發(fā)布時(shí)間：2018-05-07 07:29

  本文選題：百日咳桿菌 + 生物學(xué)作用�。� 參考：《第三軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 百日咳是由革蘭陰性桿菌百日咳桿菌(Bordertella pertussis)引起的一種具有強(qiáng)傳染性的呼吸系統(tǒng)疾病,是一種流行周期為2～5年的區(qū)域性疾病。疫苗是預(yù)防和控制百日咳流行的最有效的手段,19世紀(jì)30年代以來(lái)全細(xì)胞百日咳疫苗(whole pertussis vaccine,WPV)成功應(yīng)用于臨床,從很大程度上控制了百日咳的流行,但由于全菌疫苗的副反應(yīng)及自身的局限性,導(dǎo)致接種覆蓋率下降,百日咳發(fā)病率呈現(xiàn)明顯上升趨勢(shì)。因此,篩選、制備安全有效的抗原成分,研制新一代無(wú)細(xì)胞疫苗是目前百日咳疫苗研究的主攻方向。無(wú)細(xì)胞百日咳疫苗主要包括百日咳毒素(Bordetella pertussis toxin,PT)和絲狀血凝素,其中減毒的百日咳毒素是無(wú)細(xì)胞組分疫苗的重要成分。 百日咳毒素為發(fā)現(xiàn)的B.pertussis主要毒力因子之一,它是由6個(gè)亞單位(S1-S5)構(gòu)成的A-B型毒素,其中,A原體由一分子的S1組成,為PT的毒性所在部位,PT的ADP-核糖轉(zhuǎn)移酶活性和主要保護(hù)性抗原決定簇即位于該亞單位上,傳統(tǒng)百日咳疫苗的毒副作用與S1亞單位的酶活性有關(guān)。由于PT基因的不連續(xù)性,使其不能直接體外表達(dá)。鑒于PT的主要保護(hù)性決定簇位于S1亞單位上,而且ADP-核糖轉(zhuǎn)移酶試驗(yàn)表明,所有天然序列的rS1(recombinant S1)亞單位均具有酶活性,而部分變異的rS1亞單位均無(wú)可檢出的酶活性,因此,突變的S1可能是良好的候選抗原。 基于以上認(rèn)識(shí),本實(shí)驗(yàn)擬解決兩個(gè)問(wèn)題:一是百日咳毒素S1亞單位及其突變體高效表達(dá)問(wèn)題。在這個(gè)過(guò)程中我們嘗試過(guò)多種商品化的表達(dá)載體,如pET-22b、pET-28a、pET-11c、pQE-30等。并同時(shí)嘗試融合表達(dá)是否能提高表達(dá)量,選用教研室自主研制的一種高效融合表達(dá)載體,其含有編碼EspA伴體蛋白的核苷酸序列,使本身不表達(dá)或表達(dá)低的目的蛋白得到高效表達(dá)。EspA是腸出血性大腸桿菌O157:H7的Ⅲ型分泌系統(tǒng)相關(guān)蛋白,因此通過(guò)基因重組技術(shù)將EspA功能性肽段構(gòu)建至表達(dá)載體上,從而達(dá)到促進(jìn)外源蛋白高效表達(dá)的目的,并且EspA能夠?yàn)槟康牡鞍滋峁撛诘恼衬ぷ魟┕δ堋?二是對(duì)S1及S1突變體rS1的生物學(xué)活性進(jìn)行研究。 主要結(jié)果如下: 1. B.pertussis S1的克隆表達(dá) 采用PCR法自B.pertussis PT基因組擴(kuò)增編碼S1的約702bp基因,構(gòu)建至克隆載體pMD18-T中,通過(guò)酶切、測(cè)序鑒定陽(yáng)性重組子。應(yīng)用生物信息學(xué)軟件DNAssist和GenBank數(shù)據(jù)庫(kù)對(duì)已公布的B.pertussis S1基因序列進(jìn)行相似性分析。改變?cè)吮磉_(dá)載體和誘導(dǎo)條件優(yōu)化表達(dá)方案。將含目的基因片段的重組載體轉(zhuǎn)化至大腸桿菌中,經(jīng)IPTG誘導(dǎo)表達(dá),用AKTA-explore純化儀純化重組表達(dá)的蛋白S1獲得純度約80%的抗原蛋白。應(yīng)用Tris-Tricine電泳對(duì)表達(dá)產(chǎn)物和純化產(chǎn)物進(jìn)行分析,并采用Western blot鑒定重組蛋白的免疫原性。 2. EspA-S1融合蛋白的克隆表達(dá) 采用重疊延伸PCR的方法,以EspA做為融合蛋白前端,與S1串聯(lián),在片段間引入5個(gè)氨基酸的linker PQDPP;將融合基因構(gòu)建在原核表達(dá)載體pET-28a(+)中,經(jīng)酶切及測(cè)序鑒定后,陽(yáng)性重組子轉(zhuǎn)化宿主菌E.coli BL21(DE3),IPTG誘導(dǎo)重組工程菌表達(dá)。Tris-Tricine電泳和免疫印跡鑒定融合蛋白。 3.百日咳毒素S1突變體rS1(S1-9K/129G)的構(gòu)建表達(dá)。 運(yùn)用基因工程的定點(diǎn)突變技術(shù),構(gòu)建S1突變體rS1(S1-9K/129G)。構(gòu)建至克隆載體pMD-18T中,通過(guò)酶切、測(cè)序鑒定陽(yáng)性重組子。將含目的基因片段的重組載體pQE30-rS1轉(zhuǎn)化至大腸桿菌M15中,經(jīng)IPTG誘導(dǎo)表達(dá),用AKTA-explore純化儀純化重組表達(dá)的蛋白,獲得純度約87%的目的蛋白。應(yīng)用Tris-Tricine電泳對(duì)表達(dá)產(chǎn)物和純化產(chǎn)物進(jìn)行分析。 4.百日咳毒素S1突變體生物學(xué)作用研究。 建立CHO細(xì)胞毒性模型。選用培養(yǎng)簡(jiǎn)單、生長(zhǎng)周期較短、貼壁生長(zhǎng)的CHO細(xì)胞,培養(yǎng)至對(duì)數(shù)生長(zhǎng)期(OD600≈0.6),然后將貼壁生長(zhǎng)在蓋玻片上的CHO細(xì)胞與不同濃度的S1、rS1、PT于37℃,5%CO2共同孵育4h,姬姆薩染色及電鏡觀察。結(jié)果顯示PT只要濃度達(dá)到20ng/ml即可使CHO細(xì)胞變形超過(guò)50%,而rS1即使達(dá)到了100ng/ml也無(wú)變形,S1對(duì)CHO細(xì)胞有變形效應(yīng)。rS1與陽(yáng)性對(duì)照PT及S1組差異顯著(P0.01)。 小鼠白細(xì)胞增多(LP)試驗(yàn)。每組6只5至8周齡雌性BALB/c小鼠,分rS1、S1實(shí)驗(yàn)組,并設(shè)置陰性對(duì)照PBS組及陽(yáng)性對(duì)照PT組。皮下肌注抗原蛋白(PT 0.5μg混于PBS中,其余量為3μg ) ,5日后每組每只小鼠采尾靜脈血20μl并使用細(xì)胞計(jì)數(shù)池測(cè)定白細(xì)胞數(shù)。rS1實(shí)驗(yàn)組與PBS對(duì)照組促白細(xì)胞增多試驗(yàn)中未見(jiàn)明顯差異(P0.05),與PT陽(yáng)性對(duì)照組有顯著差異(P0.01)。S1組與PT陽(yáng)性對(duì)照組無(wú)顯著差異(P0.05),結(jié)果表明rS1實(shí)驗(yàn)組無(wú)促白細(xì)胞增多作用,而S1實(shí)驗(yàn)組仍有促白細(xì)胞增多效應(yīng)。 5. rS1作為百日咳疫苗候選抗原的免疫原性研究。 用純化后的rS1和S1重組蛋白免疫BALB/c小鼠,ELISA檢測(cè)蛋白免疫小鼠后血清特異性IgG水平。與PBS組相比,升高明顯,差異極顯著(p0.001),與疫苗免疫組相比,差異顯著(p0.01)。rS1和S1組相比,無(wú)顯著差異(p0.05)。表明重組蛋白rS1和S1均具有很強(qiáng)的免疫原性。 綜上所述,本研究成功表達(dá)了B.pertussis S1、EspA- S1和B.pertussis rS1重組蛋白,與GenBank已公布的B.pertussis菌株基因序列具有高度同源性。純化的重組蛋白rS1和S1,具有良好的免疫原性和免疫反應(yīng)性,可以作為B.pertussis基因工程疫苗候選抗原。建立了CHO細(xì)胞毒性體外模型,通過(guò)對(duì)rS1和S1重組蛋白的生物學(xué)功能研究顯示:rS1和S1均具有良好的免疫原性和免疫反應(yīng)性,但S1仍保留有PT的毒性作用,而rS1則無(wú)此毒性。因此rS1可作為一個(gè)較為理想的候選蛋白。本課題的實(shí)施為研制百日咳疫苗奠定了良好的基礎(chǔ)。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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