乙型肝炎新型植物口服疫苗的研究

发布时间：2018-09-09 16:14

【摘要】： 乙型肝炎(Hepatitis B,HB)是一种由乙型肝炎病毒(Hepatitis B virus,HBV)感染所引起的传染性疾病,我国大约有10%的人群是乙肝病毒携带者。目前仍然缺乏有效的治疗手段治疗HB,因此,主要靠接种疫苗来进行预防HB。由于现在使用商业化HBV疫苗主要通过酵母表达系统生产,存在较高的生产成本,而且需要冷藏,因此对边远和经济不发达地区人群疫苗接种普及带来困难。此外,大约有10%的接种人群对现在商业化HB疫苗不产生明显免疫反应。因此,探索研制新型价廉的HB疫苗成为疫苗研究关注的焦点。本研究在国内外已有工作基础上,对人新型HBV表面抗原融合基因SS1进行了改造,并将SS1基因转入植物中,开展了相关研究,取得以下主要结果。 1、为了分析新型乙肝表面抗原SS1(在乙肝主要表面抗原(S)的C端融合了乙肝表面抗原前体肽preS1(21-47aa))是否具有preS1的免疫原性,并在体内激起针对preS1的抗体反应,我们依照大肠杆菌B株系的密码子偏爱特性改造和合成了全长的adr血清型preS1基因(spreS1)并在大肠杆菌进行了表达。结果显示表达产物以可溶性形式存在,表达量占到总蛋白的44%,经过Ni-NTA亲和层析纯化,纯化的蛋白纯度达到98%以上,western blot和间接ELISA结果表明该重组蛋白具有preS1的抗原性,而且抗原性与现在商业化的preS1相当。这些克服了以往在表达全长preS1蛋白时遇到的包涵体、表达产物不稳定或低表达的难题。 2、为了探索生产一种新型的HB口服疫苗形式,我们选择了水稻作为新型乙肝表面抗原SS1的表达宿主。我们通过目前的文献报道选取了水稻种子表达的高效表达启动子P_(GluB-4),构建乙型肝炎抗原基因SS1的水稻种子高效表达载体p1300GSS1,通过农杆菌转化,实现了其在水稻种子中的表达。重组蛋白的表达量为31.5 ng/g DW种子,且能够自组装成病毒样颗粒结构(virus-like particles,VLP),颗粒的粒径大约为22±2 nm,沉降系数为1.25 g.cm~(-3)。Western blot结果显示同时具有S和preS1抗原性。动物免疫试验显示重组蛋白能够在小鼠体内激起针对S和preS1的抗体反应,这些为今后的临床评价提供理论基础。 3、为了进一步提高新型乙肝表面抗原SS1在植物体内的表达水平,我们首次尝试利用植物叶绿体表达系统进行这种新型乙肝表面抗原的表达。越来越多的研究表明植物叶绿体表达系统具有诸多优越性,其中外源基因的高效表达最引人注目。乙肝表面抗原是一种糖蛋白,而且它的糖基化程度与其抗原性直接相关,本研究构建了其烟草的叶绿体表达载体并进行了烟草的遗传转化,结果显示外源基因被正确地整合到叶绿体基因组中预定的位点,表达的重组蛋白经乙肝表面抗原检测试剂盒检测具有抗原性,相关的研究还在继续中。 4、为了提高口服疫苗的免疫效果,我们对目前常用的免疫佐剂分子霍乱毒素B亚基蛋白(cholera toxin B,CTB)进行了大肠杆菌表达。本研究首先克隆了去除信号肽编码序列的霍乱毒素B亚基基因CTB,构建了大肠杆菌表达载体,进行表达,结果显示表达产物以包函体的形式出现,表达量占到总蛋白的25%,经过包函体的变性和复性,纯化的蛋白纯度达到98%,体外生活性试验表明纯化的蛋白主要以五聚体的形式出现,western blot结果显示五聚体大约占到总纯化蛋白的66.8%。由于现在商业化的疫苗中还没有添加任何佐剂成份,尤其是CTB蛋白,所以口服免疫佐剂表达系统的建立为今后水稻表达的新型乙肝表面抗原的动物口服试验提供了基础。
[Abstract]:Hepatitis B (HB) is an infectious disease caused by hepatitis B virus (HBV) infection. About 10% of the population in China is hepatitis B virus carriers. There is still a lack of effective treatment for HB, so vaccination is the main way to prevent HB. It is difficult to vaccinate people in remote and economically underdeveloped areas because of the high production cost and the need for refrigeration. In addition, about 10% of the vaccinated population do not have obvious immune response to the commercial HB vaccine. Therefore, exploring new and inexpensive HB vaccine becomes epidemic. In this study, a novel human HBV surface antigen fusion gene SS1 was modified and transformed into plants based on the previous work at home and abroad.
1. In order to analyze the immunogenicity of a novel hepatitis B surface antigen SS1 (preS1 (21-47aa), fused with a hepatitis B surface antigen precursor peptide preS1 (21-47aa) at the C terminal of the main hepatitis B surface antigen (S), and to stimulate antibody response against preS1 in vivo, we modified and synthesized a full-length ADR serum according to the codon preference characteristics of E. coli B strain. PreS1 gene (spreS1) was expressed in E. coli. The results showed that the expressed product was soluble and accounted for 44% of the total protein. The purity of the purified protein was over 98% after purification by Ni-NTA affinity chromatography. Western blot and indirect ELISA showed that the recombinant protein was antigenic and antigenic to preS1. The commercialized preS1 is comparable now. These overcome the difficulties encountered in expressing full-length preS1 proteins, including inclusion bodies, unstable or low expression products.
2. In order to explore a new form of oral HB vaccine, we chose rice as the expression host of a novel hepatitis B surface antigen SS1. The recombinant protein was expressed in rice seeds by Agrobacterium tumefaciens transformation. The expression of the recombinant protein was 31.5 ng/g DW, and it could self-assemble into virus-like particles (VLP). The particle size was about 22 2 nm, and the sedimentation coefficient was 1.25 g.cm~(-3). Western blot results showed that the recombinant protein had both S and preS1 antigenicity. Immunoassay showed that the recombinant protein could stimulate antibody response against S and preS1 in mice, which provided theoretical basis for future clinical evaluation.
3. In order to further improve the expression level of the novel hepatitis B surface antigen SS1 in plants, we first attempted to use the plant chloroplast expression system to express this novel hepatitis B surface antigen. Objective. Hepatitis B surface antigen is a glycoprotein, and its glycosylation degree is directly related to its antigenicity. In this study, we constructed its tobacco chloroplast expression vector and transformed it into tobacco. The results showed that the exogenous genes were correctly integrated into the predetermined sites in the chloroplast genome, and the recombinant protein was expressed on the surface of hepatitis B. Antigen detection kit has antigenicity, and related research is continuing.
4. In order to improve the immune effect of oral vaccine, cholera toxin B subunit protein (CTB) was expressed in E. coli. In this study, the cholera toxin B subunit gene CTB was cloned to remove the coding sequence of signal peptide, and the expression vector of E. coli was constructed. The purity of the purified protein was 98% after denaturation and renaturation of the inclusion bodies. In vitro bioassay showed that the purified protein was mainly in the form of pentamer. Western blot showed that pentamer accounted for about 66.8% of the total purified protein. No adjuvant, especially CTB protein, has been added to commercial vaccines, so the establishment of an oral immune adjuvant expression system will provide a basis for future animal oral trials of novel hepatitis B surface antigen expressed in rice.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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