase pglB gene (Gene ID: 905417) and sialic acid synthesis related enzyme neuBCA gene cluster (GenBank: AF400048.1 ), ⁇ -2,6 sialyltransferase ⁇ 16psp2, 6ST gene (GenBank: AB293985.1) derived from Vibrio luminescens JT-ISH-224 (Vibrionaceae Photobacterium sp.JT-ISH-224), and sialylated N -Glycosylation-modified recombinant protein gene, cloned into E.
  • coli expression vector by gene recombination method jointly constructed into a sialic acid modified N-glycosylation recombinant protein system in E. coli, suitable for sialic acid modified N-glycosylation Recombinant E.coli containing recombinant protein is automatically induced and cultured in vivo without adding exogenous sialic acid, and sialic acid modified N-glycosylated recombinant protein is added.
  • the sialylated oligosaccharide chain is Neu5Ac- ⁇ -2,6-Gal- ⁇ -1,4-GlcNAc- ⁇ -1,3-Gal- ⁇ -1,3-GlcNAc; sialylated N-glycosyl is required
  • the genetically modified recombinant protein gene carries a recognition sequence encoding oligosaccharide transferase pglB.
  • the method of the present invention includes the following steps:
  • E. coli produces sialic acid modified N-glycosylated recombinant protein in vivo;
  • step (3) The recombinant protein obtained in step (3) is purified to obtain a sialic acid-modified N-glycosylated recombinant protein.
  • step (1) is specifically: the construction of the E. coli strain suitable for sialic acid modified N-glycosylation recombinant protein, the basic method is to use the Red homologous recombination system to knock out nanKETA in the W3110 genome of E. coli K12 origin Gene cluster, thereby blocking the bypass pathway in the process of sialic acid synthesis, the genotype of E. coli strain constructed was W3110 ⁇ nanKETA::Kan.
  • Step (2) is specifically: constructing the glycosyltransferase lsgCDEF gene cluster, sugar chain synthesis initiating enzyme WecA gene, oligosaccharide turnover enzyme pglK gene, oligosaccharide transferase pglB gene together on an E. coli expression vector to obtain expression N -Carrier of glycosylation system; sialic acid synthesis related enzyme neuBCA gene cluster, ⁇ -2,6 sialyltransferase ⁇ 16psp2,6ST gene and recombinant protein genes requiring sialylated N-glycosylation modification by gene recombination method , Cloned into an E. coli expression vector, and jointly constructed a sialic acid modified N-glycosylation recombinant protein system in the periplasmic cavity of E. coli.
  • the step (3) is specifically: transforming the sialic acid modified N-glycosylated recombinant protein expression vector constructed in step (2) into the E. coli strain W3110 obtained in step (1) ⁇ nanKETA::Kan, which is automatically induced
  • the cultivation method does not need to add exogenous sialic acid, and produces sialic acid modified N-glycosylated recombinant protein in the strain.
  • the present invention provides a method for sialic acid-modified N-glycosylated recombinant protein in recombinant E. coli strain W3110 ⁇ nanKETA::Kan
  • the method requires short time, low cost, high sialylation efficiency, and no need to add exogenous sialic acid during the cultivation process, and establishes a technical platform for the production of therapeutic sialic acid modified N-glycosylated protein drugs.
  • Figure 1 Vector map of pC15-Ara-pglB-WecA-pglK-lsgCDEF.
  • Figure 2 Vector map of pIG6-Fn3-P- ⁇ 16psp2, 6ST-neuBCA.
  • FIG. 4 Western Blotting detection results of recombinant E. coli expressing sialic acid modified N-glycosylated recombinant protein in vivo.
  • Unglycosylated Fn3 recombinant protein 2.
  • Non-sialic acid modified N-glycosylated Fn3 recombinant protein 3.
  • Sialic acid modified N-glycosylated Fn3 recombinant protein 4.
  • Fig. 5A is the result of the lectin ECA (Catalog Number: H-5901-1) lectin imprinting that specifically recognizes Gal- ⁇ -1,4-GlcNAc produced by EY Laboratories: 1. Unglycosylated Fn3 recombination Protein, 2. N-glycosylated Fn3 recombinant protein without sialic acid modification, 3. N-glycosylated Fn3 recombinant protein with sialic acid modification.
  • Figure 5B is the result of lectin blotting detection of lectin SNA-I (Catalog Number: H-6802-1) of Neu5Ac- ⁇ -2,6-Gal produced by EY Laboratories: 1. Recombination of unglycosylated Fn3 Protein, 2. N-glycosylated Fn3 recombinant protein without sialic acid modification, 3. N-glycosylated Fn3 recombinant protein with sialic acid modification.
  • FIG. 6A Sugar chain composition analysis of sialic acid modified N-glycosylated recombinant protein expressed in recombinant E. coli. 6A. HCD MS/MS spectrum, 6B. Deconvolution MS/MS spectrum.
  • the present invention relates to experimental equipment, materials, reagents, etc., which can be obtained from commercial sources.
  • the E. coli strain W3110 was purchased from Yale University-E. coli Genetic Inventory Center.
  • Example 1 Construction of E. coli strains suitable for sialic acid modified N-glycosylated recombinant protein
  • the original strain used for genetic modification was W3110 derived from Escherichia coli K12, using the Red homologous recombination system to knock out the nanKETA gene cluster (SEQ ID NO. 1) on its genome. Plasmids used for gene knockout include pKD13 and pKD46. The specific steps for knocking out the nanKETA gene cluster are as follows:
  • Two pairs of knockout primers were designed according to the nucleotide sequences on both sides of the nanKETA gene cluster on the genome of E. coli W3110 and the nucleotide sequences on both sides of the kanamycin resistance gene on pKD13:
  • NanKETA F1 5 ⁇ -gcatccgcgccagccaactccccctgcgctgccgctgcgtgtaggctggagctgctt-3 ⁇ ;
  • Second use pKD13 as a template use primers del NanKETA F1, del NanKETA R1 for the first PCR amplification; then use the first PCR product as a template, use primers del NanKETA F2, del NanKETA R2 for the second PCR amplification To obtain PCR fragments with 75 bp and 71 bp on both sides that are homologous to the genomic sequence at both ends of the nanKETA gene cluster and carry the kanamycin resistance gene. The PCR fragment was electroporated and recovered by gel cutting and then electroporated into E.
  • the transformants were spread on LB solid medium plates containing kanamycin (15 ⁇ g/mL) and incubated overnight at 30°C.
  • JDnanKETAF 5 ⁇ -cgcactggcaatcagttgtg-3 ⁇
  • JDnanKETAR 5 ⁇ -cgtcacgccgttctactatc-3 ⁇
  • LB solid medium plates were grown, and colonies that did not grow on LB solid medium plates containing ampicillin (100 ⁇ g/mL) proved that the plasmid pKD46 had been lost.
  • the E. coli strain obtained after the deletion of the nanKETA gene cluster was W3110 ⁇ nanKETA::Kan, which blocked the bypass pathway in the synthesis of sialic acid.
  • LB solid medium The formula of LB solid medium is as follows: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar powder 15g/L, and ddH 2 O.
  • LB liquid medium The formula of LB liquid medium is as follows: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and ddH 2 O.
  • glycosyltransferase lsgCDEF (GenBank: M94855.1), sugar chain synthesis initiation enzyme WecA gene (Gene ID: 948789), oligosaccharide invertase pglK gene (Gene ID: 905421 ) And oligosaccharide transferase pglB (Gene ID: 905417) are constructed on the vector pACYC184, and the expression of the above four genes is regulated by the arabinose promoter (Ara) to obtain N-glycosylation expression
  • the vector pC15-Ara-pglB-WecA-pglK-lsgCDEF of the system is shown in Figure 1, and its sequence is shown in SEQ ID NO.3.
  • the recombinant human fibronectin type III domain (Fn3) is used as the receptor protein, and sialic acid modified N-glycosylated recombinant protein is studied in the periplasmic cavity of E. coli.
  • the recombinant protein Fn3 gene carries a FLAG tag (amino acid sequence: DYKD, D for aspartic acid residues, Y for tyrosine residues, and K for lysine residues) at the 5 ⁇ end, which facilitates Western blotting detection;
  • DYKD amino acid sequence: DYKD, D for aspartic acid residues, Y for tyrosine residues, and K for lysine residues
  • DQNAT D stands for aspartic acid residues
  • Q stands for glutamine residues
  • N stands for asparagine residues
  • A stands for alanine residues
  • T Threonine residue
  • the histidine tag is used for recombinant protein separation and purification.
  • the recombinant protein Fn3 gene (SEQ ID NO. 3) and the sialic acid synthesis-related enzyme neuBCA gene cluster (GenBank: AF400048.1), Vibrio luminescens JT-ISH-224 (Vibrionaceae Photobacterium sp. JT-ISH-224) ⁇ -2,6 sialyltransferase ⁇ 16psp2, 6ST gene (GenBank: AB293985.1) and the 332bp regulatory sequence upstream from the Pgl gene cluster of Campylobacter jejuni (GenBank: Y11648.1) (Referred to as P, SEQ ID NO.
  • Example 3 Production and purification of sialic acid modified N-glycosylated recombinant protein in E. coli.
  • the vectors pC15-Ara-pglB-WecA-pglK-lsgCDEF and pIG6-Fn3-P- ⁇ 16psp2, 6ST-ne-uBCA were transformed into recombinant E. coli strain W3110 ⁇ nanKETA::Kan to obtain sialic acid-modified N-glycosylation Recombinant protein expression vector recombinant E. coli strain.
  • the transformants were inoculated on LB solid medium plates containing kanamycin (15 ⁇ g/mL), ampicillin (100 ⁇ g/mL), and chloramphenicol (34 ⁇ g/mL), and cultured overnight at 37°C for 12 hours. After screening out the single clones, inoculate them into 3mL LB liquid medium containing ampicillin (100 ⁇ g/mL) and chloramphenicol (34 ⁇ g/mL), and incubate at 220rpm and 37°C overnight.
  • the bacterial solution was inoculated into a 500 mL auto-induction medium containing ampicillin (100 ⁇ g/mL) and chloramphenicol (34 ⁇ g/mL) at a ratio of 1:100, and cultured at 220 rpm and 25°C for 40 hours, and L-arabinose (200 ⁇ g/mL) was added as an inducer every 12 hours.
  • the cells were collected by centrifugation at 4000 rpm and 4°C, and the supernatant containing N-glycosylated Fn3 recombinant protein modified by sialic acid was obtained by methods such as ultrasonic disruption and low-temperature high-speed centrifugation.
  • the eluent is collected and separated for purification
  • the recombinant protein is sialic acid modified N-glycosylated Fn3 recombinant protein, wherein the sialylated oligosaccharide chain is Neu5Ac- ⁇ -2,6-Gal- ⁇ -1,4-GlcNAc- ⁇ -1,3 -Gal- ⁇ -1,3-GlcNAc.
  • the sialic acid-modified N-glycosylated Fn3 recombinant protein purified above was desalted using a desalting column and stored at 4°C.
  • Escherichia coli strain W3110 carrying only vector pIG6-Fn3 ⁇ nanKETA::Kan and E. coli strain W3110 carrying vector pIG6-Fn3 and vector pC15-Ara-pglB-WecA-pglK-lsgCDEF were cultured as a control group, Specific steps are as follows:
  • the vector pIG6-Fn3 was transformed into E. coli W3110 ⁇ nanKETA::Kan.
  • the transformants were inoculated on LB solid medium plates containing kanamycin (15 ⁇ g/mL) and ampicillin (100 ⁇ g/mL), and cultured overnight at 37°C for 12 hours. After screening out the single clones, inoculate them into 3mL LB liquid medium containing ampicillin (100 ⁇ g/mL) and incubate at 220rpm and 37°C overnight. The next day, the bacterial solution was inoculated into a 500 mL auto-induction medium containing ampicillin (100 ⁇ g/mL) at a ratio of 1:100, and cultured at 220 rpm and 25°C for 40 hours.
  • the vector pIG6-Fn3 and the vector pC15-Ara-pglB-WecA-pglK-lsgCDEF were transformed into E. coli W3110 ⁇ nanKETA::Kan.
  • the transformants were inoculated on LB solid medium plates containing kanamycin (15 ⁇ g/mL), ampicillin (100 ⁇ g/mL), and chloramphenicol (34 ⁇ g/mL), and cultured overnight at 37°C for 12 hours. After screening out the single clones, inoculate them into 3mL LB liquid medium containing ampicillin (100 ⁇ g/mL) and chloramphenicol (34 ⁇ g/mL), and incubate at 220rpm and 37°C overnight.
  • the bacterial solution was inoculated into a 500 mL auto-induction medium containing ampicillin (100 ⁇ g/mL) and chloramphenicol (34 ⁇ g/mL) at a ratio of 1:100, and cultured at 220 rpm and 25°C for 40 hours, and L-arabinose (200 ⁇ g/mL) was added as an inducer every 12 hours.
  • LB solid medium The formula of LB solid medium is as follows: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar powder 15g/L, and ddH 2 O.
  • LB liquid medium The formula of LB liquid medium is as follows: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and ddH 2 O.
  • the formula of the automatic induction medium is as follows: tryptone 10g/L, yeast extract 5g/L, glycerol 5g/L, glucose 0.5g/L, lactose 2g/L, disodium hydrogen phosphate 7.1g/L, potassium dihydrogen phosphate 6.8g/L, ammonium sulfate 3.3g/L, sodium sulfate 0.9g/L, magnesium sulfate heptahydrate 0.25g/L, added with ddH 2 O.
  • Example 4 Detection of sialic acid modified N-glycosylated recombinant protein produced in E. coli.
  • the lectin blot detection was carried out using the lectin ECA (Catalog Number: H-5901-1) that specifically recognizes Gal- ⁇ -1,4-GlcNAc produced by EY Laboratories and the specific recognition Neu5Ac- ⁇ -2,6- Gal lectin SNA-I (Catalog Number: H-6802-1), purified and desalted non-glycosylated Fn3 recombinant protein, non-sialic acid modified N-glycosylated Fn3 recombinant protein as a negative control, The sialic acid modified N-glycosylated Fn3 recombinant protein was detected and analyzed. The results are shown in Figure 5.
  • Panel A is the result of the lectin ECA (Catalog Number: H-5901-1) lectin imprinting test that specifically recognizes Gal- ⁇ -1,4-GlcNAc produced by EY Laboratories, unglycosylated
  • the Fn3 recombinant protein and the sialic acid-modified N-glycosylated Fn3 recombinant protein showed no specific bands, while the non-sialic acid-modified N-glycosylated Fn3 recombinant protein detected a specific band, proving that the Fn3 recombinant protein was N-glycosylation modification
  • Panel B is the result of lectin blotting detection of the lectin SNA-I (Catalog Number: H-6802-1) specifically recognized by Neu5Ac- ⁇ -2,6-Gal produced by EY Laboratories, without sugar Neither the sialic acid-modified N-glycosylated Fn3 recombinant protein nor the sialic acid-modified N
  • Detection method take the purified and desalted sialic acid modified N-glycosylated Fn3 recombinant protein obtained in Example 3, add trypsin (promega V5280) and Glu-C enzyme (promega) for enzymolysis, and then use The Thermo Orbitrap Exactive HF LC/MS is used for qualitative detection. According to the secondary spectrum By ion and the corresponding modification mass matching, determine the main modified glycoforms.
  • Panel A shows the specific peaks of NeuAc (molecular weight 292.10), NeuAc-H2O (molecular weight 274.09), NeuAc(1)Hex(1)HexNAc(1) (molecular weight 657.23); after deconvolution, glycopeptide can be seen in panel B IGGGGSDQ N ATK has a specific peak of sugar chain NeuAc(1)Hex(2)HexNAc(2) (molecular weight is 2125.89).
  • glycopeptide IGGGGSDQ N ATK in figure A is obtained respectively Sugar chain Hex(2) HexNAc(2) (MW 1634.79), glycopeptide IGGGGSDQ N ATK with sugar chain Hex(1) HexNAc(1) (MW 1469.66) and glycopeptide IGGGGSDQ N ATK with sugar chain HexNAc( 1) (Molecular weight 1307.61).
  • sialic acidified oligosaccharide chain of the modified Fn3 recombinant protein is Neu5Ac-Gal- ⁇ -1,4-GlcNAc- ⁇ -1,3-Gal- ⁇ -1,3-GlcNAc.
  • the method of sialic acid modification of N-glycosylated recombinant protein in the periplasmic cavity of recombinant E. coli according to the present invention is simple, fast, efficient and low in production cost, and is more stable for further production. Establish a technology platform for therapeutic protein drugs or sugar vaccines.
  • 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 content of the present invention, according to the general technical knowledge and common methods in the field, without departing from the above basic technical ideas of the present invention, The invention can also have other embodiments. Any person who is familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solutions of the present invention and the inventive concept of equivalent replacement or change, the present invention can also make other forms of modification, replacement or change, For example, other E. coli expression vectors, replacement of sialylglycosyltransferase genes with the same function, other receptor protein expression genes, etc. can be used to produce sialic acid modified N-glycosylated recombinant proteins in E. coli. It falls within the protection scope of the present invention.

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Abstract

本发明提供一种重组大肠杆菌质周腔内唾液酸修饰N-糖基化重组蛋白方法。具体涉及在重组大肠杆菌菌株W3110ΔnanKETA::Kan体内生产唾液酸修饰的N-糖基化重组蛋白，实现大肠杆菌质周腔中唾液酸修饰N-糖基化重组蛋白，其中，唾液酸化的寡糖链为Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc。该方法时间短、效率高、成本低，且无需添加外源唾液酸，为生产治疗性唾液酸修饰N-糖基化蛋白药物建立技术平台。

Description

一种重组大肠杆菌质周腔内生产唾液酸修饰N-糖基化重组蛋白方法 技术领域
本发明具体涉及一种重组大肠杆菌质周腔内生产唾液酸修饰N-糖基化重组蛋白方法，属于生物技术领域、蛋白质工程领域。
背景技术
目前，已批准的蛋白药物中有70％是N-糖基化修饰的药物，唾液酸修饰N-糖基化药物蛋白可提高其理化性质。小分子量的药物蛋白在体内循环过程中很容易被肾脏滤掉、被肝脏非唾液酸糖蛋白受体介导清除及被外周血中蛋白酶降解。唾液酸修饰N-糖基化药物蛋白可显著增加药物蛋白的稳定性，降低免疫反应，延长药物半衰期。有报道表明，胚胎肾细胞系(HEK)293S经过基因工程改造后，可生产唾液酸化寡糖链Neu5Ac-α-2,3-Gal-β-1,4-GlcNAc修饰的重组蛋白，可降低该重组蛋白在体内的清除率，并且未产生新的免疫原性。但是，真核生物系统培养所需时间较长、成本高。虽然，在体外通过化学方法也可以获得唾液酸修饰的N-糖基化重组蛋白，但是该方法成本高、过程复杂，且产量低。在大肠杆菌质周腔内实现唾液酸修饰N-糖基化重组蛋白可降低生产唾液酸修饰N-糖基化药物蛋白成本，提高唾液酸修饰效率。利用流感嗜血杆菌(Haemophilus influenzae)来源的糖基转移酶lsg基因簇表达的糖基转移酶可在大肠杆菌质周腔内合成寡糖链Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc,并在空肠弯曲杆菌来源的寡糖转移酶pglB作用下进行N-糖基化修饰重组蛋白。但还未见在大肠杆菌质周腔中末端唾液酸化的寡糖链Neu5Ac-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc修饰重组蛋白的方法，如果能实现在大肠杆菌质周腔内末端唾液酸化的寡糖链Neu5Ac-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc修饰重组蛋白，该方法可用于在大肠杆菌质周腔内生产唾液酸修饰N-糖基化药物蛋白。
发明内容
为解决上述难题，本发明提供了一种重组大肠杆菌质周腔内唾液酸修饰N-糖基化重组蛋白方法，可快速、高效、大量生产唾液酸修饰的N-糖基化重组蛋白，无需在培养基中添加唾液酸即可低成本生产唾液酸修饰的N-糖基化重组蛋白，为唾液酸修饰N-糖基化药物蛋白的研发提供有效途径。
本发明的技术方案是利用流感嗜血杆菌(Haemophilus influenzae)来源的糖基转移酶lsgCDEF基因簇(GenBank：M94855.1)、大肠杆菌来源的糖链合成起始酶WecA基因(Gene ID：948789)、空肠弯曲杆菌(Campylobacter jejuni)来源的寡糖翻转酶pglK基因(Gene ID:905421)、寡糖转移酶pglB基因(Gene ID:905417)及唾液酸合成相关酶neuBCA基因簇(GenBank:AF400048.1),弧菌发光菌JT-ISH-224(Vibrionaceae Photobacterium sp.JT-ISH-224)来源的α-2,6唾液酸转移酶Δ16psp2,6ST基因(GenBank：AB293985.1),以及需要唾液酸化N-糖基化修饰的重组蛋白基因，通过基因重组方法，克隆到大肠杆菌表达载体上，共同构建成大肠杆菌体内唾液酸修饰N-糖基化重组蛋白系统，在适合唾液酸修饰N-糖基化重组蛋白的重组大肠杆菌体内经自动诱导培养方法，无需添加外源唾液酸，进行唾液酸修饰N-糖基化重组蛋白。
其中，唾液酸化的寡糖链为Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc；需要唾液酸化N-糖基化修饰的重组蛋白基因上携带编码寡糖转移酶pglB识别序列。
本发明所述方法包括以下步骤：
(1)适合唾液酸修饰N-糖基化重组蛋白大肠杆菌菌株的构建；
(2)唾液酸修饰N-糖基化重组蛋白表达载体的构建；
(3)大肠杆菌体内生产唾液酸修饰的N-糖基化重组蛋白；
(4)步骤(3)中获得的重组蛋白经纯化后即得唾液酸修饰的N-糖基化重组蛋白。
其中，步骤(1)具体为：所述适合唾液酸修饰N-糖基化重组蛋白大肠杆菌菌株的构建，基本方法为利用Red同源重组系统，敲除大肠杆菌K12来源的W3110基因组中的nanKETA基因簇，从而阻断唾液酸合成过程中的旁路途经，构建成大肠杆菌菌株的基因型为W3110 ΔnanKETA::Kan。
步骤(2)具体为：将糖基转移酶lsgCDEF基因簇、糖链合成起始酶WecA基因、寡糖翻转酶pglK基因、寡糖转移酶pglB基因共同构建至大肠杆菌表达载体上，获得表达N-糖基化体系的载体；将唾液酸合成相关酶neuBCA基因簇、α-2,6唾液酸转移酶Δ16psp2,6ST基因以及需要唾液酸化N-糖基化修饰的重组蛋白基因，通过基因重组方法，克隆到大肠杆菌表达载体上，共同构建成大肠杆菌 质周腔内唾液酸修饰N-糖基化重组蛋白系统。
所述步骤(3)具体为：将步骤(2)中构建的唾液酸修饰N-糖基化重组蛋白表达载体转化到步骤(1)获得的大肠杆菌菌株W3110 ΔnanKETA::Kan中，经自动诱导培养方法，无需添加外源唾液酸，在该菌株体内生产唾液酸修饰的N-糖基化重组蛋白。
与现有技术相比，本发明的有益效果为：本发明提供一种在重组大肠杆菌菌株W3110ΔnanKETA::Kan质周腔内进行唾液酸修饰N-糖基化重组蛋白的方法，实现大肠杆菌质周腔中唾液酸修饰N-糖基化重组蛋白，其中，唾液酸化的寡糖链为Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc。该方法所需时间短、成本低、唾液酸化效率高，且培养过程中无需添加外源唾液酸，为生产治疗性唾液酸修饰N-糖基化蛋白药物建立技术平台。
附图说明
图1.pC15-Ara-pglB-WecA-pglK-lsgCDEF载体图谱。
图2.pIG6-Fn3-P-Δ16psp2,6ST-neuBCA载体图谱。
图3.pIG6-Fn3载体图谱。
图4.重组大肠杆菌体内表达唾液酸修饰的N-糖基化重组蛋白的Western Blotting检测结果。1.未糖基化的Fn3重组蛋白，2.未唾液酸修饰的N-糖基化Fn3重组蛋白，3.唾液酸修饰的N-糖基化Fn3重组蛋白。
图5.重组大肠杆菌体内表达唾液酸修饰的N-糖基化重组蛋白的凝集素印记检测结果。
其中，图5A是EY Laboratories公司生产的特异识别Gal-β-1,4-GlcNAc的凝集素ECA(Catalog Number：H-5901-1)凝集素印记检测结果：1.未糖基化的Fn3重组蛋白，2.未唾液酸修饰的N-糖基化Fn3重组蛋白，3.唾液酸修饰的N-糖基化Fn3重组蛋白。
图5B是EY Laboratories公司生产的特异识别Neu5Ac-ɑ-2,6-Gal的凝集素SNA-Ⅰ(Catalog Number：H-6802-1)凝集素印迹检测结果：1.未糖基化的Fn3重组蛋白，2.未唾液酸修饰的N-糖基化Fn3重组蛋白，3.唾液酸修饰的N-糖基化Fn3重组蛋白。
图6.重组大肠杆菌体内表达唾液酸修饰的N-糖基化重组蛋白的糖链组成 分析。6A.HCD MS/MS谱图，6B.去卷积MS/MS谱图。
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。如无特殊说明，本发明涉及实验器材、材料、试剂等均可从商业途径获得。大肠杆菌菌株W3110购买于美国耶鲁大学-大肠杆菌遗传库存中心。
实施例1、适合唾液酸修饰N-糖基化重组蛋白大肠杆菌菌株的构建
用于基因改造的原始菌株是大肠杆菌K12来源的W3110，利用Red同源重组系统敲除其基因组上的nanKETA基因簇(SEQ ID NO.1)。基因敲除所用的质粒包括pKD13、pKD46。nanKETA基因簇的敲除，具体步骤如下：
根据大肠杆菌W3110基因组上nanKETA基因簇两侧碱基序列及pKD13上卡那霉素抗性基因两侧碱基序列设计两对敲除引物，分别为：
del nanKETA F1：5ˊ-gcatccgcgccagccaactccccctgcgctgccgctgcgtgtaggctggagctgctt-3ˊ；
del nanKETA R1：5ˊ-tggtgtacaacattccagccctgagtggggtaaaactctgtcaaacatgagaattaa-3ˊ；
del nanKETA F2：5ˊ-gtcaccctgcccggcgcgcgtgaaaatagttttcgcatccgcgccagccaactccccct-3ˊ；
del nanKETA R2：5ˊ-gcaattattgattcggcggatggtttgccgatggtggtgtacaacattccagccctgag-3ˊ。
先以pKD13为模板，利用引物del nanKETA F1、del nanKETA R1进行第一次PCR扩增；再以第一次的PCR产物为模板，利用引物del nanKETA F2、del nanKETA R2进行第二次PCR扩增，得到两侧分别有75bp、71bp与nanKETA基因簇两端的基因组序列同源且带有卡那霉素抗性基因的PCR片段。PCR片段经电泳、切胶回收后电击转化进入携带pKD46表达的同源重组酶的大肠杆菌W3110菌株细胞中，通过pKD46表达的同源重组酶将PCR片段整合至基因组中替换nanKETA基因簇。将转化子涂布于含有卡那霉素(15μg/mL)的LB固体培养基平板上，30℃培养过夜。利用鉴定引物JD nanKETA F：5ˊ-cgcactggcaatcagttgtg-3ˊ与JD nanKETA R：5ˊ-cgtcacgccgttctactatc-3ˊ对长出的单克隆菌落进行菌落PCR扩增鉴定，并对PCR产物进行基因测序鉴定，确定 nanKETA基因簇被敲除。
为去除质粒pKD46，挑取阳性单克隆菌落于含卡那霉素(15μg/mL)的3mL LB液体培养基中，42℃培养12小时，将菌液涂布于含卡那霉素(15μg/mL)的LB固体培养基平板上，37℃培养过夜。利用分别含氨苄青霉素(100μg/mL)、卡那霉素(15μg/mL)的LB固体培养基平板对长出的单克隆菌落进行抗性筛选，只在含卡那霉素(15μg/mL)的LB固体培养基平板上生长，而在含氨苄青霉素(100μg/mL)的LB固体培养基平板上不生长的菌落证明其质粒pKD46已丢失。获得缺失nanKETA基因簇后的大肠杆菌菌株为W3110 ΔnanKETA::Kan，该菌株阻断了唾液酸合成过程中的旁路途经。
LB固体培养基配方如下：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，琼脂粉15g/L，加ddH 2O配制而成。
LB液体培养基的配方如下：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，加ddH 2O配制而成。
实施例2、唾液酸修饰N-糖基化重组蛋白表达载体的构建
(1)N-糖基化机制的构建。具体步骤如下：
利用常规的基因重组技术，将编码糖基转移酶lsgCDEF基因簇(GenBank：M94855.1)、糖链合成起始酶WecA基因(Gene ID：948789)、寡糖翻转酶pglK基因(Gene ID:905421)及寡糖转移酶pglB(Gene ID:905417)的基因构建至载体pACYC184上，并由阿拉伯糖启动子(Arabinose promoter，简写为Ara)调控以上四种基因的表达，获得表达N-糖基化体系的载体pC15-Ara-pglB-WecA-pglK-lsgCDEF，该载体图见图1，其序列见SEQ ID NO.3。
(2)唾液酸合成及转移途径的构建。具体步骤如下：
本发明选用重组人纤维连接蛋白Ⅲ型结构域(Fn3)作为受体蛋白，在大肠杆菌质周腔内进行唾液酸修饰N-糖基化重组蛋白研究。重组蛋白Fn3基因在5ˊ端携带编码FLAG标签(氨基酸序列为：DYKD，D代表天冬氨酸残基，Y代表酪氨酸残基，K代表赖氨酸残基)序列，便于Western Blotting检测；在3ˊ端携带编码pglB寡糖转移酶识别位点DQNAT(D代表天冬氨酸残基，Q代表谷氨酰胺残基，N代表天冬酰胺残基，A代表丙氨酸残基，T代表苏氨酸残基)序列，并在其下游引入编码6个组氨酸标签序列。组氨酸标签用于重组蛋白分离纯化。 利用常规的基因重组技术，将重组蛋白Fn3基因(SEQ ID NO.3)与唾液酸合成相关酶neuBCA基因簇(GenBank:AF400048.1)、弧菌发光菌JT-ISH-224(Vibrionaceae Photobacterium sp.JT-ISH-224)来源的α-2,6唾液酸转移酶Δ16psp2,6ST基因(GenBank：AB293985.1)及来源于空肠弯曲杆菌Pgl基因簇(GenBank:Y11648.1)上游的332bp的调控序列(简称为P,SEQ ID NO.4)克隆至载体pIG6，获得表达重组蛋白Fn3、唾液酸合成与转移相关酶的载体pIG6-Fn3-P-Δ16psp2,6ST-neuBCA，该载体图见图2，其序列见SEQ ID NO.5。另外，为了添加对照组，将重组蛋白Fn3基因单独克隆至载体pIG6，获得表达重组蛋白Fn3的载体pIG6-Fn3，该载体图见图3，其序列见SEQ ID NO.6。
实施例3、大肠杆菌体内生产唾液酸修饰的N-糖基化重组蛋白及纯化。
将载体pC15-Ara-pglB-WecA-pglK-lsgCDEF和pIG6-Fn3-P-Δ16psp2,6ST-ne-uBCA共同转化进入重组大肠杆菌菌株W3110 ΔnanKETA::Kan，获得携带唾液酸修饰N-糖基化重组蛋白表达载体的重组大肠杆菌菌株。
将转化子接种至含有卡那霉素(15μg/mL)、氨苄青霉素(100μg/mL)、氯霉素(34μg/mL)的LB固体培养基平板上，37℃过夜培养12小时。筛选出单克隆后将其接种到含有氨苄青霉素(100μg/mL)和氯霉素(34μg/mL)的3mL LB液体培养基中，220rpm、37℃过夜培养。次日，以1:100的比例将菌液接种到含有氨苄青霉素(100μg/mL)、氯霉素(34μg/mL)的500mL自动诱导培养基中，220rpm、25℃条件下培养40小时，并每隔12小时加入L-阿拉伯糖(200μg/mL)作为诱导剂。4000rpm、4℃离心收集菌体，通过超声破碎、低温高速离心等方法获得包含唾液酸修饰的N-糖基化Fn3重组蛋白的上清液。用平衡缓冲液平衡镍柱10个柱体积，将上述所得上清液平衡后低速上柱。样品上柱完毕后，用20mM咪唑浓度的缓冲液洗脱20个柱体积，接着分别用含有40、60、120、240、500mM咪唑浓度的缓冲液进行梯度洗脱，收集洗脱液，分离纯化的重组蛋白即为唾液酸修饰的N-糖基化Fn3重组蛋白，其中，唾液酸化的寡糖链为Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc。以上纯化后的唾液酸修饰的N-糖基化Fn3重组蛋白采用脱盐柱除盐后，于4℃保存。
同时，培养只携带载体pIG6-Fn3的大肠杆菌菌株W3110 ΔnanKETA::Kan与携带载体pIG6-Fn3和载体pC15-Ara-pglB-WecA-pglK-lsgCDEF的大肠杆菌菌株 W3110 ΔnanKETA::Kan作为对照组，具体步骤如下：
将载体pIG6-Fn3转化进入大肠杆菌W3110 ΔnanKETA::Kan。将转化子接种至含有卡那霉素(15μg/mL)、氨苄青霉素(100μg/mL)的LB固体培养基平板上，37℃过夜培养12小时。筛选出单克隆后将其接种到含有氨苄青霉素(100μg/mL)的3mL LB液体培养基中，220rpm、37℃过夜培养。次日，以1:100的比例将菌液接种到含有氨苄青霉素(100μg/mL)的500mL自动诱导培养基中，220rpm、25℃条件下培养40小时。
将载体pIG6-Fn3和载体pC15-Ara-pglB-WecA-pglK-lsgCDEF共同转化进入大肠杆菌W3110 ΔnanKETA::Kan。将转化子接种至含有卡那霉素(15μg/mL)、氨苄青霉素(100μg/mL)、氯霉素(34μg/mL)的LB固体培养基平板上，37℃过夜培养12小时。筛选出单克隆后将其接种到含有氨苄青霉素(100μg/mL)和氯霉素(34μg/mL)的3mL LB液体培养基中，220rpm、37℃过夜培养。次日，以1:100的比例将菌液接种到含有氨苄青霉素(100μg/mL)、氯霉素(34μg/mL)的500mL自动诱导培养基中，220rpm、25℃条件下培养40小时，并每隔12小时加入L-阿拉伯糖(200μg/mL)作为诱导剂。
运用与以上获得唾液酸修饰的N-糖基化Fn3重组蛋白的相同菌体收集、纯化及脱盐方法，获得未糖基化的Fn3重组蛋白与未唾液酸修饰的N-糖基化Fn3重组蛋白。
LB固体培养基配方如下：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，琼脂粉15g/L，加ddH 2O配制而成。
LB液体培养基的配方如下：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，加ddH 2O配制而成。
自动诱导培养基的配方如下：胰蛋白胨10g/L，酵母提取物5g/L，甘油5g/L，葡萄糖0.5g/L，乳糖2g/L，磷酸氢二钠7.1g/L，磷酸二氢钾6.8g/L，硫酸铵3.3g/L，硫酸钠0.9g/L，七水硫酸镁0.25g/L，加ddH 2O配制而成。
实施例4、大肠杆菌体内生产唾液酸修饰N-糖基化重组蛋白的检测。
利用Western Blotting检测、凝集素印迹检测及质谱检测等方法检测重组蛋白的唾液酸修饰情况。
(1)Western Blotting检测采用Sigma公司生产的抗FLAG M1单克隆抗体作 为一抗、Solarbio公司生产的辣根过氧化物酶标记的羊抗小鼠IgG作为二抗，以纯化并脱盐后的未糖基化的Fn3重组蛋白、未唾液酸修饰的N-糖基化Fn3重组蛋白作为阴性对照，对唾液酸修饰的N-糖基化Fn3重组蛋白进行检测分析。通过对比分子迁移率，确定Fn3重组蛋白的糖基化及唾液酸修饰情况。结果图见图4，Fn3重组蛋白经N-糖基化修饰后分子量增大，经唾液酸化N-糖基化修饰后分子量进一步增大。
(2)凝集素印迹检测分别采用EY Laboratories公司生产的特异识别Gal-β-1,4-GlcNAc的凝集素ECA(Catalog Number：H-5901-1)、特异识别Neu5Ac-ɑ-2,6-Gal的凝集素SNA-Ⅰ(Catalog Number：H-6802-1)，以纯化并脱盐后的未糖基化的Fn3重组蛋白、未唾液酸修饰的N-糖基化Fn3重组蛋白作为阴性对照，对唾液酸修饰的N-糖基化Fn3重组蛋白进行检测分析。结果图见图5，A图为EY Laboratories公司生产的特异识别Gal-β-1,4-GlcNAc的凝集素ECA(Catalog Number：H-5901-1)凝集素印记检测结果，未糖基化的Fn3重组蛋白及唾液酸修饰的N-糖基化Fn3重组蛋白都没有特异性条带出现，而未唾液酸修饰的N-糖基化Fn3重组蛋白检测到特异性条带，证明Fn3重组蛋白被N-糖基化修饰；B图为EY Laboratories公司生产的特异识别Neu5Ac-ɑ-2,6-Gal的凝集素SNA-Ⅰ(Catalog Number：H-6802-1)凝集素印迹检测结果，未糖基化的Fn3重组蛋白及未唾液酸修饰的N-糖基化Fn3重组蛋白都没有特异性条带出现，而唾液酸修饰的N-糖基化Fn3重组蛋白检测到特异性条带，证明Fn3重组蛋白被唾液酸化N-糖基化修饰。
(3)唾液酸修饰N-糖基化Fn3重组蛋白糖链组成分析。
检测方法：取实施例3中获得的纯化并脱盐后的唾液酸修饰的N-糖基化Fn3重组蛋白，加入胰酶Trypsin(promega V5280)、Glu-C酶(promega)进行酶解后，采用Thermo Orbitrap Exactive HF型液质联用质谱仪进行定性检测。根据二级谱图By离子及相应修饰质量数匹配情况，确定主要的修饰糖型。
检测器：Thermol Orbitrap Exactive HF mass spectrometer(Thermo fisher)
流动相：A液，0.1％甲酸+99％水；B液，99.9％乙腈+0.1％甲酸；流速:0.6μL/min。结果图见6，糖肽为IGGGGSDQ NATK(分子量为1104.1)，I代表异亮氨酸残基，G代表甘氨酸残基，S代表丝氨酸残基，D代表天冬氨酸残基，Q代 表谷氨酰胺残基，N代表天冬酰胺残基，A代表丙氨酸残基，T代表苏氨酸残基，K代表赖氨酸残基，修饰位点位于天冬酰胺残基。A图可见NeuAc(分子量为292.10)、NeuAc-H2O(分子量为274.09)、NeuAc(1)Hex(1)HexNAc(1)(分子量为657.23)的特异峰；去卷积后，B图可见糖肽IGGGGSDQ NATK带有糖链NeuAc(1)Hex(2)HexNAc(2)(分子量为2125.89)的特异峰，其去除NeuAc、Hex或HexNAc后，分别得到A图中的糖肽IGGGGSDQ NATK带有糖链Hex(2)HexNAc(2)(分子量为1634.79)、糖肽IGGGGSDQ NATK带有糖链Hex(1)HexNAc(1)(分子量为1469.66)及糖肽IGGGGSDQ NATK带有糖链HexNAc(1)(分子量为1307.61)。以上质谱分析结果，结合Western Blotting检测、凝集素印迹检测结果，确定修饰Fn3重组蛋白的末端唾液酸化的寡糖链为Neu5Ac-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc。
根据以上具体实施例可得出结论，本发明所述的一种重组大肠杆菌质周腔内唾液酸修饰N-糖基化重组蛋白方法简单、快速、高效且生产成本低，为进一步生产更加稳定的治疗性蛋白药物或糖疫苗建立技术平台。
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杆菌表达载体、更换具有相同功能的唾液酸糖基转移酶基因、其他受体蛋白表达基因等类似方法，用大肠杆菌生产唾液酸修饰的N-糖基化重组蛋白，均落在本发明权利保护范围之内。

Claims (6)

  1. 一种重组大肠杆菌质周腔内唾液酸修饰N-糖基化重组蛋白方法，其特征在于：利用流感嗜血杆菌(Haemophilus influenzae)来源的糖基转移酶lsgCDEF基因簇、大肠杆菌来源的糖链合成起始酶WecA基因、空肠弯曲杆菌(Campylobacter jejuni)来源的寡糖翻转酶pglK基因、寡糖转移酶pglB基因、唾液酸合成相关酶neuBCA基因簇及弧菌发光菌JT-ISH-224(Vibrionaceae Photobacterium sp.JT-ISH-224)来源的α2,6唾液酸转移酶Δ16psp2,6ST基因，在重组大肠杆菌体内共同构建成唾液酸修饰N-糖基化重组蛋白系统，经自动诱导培养方法，进行大肠杆菌质周腔内唾液酸修饰N-糖基化重组蛋白。
  2. 根据权利要求1所述的方法，其特征在于，唾液酸化的寡糖链为Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc。
  3. 根据权利要求1所述的方法，其特征在于，具体包括以下步骤：
    (1)构建适合唾液酸修饰N-糖基化重组蛋白大肠杆菌菌株；
    (2)利用权利要求1所述的基因构建唾液酸修饰N-糖基化重组蛋白表达载体；
    (3)重组大肠杆菌体内生产唾液酸修饰的N-糖基化重组蛋白；
    (4)步骤(3)中获得的重组蛋白经纯化后即得唾液酸修饰的N-糖基化重组蛋白。
  4. 根据权利要求1所述的方法，其特征在于，步骤(1)具体为：所述适合唾液酸修饰N-糖基化重组蛋白大肠杆菌菌株的构建，基本方法为利用Red同源重组系统，敲除大肠杆菌K12来源的W3110基因组中的nanKETA基因簇，从而阻断唾液酸合成过程中的旁路途经，构建成大肠杆菌菌株的基因型为W3110ΔnanKETA::Kan。
  5. 根据权利要求1所述的方法，其特征在于，步骤(2)具体为：利用糖基转移酶lsgCDEF基因簇、糖链合成起始酶WecA基因、寡糖翻转酶pglK基因、寡糖转移酶pglB基因构建至大肠杆菌表达载体上，获得表达N-糖基化体系的载体；将唾液酸合成相关酶neuBCA基因簇及α-2,6唾液酸转移酶Δ16psp2,6ST基因以及需要唾液酸化N-糖基化修饰的重组蛋白基因，通过基因重组方法，克隆至大肠杆菌表达载体上，共同构建成大肠杆菌体内唾液酸修饰N-糖基化重组蛋白系统。
  6. 根据权利要求1所述的方法，其特征在于，将步骤(2)中构建的唾液酸修饰N-糖基化重组蛋白表达载体转化到步骤(1)获得的大肠杆菌菌株W3110ΔnanKETA::Kan中，经自动诱导培养方法，无需添加外源唾液酸，在该菌株体内生产唾液酸修饰的N-糖基化重组蛋白。
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