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重组百日咳杆菌毒素S1亚单位及其突变体的克隆表达和生物学性质研究

发布时间:2018-05-07 07:29

  本文选题:百日咳杆菌 + 生物学作用 ; 参考:《第三军医大学》2007年硕士论文


【摘要】: 百日咳是由革兰阴性杆菌百日咳杆菌(Bordertella pertussis)引起的一种具有强传染性的呼吸系统疾病,是一种流行周期为2~5年的区域性疾病。疫苗是预防和控制百日咳流行的最有效的手段,19世纪30年代以来全细胞百日咳疫苗(whole pertussis vaccine,WPV)成功应用于临床,从很大程度上控制了百日咳的流行,但由于全菌疫苗的副反应及自身的局限性,导致接种覆盖率下降,百日咳发病率呈现明显上升趋势。因此,筛选、制备安全有效的抗原成分,研制新一代无细胞疫苗是目前百日咳疫苗研究的主攻方向。无细胞百日咳疫苗主要包括百日咳毒素(Bordetella pertussis toxin,PT)和丝状血凝素,其中减毒的百日咳毒素是无细胞组分疫苗的重要成分。 百日咳毒素为发现的B.pertussis主要毒力因子之一,它是由6个亚单位(S1-S5)构成的A-B型毒素,其中,A原体由一分子的S1组成,为PT的毒性所在部位,PT的ADP-核糖转移酶活性和主要保护性抗原决定簇即位于该亚单位上,传统百日咳疫苗的毒副作用与S1亚单位的酶活性有关。由于PT基因的不连续性,使其不能直接体外表达。鉴于PT的主要保护性决定簇位于S1亚单位上,而且ADP-核糖转移酶试验表明,所有天然序列的rS1(recombinant S1)亚单位均具有酶活性,而部分变异的rS1亚单位均无可检出的酶活性,因此,突变的S1可能是良好的候选抗原。 基于以上认识,本实验拟解决两个问题:一是百日咳毒素S1亚单位及其突变体高效表达问题。在这个过程中我们尝试过多种商品化的表达载体,如pET-22b、pET-28a、pET-11c、pQE-30等。并同时尝试融合表达是否能提高表达量,选用教研室自主研制的一种高效融合表达载体,其含有编码EspA伴体蛋白的核苷酸序列,使本身不表达或表达低的目的蛋白得到高效表达。EspA是肠出血性大肠杆菌O157:H7的Ⅲ型分泌系统相关蛋白,因此通过基因重组技术将EspA功能性肽段构建至表达载体上,从而达到促进外源蛋白高效表达的目的,并且EspA能够为目的蛋白提供潜在的粘膜佐剂功能。 二是对S1及S1突变体rS1的生物学活性进行研究。 主要结果如下: 1. B.pertussis S1的克隆表达 采用PCR法自B.pertussis PT基因组扩增编码S1的约702bp基因,构建至克隆载体pMD18-T中,通过酶切、测序鉴定阳性重组子。应用生物信息学软件DNAssist和GenBank数据库对已公布的B.pertussis S1基因序列进行相似性分析。改变原核表达载体和诱导条件优化表达方案。将含目的基因片段的重组载体转化至大肠杆菌中,经IPTG诱导表达,用AKTA-explore纯化仪纯化重组表达的蛋白S1获得纯度约80%的抗原蛋白。应用Tris-Tricine电泳对表达产物和纯化产物进行分析,并采用Western blot鉴定重组蛋白的免疫原性。 2. EspA-S1融合蛋白的克隆表达 采用重叠延伸PCR的方法,以EspA做为融合蛋白前端,与S1串联,在片段间引入5个氨基酸的linker PQDPP;将融合基因构建在原核表达载体pET-28a(+)中,经酶切及测序鉴定后,阳性重组子转化宿主菌E.coli BL21(DE3),IPTG诱导重组工程菌表达。Tris-Tricine电泳和免疫印迹鉴定融合蛋白。 3.百日咳毒素S1突变体rS1(S1-9K/129G)的构建表达。 运用基因工程的定点突变技术,构建S1突变体rS1(S1-9K/129G)。构建至克隆载体pMD-18T中,通过酶切、测序鉴定阳性重组子。将含目的基因片段的重组载体pQE30-rS1转化至大肠杆菌M15中,经IPTG诱导表达,用AKTA-explore纯化仪纯化重组表达的蛋白,获得纯度约87%的目的蛋白。应用Tris-Tricine电泳对表达产物和纯化产物进行分析。 4.百日咳毒素S1突变体生物学作用研究。 建立CHO细胞毒性模型。选用培养简单、生长周期较短、贴壁生长的CHO细胞,培养至对数生长期(OD600≈0.6),然后将贴壁生长在盖玻片上的CHO细胞与不同浓度的S1、rS1、PT于37℃,5%CO2共同孵育4h,姬姆萨染色及电镜观察。结果显示PT只要浓度达到20ng/ml即可使CHO细胞变形超过50%,而rS1即使达到了100ng/ml也无变形,S1对CHO细胞有变形效应。rS1与阳性对照PT及S1组差异显著(P0.01)。 小鼠白细胞增多(LP)试验。每组6只5至8周龄雌性BALB/c小鼠,分rS1、S1实验组,并设置阴性对照PBS组及阳性对照PT组。皮下肌注抗原蛋白(PT 0.5μg混于PBS中,其余量为3μg ) ,5日后每组每只小鼠采尾静脉血20μl并使用细胞计数池测定白细胞数。rS1实验组与PBS对照组促白细胞增多试验中未见明显差异(P0.05),与PT阳性对照组有显著差异(P0.01)。S1组与PT阳性对照组无显著差异(P0.05),结果表明rS1实验组无促白细胞增多作用,而S1实验组仍有促白细胞增多效应。 5. rS1作为百日咳疫苗候选抗原的免疫原性研究。 用纯化后的rS1和S1重组蛋白免疫BALB/c小鼠,ELISA检测蛋白免疫小鼠后血清特异性IgG水平。与PBS组相比,升高明显,差异极显著(p0.001),与疫苗免疫组相比,差异显著(p0.01)。rS1和S1组相比,无显著差异(p0.05)。表明重组蛋白rS1和S1均具有很强的免疫原性。 综上所述,本研究成功表达了B.pertussis S1、EspA- S1和B.pertussis rS1重组蛋白,与GenBank已公布的B.pertussis菌株基因序列具有高度同源性。纯化的重组蛋白rS1和S1,具有良好的免疫原性和免疫反应性,可以作为B.pertussis基因工程疫苗候选抗原。建立了CHO细胞毒性体外模型,通过对rS1和S1重组蛋白的生物学功能研究显示:rS1和S1均具有良好的免疫原性和免疫反应性,但S1仍保留有PT的毒性作用,而rS1则无此毒性。因此rS1可作为一个较为理想的候选蛋白。本课题的实施为研制百日咳疫苗奠定了良好的基础。
[Abstract]:Pertussis is a highly infectious respiratory system disease caused by gram-negative bacillus pertussis (Bordertella pertussis). It is a regional disease with a period of 2~5 years. Vaccine is the most effective means to prevent and control pertussis epidemic. All cell pertussis vaccine (whole pertussi) since 1830s S vaccine, WPV) was successfully applied to clinical and controlled the epidemic of pertussis to a great extent. However, due to the side effects of the vaccine and its own limitations, the coverage rate of the inoculation decreased and the incidence of pertussis showed an obvious upward trend. Therefore, screening, preparing the safe and effective antigen components and developing a new generation of acellular vaccine are now 100%. No cell-free pertussis vaccine mainly includes Bordetella pertussis toxin (PT) and filamentous hemagglutinin, and the attenuated pertussis toxin is an important component of the cell free vaccine.
Pertussis toxin is one of the major B.pertussis virulence factors found. It is a A-B toxin made up of 6 subunits (S1-S5). Among them, the A mycoplasma is composed of a molecule of S1, which is the toxic site of PT. The PT ADP- ribonuclease activity and the main protective antigen determinant are located on the subunit, and the toxic pair of the traditional pertussis vaccine. The action is related to the enzyme activity of the subunit of S1. Due to the discontinuity of the PT gene, it can not be expressed directly in vitro. Given that the main protective determinant of PT is located on the subunit of S1, and the ADP- ribose transferase test shows that all the rS1 (recombinant S1) subunits of all natural sequences have enzyme activity, and the partial variant of the rS1 subunit is none. The enzyme activity can be detected. Therefore, mutant S1 may be a good candidate antigen.
Based on the above understanding, this experiment will solve two problems: one is the high expression of pertussis toxin S1 subunit and its mutant. In this process, we have tried a variety of commercialized expression vectors, such as pET-22b, pET-28a, pET-11c, pQE-30 and so on. At the same time, we try to integrate the table to improve the expression and choose the independent development of the teaching and research department. A highly efficient fusion expression vector containing the nucleotide sequence encoding the EspA body protein to express.EspA as a type III secreting system associated protein of entero hemorrhagic Escherichia coli O157:H7, which is not expressed or expressed as low target protein. Therefore, the functional peptide segment of EspA is constructed to the expression vector by gene recombination technology. It can promote the efficient expression of foreign protein, and EspA can provide potential mucosal adjuvant function for target protein.
The two is to study the biological activity of S1 and S1 mutant rS1.
The main results are as follows:
Cloning and expression of 1. B.pertussis S1
Approximately 702bp gene encoding S1 from B.pertussis PT genome was amplified by PCR method and constructed to clone vector pMD18-T. The positive recombinant was identified by enzyme digestion and sequencing. The bioinformatics software DNAssist and GenBank database were used to analyze the published B.pertussis S1 gene sequences. The prokaryotic expression vector and induction conditions were changed. The recombinant vector containing the target gene fragment was transformed into Escherichia coli and was induced by IPTG, and the recombinant protein S1 was purified by AKTA-explore purify to obtain the antigen protein of the purity about 80%. The expression products and purified products were analyzed by Tris-Tricine electrophoresis, and the recombinant protein was identified by Western blot. Immunogenicity.
Cloning and expression of 2. EspA-S1 fusion protein
Using the method of overlapping extended PCR, EspA is used as the front end of the fusion protein, in series with S1, and the linker PQDPP of 5 amino acids is introduced into the fragment. The fusion gene is constructed in the prokaryotic expression vector pET-28a (+). The positive recombinant is transformed into E.coli BL21 (DE3) by enzyme digestion and sequencing, and IPTG induces the.Tris-Tricine expression of recombinant engineering bacteria. Fusion protein was identified by electrophoretic and immunoblotting.
Construction and expression of 3. pertussis toxin S1 mutant rS1 (S1-9K/129G).
The S1 mutant rS1 (S1-9K/129G) was constructed by the site directed mutation technique of genetic engineering. The recombinant plasmid was constructed into the clone vector pMD-18T, and the recombinant plasmid was sequenced by enzyme digestion. The recombinant vector containing the target gene fragment, pQE30-rS1, was transformed into the Escherichia coli M15, induced by IPTG, and the recombinant protein was purified by the AKTA-explore purify instrument. The purity of protein was about 87%. The expression product and purified product were analyzed by Tris-Tricine electrophoresis.
Study on the biological effect of 4. pertussis toxin S1 mutant.
The CHO cell toxicity model was established. CHO cells with simple culture, short growth cycle and adherent growth were cultured to logarithmic growth period (OD600 0.6). Then, the CHO cells on the cover glass were incubated with different concentrations of S1, rS1, PT at 37, 5%CO2, and 4h, Giemsa staining and electron microscopy. The results showed PT as long as 20n concentration reached 20n. G/ml could make CHO cells deformed more than 50%, while rS1 had no deformation even when it reached 100ng/ml. The deformation effect of S1 on CHO cell.RS1 was significantly different from that of the positive control PT and the S1 group (P0.01).
Mouse leukocytosis (LP) test. 6 female BALB/c mice of 5 to 8 weeks of age in each group were divided into rS1, S1 experimental group and negative control PBS group and positive control PT group. Subcutaneous injection of antigen protein (PT 0.5 mu g was mixed in PBS, the remaining amount was 3 micron g). 5 days later, each group of each mouse had 20 mu L and the cell count pool was used to determine the number.RS1. There was no significant difference between the experimental group and the PBS control group (P0.05), and there was significant difference with the PT positive control group (P0.01) there was no significant difference between the.S1 group and the PT positive control group (P0.05). The results showed that the rS1 experimental group had no leukocytosis, but the S1 experimental group still had the leukocytosis effect.
5. immunogenicity of rS1 as a candidate antigen for pertussis vaccine.
BALB/c mice were immunized with purified rS1 and S1 recombinant protein, and ELISA detected the serum specific IgG levels after immunization with the mice. Compared with the PBS group, the difference was very significant (p0.001). Compared with the vaccine group, the difference was significant (P0.01) between.RS1 and S1 groups (P0.05). Phytophthora.
To sum up, the recombinant protein of B.pertussis S1, EspA- S1 and B.pertussis rS1 has been successfully expressed, which is highly homologous with the gene sequence of B.pertussis strain released by GenBank. The purified recombinant protein rS1 and S1, with good immunogenicity and immunoreactivity, can be used as the candidate antigen of the B.pertussis gene engineering vaccine. An in vitro model of CHO cytotoxicity was established. The biological function of the recombinant protein of rS1 and S1 showed that both rS1 and S1 had good immunogenicity and immunoreactivity, but S1 still retained the toxic effect of PT, but rS1 had no toxicity. Therefore rS1 could be a more ideal candidate protein. The implementation of this topic was to develop pertussis pestilence The seedling lay a good foundation.

【学位授予单位】:第三军医大学
【学位级别】:硕士
【学位授予年份】:2007
【分类号】:R392

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5 王雅英;百日咳杆菌粘着素和菌毛的克隆、表达及免疫原性和保护性研究[D];中国药品生物制品检定所;2006年

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9 李晓军;铜绿微囊藻人工培养及藻毒素粗提物对棉铃虫的生物学作用的研究[D];山西农业大学;2003年

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