WO2023109383A1 - 百日咳抗原重组表达载体及其工程菌和应用 - Google Patents

百日咳抗原重组表达载体及其工程菌和应用 Download PDF

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WO2023109383A1
WO2023109383A1 PCT/CN2022/131069 CN2022131069W WO2023109383A1 WO 2023109383 A1 WO2023109383 A1 WO 2023109383A1 CN 2022131069 W CN2022131069 W CN 2022131069W WO 2023109383 A1 WO2023109383 A1 WO 2023109383A1
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nucleic acid
pertussis
gene
acid fragment
protein
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French (fr)
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葛君
李建强
李玲
张大燕
王玺
石应辉
任苏林
周童
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远大赛威信生命科学(南京)有限公司
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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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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biopharmaceuticals, and relates to a pertussis antigen recombinant expression vector, engineering bacteria and application thereof. Specifically, the present invention relates to a recombinant expression vector for overexpressing pertussis adhesin antigen and simultaneously expressing pertussis toxin antigen, an engineering bacterium containing it and the application of the two in preparing pertussis adhesin and pertussis toxin antigen.
  • pertussis vaccine acellular pertussis vaccine, aPV
  • aPV pertussis vaccine
  • Bacillus pertussis produces various antigens such as pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN) and lipopolysaccharide (LPS) during the growth process.
  • PT pertussis toxin
  • FHA filamentous hemagglutinin
  • PRN pertactin
  • LPS lipopolysaccharide
  • the domestic aPV marketed in China is made by purifying pertussis antigen solution by co-purification method, and absorbing aluminum adjuvant after treatment with detoxification agent. Since the different antigenic components of pertussis were not separated separately, the proportions of various antigens in different production batches of pertussis antigenic liquid were quite different, which affected the stability of vaccine quality.
  • Imported aPV uses more advanced column chromatography to extract antigens such as PT and FHA and detoxify them separately, and then mix these antigen components in a certain proportion to make a vaccine. This process ensures that the quality of the vaccine is consistent between batches, and the purity of the antigen is 90% to 95%, which is suitable for the development of a combined vaccine based on DTacP (Hu Yeqin et al., Establishment of a new purification process for component acellular pertussis vaccine).
  • Chinese patent CN102793915B provides a three-component pertussis vaccine composition, that is, separately purified to obtain PT, PRN and FHA, and then mixed according to a certain ratio.
  • the inventors improved the pertussis antigen production strain by constructing a pertussis antigen recombinant expression vector, thereby providing a recombinant engineering in which the expression of FHA protein is blocked bacteria.
  • the strain can simultaneously express the PRN protein and the PT protein, and the fermentation time is short, and the content of the two proteins produced is also significantly higher than that of the wild strain, which is convenient for separation and purification.
  • the present invention provides a pertussis antigen recombinant expression vector, which comprises pertussis adhesin protein expression cassette, resistance selection gene, filamentous hemagglutinin gene upstream recombinant nucleic acid fragment and downstream recombinant nucleic acid fragment, wherein the pertussis adhesin
  • the protein expression frame is located between the upstream recombinant nucleic acid fragment and the downstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene, and the upstream recombinant nucleic acid fragment and the downstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene can be combined with the filamentous hemagglutinin gene respectively Homologous recombination is performed upstream and downstream of the gene gene.
  • the filamentous hemagglutinin gene is shown in SEQ ID NO:1.
  • the expression vector according to the present invention may be plasmid pUC57, pEASY-Blunt cloning vector or pBBR series vector, preferably plasmid pUC57.
  • the upstream recombined nucleic acid fragment of the filamentous hemagglutinin gene may comprise or be a nucleic acid fragment consisting of the m-1000th consecutive bases in the nucleotide sequence shown in SEQ ID NO: 2, wherein m is a natural number ⁇ 57 .
  • the upstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene is a nucleic acid fragment consisting of the 57th-1000th consecutive bases in the nucleotide sequence shown in SEQ ID NO:2.
  • nucleotide sequence of the pertussis adhesin expression cassette is shown in SEQ ID NO:3.
  • the downstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene according to the present invention may comprise or be a nucleic acid fragment composed of 1-n consecutive bases in the nucleotide sequence shown in SEQ ID NO.4, wherein n is ⁇ 798-1000 Natural number.
  • the recombinant nucleic acid fragment downstream of the filamentous hemagglutinin gene is a nucleic acid fragment consisting of the 1-798th consecutive bases in the nucleotide sequence shown in SEQ ID NO:4.
  • the resistance selection gene according to the present invention can be selected from one or more of kanamycin resistance selection gene, tetracycline resistance selection gene, ampicillin resistance selection gene or chloramphenicol resistance selection gene, preferably Kanamycin resistance selection gene.
  • the nucleotide sequence of the kanamycin resistance selection gene is shown in SEQ ID NO: 5 (Cloning vector pSN-caSAT1, genebank: MT001914.1); the nucleoside sequence of the tetracycline resistance selection gene
  • the acid sequence is shown in SEQ ID NO: 6 (Cloning vector pJC24, Genebank: KC442291.1); the nucleotide sequence of the ampicillin resistance screening gene is shown in SEQ ID NO: 7 (Cloning vector pcDNA3-WSN- PB2, Genebank: MT966986.1);
  • the nucleotide sequence of the chloramphenicol resistance screening gene is shown in SEQ ID NO: 8 (Cloning vector pMYC, Genebank:
  • the present invention also provides a pertussis antigen recombinant expression engineering bacterium, which comprises the recombinant expression vector according to the present invention.
  • the present invention also provides a preparation method of the pertussis antigen recombinant expression engineered bacterium according to the present invention, which comprises transforming wild-type B. pertussis competent cells with the recombinant expression vector according to the present invention.
  • the wild-type B. pertussis is wild-type B. pertussis strain ATCC-BAA-589.
  • said conversion is electrotransformation.
  • the electrotransformation may include linearizing the recombinant expression vector, and electrotransforming wild-type B. pertussis competent cells under the conditions of 2500V and 5ms.
  • the preparation method further includes screening the transformed wild-type B. pertussis competent cells by using the resistance selection gene.
  • the present invention also provides a method for preparing a pertussis antigen, which comprises fermenting the pertussis antigen recombinant expression engineered bacterium according to the present invention.
  • the fermentation comprises the steps of:
  • step (2) inoculate the thalline obtained in step (1) in a shake flask comprising MSS medium (Modified Stainer Scholte medium), and cultivate for 22.5-25 hours;
  • MSS medium Modified Stainer Scholte medium
  • step (3) Inoculate the bacterial solution obtained in step (2) into a fermenter containing MSS medium, and cultivate for 28-30 hours.
  • the pertussis antigen is pertussis adhesin (PRN, whose amino acid sequence is shown in SEQ ID NO: 9) and/or pertussis toxin (PT, whose amino acid sequence is shown in SEQ ID NO: 10).
  • PRN pertussis adhesin
  • PT pertussis toxin
  • the present invention obtains a genetic engineering bacterium of Bacillus pertussis, whose FHA protein gene is knocked out and the copy of the PRN protein gene is increased, so that it can efficiently overexpress the PRN protein antigen and simultaneously express PT protein.
  • the PRN protein antigen and PT protein expressed by the genetically engineered bacteria of B. pertussis provided by the present invention exist in the bacterial body and the supernatant of the fermentation broth respectively, which is more conducive to the subsequent separation and purification of antigens.
  • the pertussis engineering bacterium OEPRN-PT provided by the present invention can simultaneously obtain higher expression levels of PRN protein and PT protein, showing significant advantages.
  • Connor's laboratory prepared two copies of the expression strain of the PRN gene initiated by the FHA promoter, and the final PRN protein concentration was about 186.7 ⁇ g/ml (Loosmore S M, Yacoob R K, Zealey G R, et al ., Hybrid genes over-express pertactin from Bordetella pertussis[J].Vaccine, 1995,13(6):571), and the PRN expression concentration produced by the engineering bacteria OEPRN-PT provided by the present invention is at 418 ⁇ 555 ⁇ g/ml between.
  • the yield of PRN is significantly improved.
  • the PRN protein content of the present invention is significantly improved.
  • the engineering bacteria OEPRN-PT can complete the fermentation and growth in about 28-30 hours, and the time is shortened by more than 25%.
  • the PRN protein expressed by the engineering bacteria OEPRN-PT of the present invention is concentrated in the thalline, and the PT protein is then concentrated in the supernatant of the bacterial liquid, while the two types of proteins produced by the wild strain are all concentrated in the supernatant of the bacterial liquid.
  • the protein produced by the invented engineered bacteria OEPRN-PT is more suitable for subsequent separation and purification.
  • the present invention can simultaneously produce high levels of PRN protein and PT protein through the same strain of pertussis engineered bacteria, and the proteins are easier to separate and purify, which meets the needs of industrialized production of antigens.
  • Figure 1 is a schematic diagram of the plasmid construction of the recombinant vector pUC57-Fhup-Prn-Fhdown-Kan;
  • Figure 2 shows the SDS-PAGE electrophoresis analysis results of enzyme digestion identification after the resistance gene Kan is connected to the downstream of the carrier fragment Fhup-Prn-Fhdown, the resistance gene Kan is about 963bp; among them, M: Marker; lane 1#, lane 2#: Parallel samples carrying the resistance gene Kan;
  • Figure 3 shows the identification of upstream and downstream insertions in recombinant expression vectors through DNA-level verification; where, M: Marker; lanes 1-6: confirmation of upstream insertion sites; lanes 1'-6': confirmation of downstream insertion sites;
  • Figure 4 is an electropherogram of the identification of FHA gene knockout through DNA level verification; wherein, M: Marker; lanes 1-6: FHA gene detection;
  • Figure 5 shows the result of the PRN protein produced by the fermentation of engineering bacteria OEPRN-PT through the QHP chromatography column and eluted with Buffer B;
  • Figure 6 shows the results of HPLC detection of PRN protein purified by QHP chromatography column
  • Figure 7 shows the results of the PT protein produced by engineering bacteria OEPRN-PT fermentation through SP-inspire chromatography column and eluted with Buffer D;
  • Figure 8 shows the results of purification of PT protein produced by wild strain WT fermentation using SP-inspire chromatography column and elution with Buffer D.
  • the recombinant vector pUC57-Fhup-Prn-Kan-Fhdown was constructed according to the schematic diagram of plasmid construction shown in Figure 1 .
  • each fragment was amplified, and then each fragment was spliced by overlapping PCR, which can be used to obtain a double copy of the PRN gene.
  • Table 1 constructs each fragment and its primer required for Fhup-Prn-Kan-Fhdown
  • wild strain WT The source of the wild-type B. pertussis strain (hereinafter referred to as the wild strain WT) used in this example: the wild-type B. pertussis strain ATCC-BAA-589, which was purchased from Beina Biotech.
  • Competent preparation collect the cells prepared in step 2.1, centrifuge at 4°C for 15 minutes, resuspend in 100ml distilled water pre-cooled at 4°C, wash the cells twice, and then centrifuge at 4°C for 15 minutes to collect the cells. Resuspend and wash the cells with an appropriate volume of 10% sterile glycerol, centrifuge at 4°C for 15 minutes, collect the cells, and resuspend the cells with 1ml of 10% sterile glycerol to obtain the wild-type strain ATCC-BAA-589. The cells were stored in a -70°C freezer.
  • Electrotransformation and homologous recombination transfer 2 ⁇ g of linear fragment DNA into the competent cells of the wild-type strain ATCC-BAA-589 prepared in step 2.2 under the conditions of 2500V and 5ms, and spread it on the cells containing card after 24 hours of liquid culture.
  • Namycin accordinging to the resistance gene can be replaced by tetracycline, ampicillin or chloramphenicol, etc.
  • solid medium cultivated for 3-5 days, picked a single clone, for verification.
  • Example 2 Pick the monoclonal prepared in Example 2 into a 1.5ml EP tube containing 10 ⁇ l of MSS, draw 2 ⁇ l as a template for PCR verification, and streak the monoclonal at the same time.
  • the verification result shows that the upstream insertion site and the downstream insertion site are all correct (Fig. 3), and the identified FHA gene is knocked out (Fig. 4), that is, the engineering bacterium OEPRN-PT is constructed successfully (engineering bacterium OEPRN-PT of the present invention
  • the PRN gene was increased by recombination, and the FHA gene was partially knocked out, and the FHA protein was not expressed).
  • the bacterial lawn was scraped from the plate and inoculated in 300ml MSS medium.
  • the measured OD 600 value was 2.12, and there was no bacteria in the microscopic examination.
  • the initial conditions of fermentation were 35°C, 150rpm, ventilation rate 2L/min, and the dissolved oxygen was controlled at 40% during the fermentation process. When the fermentation reaches 29 hours, the culture is terminated according to the recovery of dissolved oxygen, and the bacteria precipitate is collected.
  • the concentration of PRN protein produced by engineering bacteria OEPRN-PT is between 418-555 ⁇ g/ml, PRN protein is not detected when wild strains are harvested, and the concentration of PT protein produced by engineering bacteria OEPRN-PT is about 6.23-8.98 ⁇ g/ml , compared with the 3.5 ⁇ g/ml PT protein concentration of the wild strain, there was a significant increase.
  • Bacteria-breaking buffer 10mM Tris-HCl, 150mM NaCl, 1mM PMSF (phenylmethylsulfonyl fluoride)
  • Reconstitution buffer 35mM NaCl, 25mM Tris-HCl
  • Buffer A 50mM Tris-HCl
  • Buffer B 50mM Tris-HCl, 1M NaCl
  • Example 3 Take the engineering bacteria OEPRN-PT fermented liquid obtained in Example 3 and centrifuge at 4°C for 30 minutes to obtain a bacterial cell precipitate, then redissolve the bacterial cell pellet in 1000ml of bacteriostasis buffer, incubate at 60°C for 1 hour, and then centrifuge at 4°C After 30 minutes, the supernatant was collected, and a total of 950ml of the extract was collected. Add ammonium sulfate aqueous solution to the extract solution for precipitation for 2 hours, let stand at room temperature for 1 hour, and then centrifuge at 4°C for 50 minutes to collect the precipitate.
  • the precipitate obtained by salting out with ammonium sulfate was redissolved with 600ml of reconstitution buffer, centrifuged at 4°C for 50min after overnight redissolution, and a total of 500ml of supernatant was collected.
  • the supernatant was ultrafiltered through a Pellicon XL PXB10C50 ultrafiltration membrane and replaced with 50mM Tris-HCl to obtain 260ml of ultrafiltrate.
  • the QHP chromatography column was washed and regenerated with 0.5M NaOH aqueous solution and Buffer B in sequence, and then 5 column volumes were equilibrated with Buffer A at a flow rate of 2ml/min; the ultrafiltrate obtained in step 4.2.1 was loaded on the QHP column at a flow rate of It is 2ml/min. Continue to wash with Buffer A for 5 column volumes after the breakthrough.
  • the results of BCA detection are shown in Table 5.
  • the concentration of PRN protein changes with the collection time, and the highest peak can reach 655.5223 ( ⁇ g/ml), and it is basically higher than 194 ⁇ g/ml at each stage.
  • the results of HPLC showed (see FIG. 6 ) that the concentration of PRN could reach 97.52%.
  • Buffer C 50mM PB (phosphate buffer) + 2M urea
  • Buffer D 50mM PB+1M NaCl+2M urea
  • the SP-inspire chromatography column was washed and regenerated with 0.5M NaOH aqueous solution and Buffer D in sequence, and then equilibrated to the baseline with Buffer C at a flow rate of 2ml/min. Take 145ml of the ultrafiltrate and load it on the SP-inspire chromatography column at a flow rate of 2ml/min. Continue to wash with Buffer C to the baseline after the flow-through, and wash with 8% (volume ratio) Buffer D.
  • Buffer D elutes the protein, collects the elution peak, and the PT protein in this elution peak is named SP-20%B.
  • the purification results of the PT protein produced by the engineered strain OEPRN-PT and the PT protein produced by the wild strain WT are shown in Figure 7 and Figure 8, respectively. It can be clearly seen from Figures 7 and 8 that the PT protein was obtained by purifying the engineering bacteria OEPRN-PT by ion chromatography, and there was no FHA protein contamination in the eluted sample, and the PT content was relatively high. However, using ion chromatography to purify the WT strain to obtain PT, the eluted sample will be contaminated by FHA protein, and the PT content is also low.
  • the present invention provides a pertussis engineered bacterium OEPRN-PT overexpressing PRN protein by constructing a recombinant expression vector for blocking the expression of FHA protein and introducing it into a wild-type pertussis strain, which can simultaneously obtain high expression levels PRN protein and PT protein.
  • the fermentation time of this strain is short, and the content of PRN protein and PT protein is obviously higher than that of the wild strain.
  • the two proteins are respectively produced in the bacterial cell precipitate and the bacterial liquid supernatant, which is convenient for separation and purification, and meets the needs of industrial production.

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Abstract

提供了百日咳抗原重组表达载体及其工程菌和应用，所述百日咳抗原重组表达载体包含百日咳黏附素蛋白表达框、抗性筛选基因、丝状血凝素基因上游重组核酸片段和下游重组核酸片段，其中所述百日咳黏附素蛋白表达框位于所述丝状血凝素基因上游重组核酸片段和下游核酸重组片段之间，且所述丝状血凝素基因上游重组核酸片段和下游重组核酸片段能够分别与丝状血凝素基因的上游和下游进行同源重组。所述重组表达载体中的FHA蛋白基因被敲除并增加了PRN蛋白基因拷贝，使得所述百日咳杆菌基因工程菌能够高效地过表达PRN蛋白抗原以及PT蛋白，而且表达的PRN蛋白抗原和PT蛋白分别存在于菌体和上清中，有利于后续的抗原分离纯化。

Description

百日咳抗原重组表达载体及其工程菌和应用 技术领域
本发明属于生物制药领域，涉及一种百日咳抗原重组表达载体及其工程菌和应用。具体地，本发明涉及一种过表达百日咳黏附素抗原，同时表达百日咳毒素抗原的重组表达载体以及包含它的工程菌和两者在制备百日咳黏附素和百日咳毒素抗原中的应用。
背景技术
百日咳(whooping cough)是由百日咳杆菌(Bordetella pertussis)引起的一种烈性呼吸道传染病，对婴幼儿危害极大。临床特征为咳嗽逐渐加重，呈典型的阵发性、痉挛性咳嗽，咳嗽终末出现深长的鸡啼样吸气性吼声，病程长达2～3个月，故有百日咳之称。
目前针对百日咳的预防主要采用注射疫苗，初代全细胞百日咳疫苗(whole cell pertussis vaccine，wPV)使该疾病得到了良好的控制，但因接种会引起较高的不良反应，逐渐被更安全的无细胞百日咳疫苗(acellular pertussis vaccine，aPV)取代。但自2010年以来，成人和青少年百日咳发病率又有所上升，分析原因主要是因为aPV效力快速下降、疫苗选择压力下的菌株变异等。因此，对百日咳疫苗的持续改进是应对百日咳传播的有效方法。
百日咳杆菌在生长过程中会产生百日咳毒素(Pertussis Toxin，PT)、丝状血凝素(Filamentous Haemagglutinin，FHA)、百日咳黏附素(Pertactin，PRN)和脂多糖(Lipopolysaccharide，LPS)等多种抗原。国内上市的国产aPV都是利用共纯化的方法纯化出百日咳抗原溶液，经脱毒剂处理后吸附铝佐剂而成。由于没有对百日咳的不同抗原成分单独进行分离，不同生产批次的百日咳抗原液中各种抗原的比例差异较大，影响了疫苗质量的稳定性。
进口的aPV是利用技术更先进的柱层析法，将PT和FHA等抗原单独提取出来分别脱毒，再将这些抗原组分按一定的比例混合制成疫苗。该工艺保证了疫苗的批间质量一致，而且抗原纯度为90％～95％，适合于开发以DTacP为基础的联合疫苗(胡业勤等，组分无细胞百日咳疫苗纯化新工艺的建立)。例如，中国专利CN102793915B提供了三种组分的百日咳疫苗组合物，即分别纯化以获得PT、PRN和FHA，再将其按照一定比例混合。资料显示，FHA抗原易降解，为纯化效果的判定带来干扰。多篇文献(如 吴腾捷，张斌，张青等，无细胞百日咳疫苗纯化新工艺的建立[J]，中国生物制品学杂志，26(01)：5-8)都曾报道将FHA蛋白与PT蛋白分离的纯化方法，而单独提取PT蛋白的步骤复杂。
上述方法都存在着抗原蛋白表达量少、分离纯化繁琐的问题。因此，亟需开发一种能够高效表达抗原组分，且分离纯化程序简化的百日咳抗原制备方法。
发明内容
为了更简便地获得单独组分的PRN蛋白及PT蛋白，本发明人通过构建一种百日咳抗原重组表达载体对百日咳抗原生产菌株进行了改进，从而提供了一种FHA蛋白表达被阻断的重组工程菌。该菌株可同时表达PRN蛋白和PT蛋白，且发酵时间短，产生的这两种蛋白含量也显著高于野生菌株，便于分离纯化。
一方面，本发明提供了一种百日咳抗原重组表达载体，其包含百日咳黏附素蛋白表达框、抗性筛选基因、丝状血凝素基因上游重组核酸片段和下游重组核酸片段，其中所述百日咳黏附素蛋白表达框位于所述丝状血凝素基因上游重组核酸片段和下游重组核酸片段之间，且所述丝状血凝素基因上游重组核酸片段和下游重组核酸片段能够分别与丝状血凝素基因的上游和下游进行同源重组。其中，所述丝状血凝素基因如SEQ ID NO：1所示。
根据本发明的表达载体可以为质粒pUC57、pEASY-Blunt克隆载体或pBBR系列载体，优选为质粒pUC57。
根据本发明的丝状血凝素基因上游重组核酸片段可以包含或为SEQ ID NO：2所示核苷酸序列中第m-1000位连续碱基组成的核酸片段，其中m为≤57的自然数。优选地，所述丝状血凝素基因上游重组核酸片段为SEQ ID NO：2所示核苷序列中第57-1000位连续碱基组成的核酸片段。
根据本发明的一个实施方案，所述百日咳黏附素表达框的核苷酸序列如SEQ ID NO：3所示。
根据本发明的丝状血凝素基因下游重组核酸片段可以包含或为SEQ ID NO.4所示核苷序列中第1-n位连续碱基组成的核酸片段，其中n为≥798-1000的自然数。优选地，所述丝状血凝素基因下游重组核酸片段为SEQ ID NO：4所示核苷序列中第1-798位连续碱基组成的核酸片段。
根据本发明的抗性筛选基因可以选自卡那霉素抗性筛选基因、四环素抗性筛选基因、氨苄青霉素抗性筛选基因或氯霉素抗性筛选基因中的一种或多种，优选为卡那霉素抗性筛选基因。具体地，所述卡那霉素抗性筛选基因的核苷酸序列如SEQ ID NO：5所示(Cloning vector pSN-caSAT1， genebank:MT001914.1)；所述四环素抗性筛选基因的核苷酸序列如SEQ ID NO：6所示(Cloning vector pJC24，Genebank:KC442291.1)；所述氨苄青霉素抗性筛选基因的核苷酸序列如SEQ ID NO：7所示(Cloning vector pcDNA3-WSN-PB2，Genebank:MT966986.1)；所述氯霉素抗性筛选基因的核苷酸序列如SEQ ID NO：8所示(Cloning vector pMYC，Genebank:MT572316.1)。
另一方面，本发明还提供了一种百日咳抗原重组表达工程菌，其包含根据本发明的重组表达载体。
再一方面，本发明还提供了一种根据本发明的百日咳抗原重组表达工程菌的制备方法，其包括采用根据本发明的重组表达载体转化野生型百日咳杆菌感受态细胞。
根据本发明的另一个实施方案，所述野生型百日咳杆菌为野生型百日咳杆菌菌株ATCC-BAA-589。
根据本发明的另一个实施方案，所述转化为电转化。具体地，所述电转化可以包括将所述重组表达载体线性化，在2500V和5ms的条件下电转化野生型百日咳杆菌感受态细胞。
根据本发明的另一个实施方案，所述制备方法还包括利用所述抗性筛选基因筛选经转化的野生型百日咳杆菌感受态细胞。
又一方面，本发明还提供了一种百日咳抗原的制备方法，其包括采用根据本发明的百日咳抗原重组表达工程菌进行发酵。
根据本发明的另一个实施方案，所述发酵包括以下步骤：
(1)将根据本发明的百日咳抗原重组表达工程菌接种于包含包姜氏血琼脂培养基的平皿中，培养40-72小时，优选48小时；
(2)将步骤(1)获得的菌体接种于包含MSS培养基(Modified Stainer Scholte培养基)的摇瓶中，培养22.5-25小时；
(3)将步骤(2)获得的菌液接种于包含MSS培养基的发酵罐中，培养28-30小时。
具体地，所述百日咳抗原为百日咳黏附素(PRN，其氨基酸序列如SEQ ID NO：9所示)和/或百日咳毒素(PT，其氨基酸序列如SEQ ID NO：10所示)。
SEQ ID NO：9：
Figure PCTCN2022131069-appb-000001
Figure PCTCN2022131069-appb-000002
SEQ ID NO： 10：
Figure PCTCN2022131069-appb-000003
Figure PCTCN2022131069-appb-000004
本发明通过构建和使用百日咳抗原重组表达载体，获得了一种百日咳杆菌基因工程菌，其FHA蛋白基因被敲除并增加了PRN蛋白基因拷贝，使得其能够高效地过表达PRN蛋白抗原并且同时表达PT蛋白。此外，本发明所提供的百日咳杆菌基因工程菌所表达的PRN蛋白抗原和PT蛋白分别存在于菌体和发酵液上清中，更有利于后续的抗原分离纯化。
与一些现有报道相比，本发明所提供的百日咳工程菌OEPRN-PT可以同时获得更高表达量的PRN蛋白和PT蛋白，显示出显著的优势。例如，康纳实验室制备了两个拷贝的以FHA启动子启动的PRN基因的表达菌株，其最终PRN蛋白浓度为186.7μg/ml左右(Loosmore S M,Yacoob R K,Zealey G R,et al.,Hybrid genes over-express pertactin from Bordetella pertussis[J].Vaccine,1995,13(6):571)，而本发明提供的工程菌OEPRN-PT所产生的PRN表达量浓度在418～555μg/ml之间。
本发明通过以下实施例证明了以下有益效果：
1.通过敲除FHA基因，阻断FHA的表达，解决了百日咳菌株在抗原纯化过程中，FHA蛋白对PT蛋白的影响，对比野生株，提高了PT蛋白含量，同时纯化的PT蛋白中的杂蛋白少。
2.通过增加PRN拷贝数，显著提高了PRN产量，对比现有报道中的双拷贝菌株的PRN含量，本发明的PRN蛋白含量显著提高。
3.与野生型相比，工程菌OEPRN-PT在28～30小时左右即可完成发酵生长，时间缩短了25％以上。
4.本发明的工程菌OEPRN-PT表达的PRN蛋白集中在菌体中，PT蛋白则集中在菌液上清中，而野生株产生的这两类蛋白均集中在菌液上清中，本发明的工程菌OEPRN-PT所产生的蛋白更适合后续的分离纯化。
综上所述，本发明通过同一株百日咳工程菌可以同时产生高含量的PRN蛋白和PT蛋白，并且蛋白更易于分离和纯化，满足了抗原工业化生产的需要。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为重组载体pUC57-Fhup-Prn-Fhdown-Kan的质粒构建示意图；
图2显示将抗性基因Kan连接到载体片段Fhup-Prn-Fhdown下游后， 酶切鉴定的SDS-PAGE电泳分析结果，抗性基因Kan约为963bp；其中，M：Marker；泳道1#、泳道2#：携带抗性基因Kan的平行样品；
图3显示通过DNA水平验证来鉴定重组表达载体中的上游插入和下游插入；其中，M：Marker；泳道1-6：上游插入位点确认；泳道1’-6’：下游插入位点确认；
图4为通过DNA水平验证来鉴定FHA基因敲除的电泳图；其中，M：Marker；泳道1-6：FHA基因检测；
图5显示由工程菌OEPRN-PT发酵产生的PRN蛋白经QHP层析柱并用Buffer B洗脱纯化的结果；
图6显示经QHP层析柱纯化后的PRN蛋白使用HPLC检测的结果；
图7显示由工程菌OEPRN-PT发酵产生的PT蛋白经SP-inspire层析柱并用Buffer D洗脱纯化的结果；
图8显示由野生株WT发酵产生的PT蛋白使用SP-inspire层析柱并用Buffer D洗脱纯化的结果。
实施发明的最佳方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着这样的描述更为清楚。
本发明所述的实验方法，若无特殊说明，均为常规方法；所述的生物材料，若无特殊说明，均可从商业途径获得。
实施例1重组载体pUC57-Fhup-Prn-Kan-Fhdown的构建
依照图1所示质粒构建示意图构建重组载体pUC57-Fhup-Prn-Kan-Fhdown。
1.1构建载体片段Fhup-Prn-Kan-Fhdown
构建载体片段Fhup-Prn-Kan-Fhdown所需的各片段序列和其引物如表1所示，先各自扩增，然后将各片段通过overlap PCR拼接，可用于获得双拷贝PRN基因。
1.2构建重组载体pUC57-Fhup-Prn-Kan-Fhdown
采用内切酶HindIII、BamHI酶切载体pUC57，酶切体系：200μl(购自NEB)，10X 2.1buffer 10μl，质粒15μg，HindIII和BamHI各4μl，H 2O补至200μl，37℃酶切2h，然后用1％的琼脂糖电泳检测，并且回收目的条带。使用重组酶(购自诺唯赞)将酶切后的pUC57与步骤1.1制备的Fhup-Prn-Kan-Fhdown连接，将构建好的重组载体pUC57-Fhup-Prn-Kan-Fhdown转化到大肠杆菌DH5a感受态(购自北京全式 金生物)中，并通过菌落PCR检测，引物分别为pt-Kan-F和pt-Kan-CZR(见表2)。结果(见图2)表明，Kan抗性片段连接正确，约为963bp，说明重组表达载体pUC57-Fhup-Prn-Kan-Fhdown构建成功。最后，将菌落PCR鉴定正确的阳性克隆送到上海生工进行测序，并将核酸序列与目的基因序列完全一致的克隆保存于-80℃。
表1构建Fhup-Prn-Kan-Fhdown所需的各片段和其引物
Figure PCTCN2022131069-appb-000005
表2菌落PCR引物
Figure PCTCN2022131069-appb-000006
实施例2百日咳杆菌基因工程菌的制备
本实施例采用的野生型百日咳杆菌菌株(以下简称野生株WT)的来源：百日咳杆菌野生型菌株ATCC-BAA-589，购自北纳生物。
2.1培养菌体：活化预备成为感受态的野生型菌株ATCC-BAA-589甘油菌，取适量的野生型菌株ATCC-BAA-589菌液涂布于包姜氏培养基平板上，置于37℃培养箱中培养48h取出，使用取样棒将表面的菌斑刮下，接种于100ml MSS培养基中，培养24h后，测定其OD 600值，控制OD 600处于1-3之间。
2.2制备感受态：收集步骤2.1制备的菌体，4℃下离心15min，用4℃预冷的100ml蒸馏水重悬，清洗菌体两遍，然后4℃下离心15min，收集菌体。用适量体积的10％的无菌甘油重悬清洗菌体，4℃下离心15min，收集菌体，用1ml 10％的无菌甘油重新悬起菌体，得到野生型菌株ATCC-BAA-589感受态细胞，将其保存至-70℃冰箱中。
2.3制备转化片段：使用HindIII内切酶将实施例1制备的重组表达载体pUC57-Fhup-Prn-Fhdown-Kan在Fhup片段的上游进行线性化。
2.4电转化和同源重组：将2μg的线性片段DNA在2500V、5ms的条件下转入步骤2.2制备好的野生型菌株ATCC-BAA-589感受态细胞中，液体培养24h后涂布于含有卡那霉素(根据抗性基因可相应地替换为四环素、氨苄青霉素或氯霉素等)的固体培养基中，培养3-5天，挑取单克隆，进行验证。
实施例3百日咳杆菌基因工程菌的鉴定
3.1 DNA水平验证
3.1.1实验步骤
挑取实施例2制备的单克隆于含10μl的MSS的1.5ml EP管中，吸取2μl做模板进行PCR验证，同时划线单克隆。
表3 DNA验证采用的引物
Figure PCTCN2022131069-appb-000007
3.1.2实验结果
验证结果说明，上游插入位点和下游插入位点均正确(图3)，以及经鉴定FHA基因被敲除(图4)，即工程菌OEPRN-PT构建成功(本发明的工程菌OEPRN-PT通过重组增加了PRN基因，且FHA基因被部分敲除，FHA蛋白不表达)。
3.2生长发酵情况验证
3.2.1实验步骤
取步骤3.1中经验证正确的工程菌OEPRN-PT 100μL接种于两块(平行操作)具有包姜氏血琼脂培养基的平皿中，37℃下培养约48h，镜检无染菌现象。从平皿刮取菌苔，接种于300ml MSS培养基中，测得OD 600值为2.12，镜检无染菌。取培养后的菌液300ml，接种于装有3L MSS培养基的发酵罐中。发酵初始条件为35℃、150rpm、通气量2L/min，发酵过程控制溶氧40％。当发酵至29h时，根据溶氧回升情况终止培养，收取菌体沉淀。
3.2.2实验结果
将工程菌OEPRN-PT与野生菌WT分别进行发酵，并对比分析结果(见表4)，其中野生菌WT的生长时间则为36-42小时左右，工程菌OEPRN-PT在发酵罐阶段的生长时间在28-30小时左右，缩短了25％以上。工程菌OEPRN-PT产生的PRN蛋白的浓度在418-555μg/ml之间，野生株收菌时 PRN蛋白没有检测到，工程菌OEPRN-PT产生的PT蛋白的浓度为6.23-8.98μg/ml左右，相比野生株的3.5μg/ml PT蛋白浓度有显著提高。
表4百日咳杆菌发酵过程各级培养参数和培养结果对比
Figure PCTCN2022131069-appb-000008
上述结果说明，本发明得到的工程菌OEPRN-PT大大缩短了发酵时间，发酵终止时发酵液中有更高的目的蛋白的表达量，其中PRN蛋白具有很高的表达量，表达的PT蛋白浓度也较野生菌WT更高。
实施例4工程菌OEPRN-PT获得的PRN蛋白的纯化
4.1实验材料
破菌缓冲液：10mM Tris-HCl，150mM NaCl，1mM PMSF(苯甲基磺酰氟)
复溶缓冲液：35mM NaCl，25mM Tris-HCl
Buffer A：50mM Tris-HCl
Buffer B：50mM Tris-HCl，1M NaCl
4.2实验步骤
4.2.1 PRN蛋白的粗提纯
取实施例3获得的工程菌OEPRN-PT发酵液在4℃下离心30min，得到菌体沉淀，再将菌体沉淀复溶于1000ml破菌缓冲液中，60℃下孵育1h后4℃下离心30min，收集上清，共收集浸提液950ml。浸提液加入硫酸铵水溶液进行沉淀2h，室温下静置1h后4℃下离心50min，收集沉淀。
采用600ml的复溶缓冲液复溶经硫酸铵盐析得到的沉淀物，过夜复溶后4℃下离心50min，收集上清共500ml。将上清通过Pellicon XL PXB10C50超滤膜包超滤并换液至50mM Tris-HCl中，获得超滤液260ml。
4.2.2 PRN蛋白的精制
QHP层析柱依次用0.5M的NaOH水溶液和Buffer B清洗再生，接着用Buffer A平衡5个柱体积，流速为2ml/min；取步骤4.2.1获得的超滤液上样于QHP柱，流速为2ml/min。流穿结束后继续用Buffer A冲洗5个柱体积。
然后使用13％(体积比)Buffer B洗脱，收集洗脱峰，PRN蛋白在此洗脱峰中，命名为QHP-13％B(图5)。洗脱样品中几乎没有杂蛋白。分别使用BCA法和HPLC对收集到的蛋白进行检测。
4.3实验结果
BCA检测结果见表5，PRN蛋白的浓度随收集时间变化，最高时可达655.5223(μg/ml)，各阶段基本都高于194μg/ml。HPLC结果显示(见图6)，PRN的浓度可达97.52％。
表5 BCA测定的纯化后的PRN浓度
蛋白名称 蛋白浓度(μg/ml)
管1 194.5504
管2 655.5223
管3 249.784
管4 194.1351
实施例5工程菌OEPRN-PT获得的PT蛋白的纯化
5.1实验步骤
试验材料：
Buffer C：50mM PB(磷酸缓冲液)+2M尿素
Buffer D：50mM PB+1M NaCl+2M尿素
5.1.1 PT蛋白的粗提纯
取实施例3获得的工程菌OEPRN-PT发酵菌液，4℃下30min离心，保留上清，将过滤后的上清用10kDa的膜包浓缩后通过滤膜除去杂质，再超滤换液至50mM PB中，共获得超滤液300ml。
5.1.2 PT蛋白的精制
SP-inspire层析柱依次用0.5M的NaOH水溶液和Buffer D清洗再生，接着用Buffer C平衡至基线，流速为2ml/min。取145ml的超滤液上样于SP-inspire层析柱，流速为2ml/min。流穿结束后继续用Buffer C冲洗至基线，8％(体积比)Buffer D洗杂。
20％(体积比)Buffer D洗脱蛋白，收集洗脱峰，PT蛋白在此洗脱峰中，命名为SP-20％B。
野生株WT获得的PT蛋白的纯化：
取野生株WT发酵后的发酵菌液，纯化步骤同上述5.1.1和5.1.2的操作。
5.2实验结果
工程菌OEPRN-PT产生的PT蛋白、野生株WT产生的PT蛋白的纯化结果分别见图7和图8。从图7和8中可明显看出，使用离子层析方法纯化工程菌OEPRN-PT得到PT蛋白，洗脱样品中没有FHA蛋白污染，PT含量较高。而使用离子层析方法纯化WT菌株得到PT，洗脱样品中会有FHA蛋白污染，PT含量也较低。
综上所述，本发明通过构建用于阻断FHA蛋白表达的重组表达载体并将其导入野生型百日咳菌株，提供了PRN蛋白过表达的百日咳工程菌OEPRN-PT，其可以同时获得高表达量的PRN蛋白和PT蛋白。该菌株的发酵时间短，PRN蛋白和PT蛋白含量明显高于野生株。而且，两种蛋白分别产生在菌体沉淀和菌液上清中，便于分离，方便纯化，满足工业化生产的需要。
尽管以上已经对本发明作了详细描述，但是本领域技术人员应理解，在不偏离本发明的精神和范围的前提下，可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。
以上所述仅是本发明的优选实施例而已，并非对本发明做出的任何形式的限制，虽然本发明已经以如上的优选实施例进行了揭示，然而并非用其限定本发明。任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，可利用上述揭示的技术内容作出些许更改或修饰而形成等同变化的等效实施例。但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作出的任何修改或等同变化与修饰均仍属于本发明技术方案的范围内。

Claims (11)

  1. 一种百日咳抗原重组表达载体，其包含百日咳黏附素蛋白表达框、抗性筛选基因、丝状血凝素基因上游重组核酸片段和下游重组核酸片段，其中所述百日咳黏附素蛋白表达框位于所述丝状血凝素基因上游重组核酸片段和下游重组核酸片段之间，且所述丝状血凝素基因上游重组核酸片段和下游重组核酸片段能够分别与丝状血凝素基因的上游和下游进行同源重组。
  2. 根据权利要求1所述的重组表达载体，其中，所述表达载体为质粒pUC57、pEASY-Blunt克隆载体或pBBR系列载体，优选为质粒pUC57。
  3. 根据权利要求1或2所述的重组表达载体，其中，所述丝状血凝素基因上游重组核酸片段包含或为SEQ ID NO：2所示核苷酸序列中第m-1000位连续碱基组成的核酸片段，其中m为≤57的自然数；优选地，所述丝状血凝素基因上游重组核酸片段为SEQ ID NO：2所示核苷序列中第57-1000位连续碱基组成的核酸片段；
    优选地，所述丝状血凝素基因下游重组核酸片段包含或为SEQ ID NO：4所示核苷酸序列中第1-n位连续碱基组成的核酸片段，其中n为≥798-1000的自然数；更优选地，所述丝状血凝素基因下游重组核酸片段为SEQ ID NO：4所示核苷酸序列中第1-798位连续碱基组成的核酸片段。
  4. 根据权利要求1至3中任一项所述的重组表达载体，其中，所述百日咳黏附素表达框的核苷酸序列如SEQ ID NO：3所示。
  5. 根据权利要求1至4中任一项所述的重组表达载体，其中，所述抗性筛选基因选自卡那霉素抗性筛选基因、四环素抗性筛选基因、氨苄青霉素抗性筛选基因或氯霉素抗性筛选基因中的一种或多种，优选为卡那霉素抗性筛选基因；
    优选地，所述卡那霉素抗性筛选基因的核苷酸序列如SEQ ID NO：5所示；所述四环素抗性筛选基因的核苷酸序列如SEQ ID NO：6所示；所述氨苄青霉素抗性筛选基因的核苷酸序列如SEQ ID NO：7所示；所述氯霉素抗性筛选基因的核苷酸序列如SEQ ID NO：8所示。
  6. 一种百日咳抗原重组表达工程菌，其包含根据权利要求1至5中任一项所述的重组表达载体。
  7. 根据权利要求6所述的百日咳抗原重组表达工程菌的制备方法，其包括采用根据权利要求1至5中任一项所述的重组表达载体转化野生型百日咳杆菌感受态细胞；
    优选地，所述野生型百日咳杆菌为野生型百日咳杆菌菌株ATCC-BAA-589；
    优选地，所述转化为电转化；更优选地，所述电转化包括将根据权利要求1至5中任一项所述的重组表达载体线性化，在2500V和5ms的条件下电转化野生型百日咳杆菌感受态细胞。
  8. 根据权利要求7所述的制备方法，其还包括利用所述抗性筛选基因筛选经转化的野生型百日咳杆菌感受态细胞。
  9. 一种百日咳抗原的制备方法，其包括采用根据权利要求6所述的百日咳抗原重组表达工程菌进行发酵。
  10. 根据权利要求9所述的制备方法，其中，所述发酵包括以下步骤：
    (1)将根据权利要求6所述的百日咳抗原重组表达工程菌接种于包含包姜氏血琼脂培养基的平皿中，培养40-72小时，优选48小时；
    (2)将步骤(1)获得的菌体接种于包含MSS培养基的摇瓶中，培养22.5-25小时；
    (3)将步骤(2)获得的菌液接种于包含MSS培养基的发酵罐中，培养28-30小时。
  11. 根据权利要求9或10所述的制备方法，其中，所述百日咳抗原为百日咳黏附素和/或百日咳毒素。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101074442A (zh) * 2007-04-29 2007-11-21 中国药品生物制品检定所 百日咳疫苗保护性抗原的重组表达及其应用
EP3459966A1 (en) * 2012-10-17 2019-03-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Novel recombinant bordetella strains

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101074442A (zh) * 2007-04-29 2007-11-21 中国药品生物制品检定所 百日咳疫苗保护性抗原的重组表达及其应用
EP3459966A1 (en) * 2012-10-17 2019-03-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Novel recombinant bordetella strains

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 8 December 2002 (2002-12-08), ANONYMOUS : "Bordetella pertussis strain MO-1503 pertactin outer membrane protein (prnA) gene, prnA(1) allele, complete cds", XP093072222, retrieved from NCBI Database accession no. AF456359.1 *
WASIN BUASRI ET AL.: "Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange", BMC MICROBIOLOGY, vol. 12, 23 April 2012 (2012-04-23), XP021124669, DOI: 10.1186/1471-2180-12-61 *
YA-JUN TAN, WU LI-JIE ,WANG LI-CHAN, HOU QI-MING, ZHANG SHU-MIN: "Prokaryotic expression of pertactin of Bordetella pertussis and preparation of its monoclonal antibody", CHINESE JOURNAL OF BIOLOGICALS, vol. 25, no. 10, 17 October 2012 (2012-10-17), pages 1344 - 1348, XP093072235, DOI: 10.13200/j.cjb.2012.10.105.xuyh.011 *

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