乙型肝炎新型植物口服疫苗的研究

【作者】 钱炳俊；

【导师】 曹凯鸣； 张大兵；

【作者基本信息】 复旦大学 ， 分子生物学与生物化学， 2007， 博士

【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。更多还原

【Abstract】 Hepatitis B is a severe infectious disease caused by Hepatitis B virus （HBV） infection. About 10% of peoples are HBV carriers in China. Currently, there is no effective treatment for this disease, and prevention of it mainly depends on vaccine. But, commercial vaccine produced in yeast expression system is expensive for its higher production costs, and needs refrigeration. This made the universal vaccination difficult in remote areas and economically underdeveloped regions. In addition, about 10% of the population had no immunological response to the commercial vaccine. Thus, it’s the exploration of a new form of HB vaccine that comes to people’s attention. In sum up the work previously studied, the novel modified Hepatitis B surface antigen gene SS1 was expressed by an optimistic plant production system in this study, and some important results were discribed below.1, To detect wheher the the new hepatitis B antigen SS1 studied in this study, in which is the precursor peptide preS1 （21-47aa） was fused to the C-terminal of the hepatitis B major surface antigen （S）, could arouse anti-preS1 antibody protection reaction in vivo through its immunization except for anti-S anitibodies, the full length preS1 gene （adr serotype） with codon preference in accordance with the E. coli strain B was synthesized and expressed in E.coli for preparetion for detection of anti-preS1 antibodies. The result showed that the recombinant proteins were in soluble form, accounting for about 44% of total protein. After purification with Ni-NTA affinity chromatography, the purity of purified protein reached to 98%. The result of Western blot and indirect ELISA showed that protein is stable and has preS1 antigenicity which was comparable to that of the commercial preS1. Results showed that we have overcome the problems encountered in the previous studies on full-length preS1 protein expression, such as products in the inclusive body form, protein unstable, or low expression. It provides the basis for the animals’ clinical study of this new hepatitis B vaccine.2, For exploration of a new form of HB oral vaccine, the rice was used as an expression host of the recombinant protein attributing to that more and more studies showed that grain will be the optimistic expression system for producing the plant vaccine in the future. In addition, rice is the major food source for people in Asia, and is a self-pollinated crop that would relieve the transgene contamination caused by target gene flowing. In this study, we selected promoter of GluB-4 gene with higher activity to drive the foreign gene SS1 according to the reported papers and constructed a rice seeds-specific expression vector p1300GSS1 for transformation. Through transformation by Agrobacterium-media, the SS1 gene was inserted into the rice genome, and the highest expression level of the recombinant protein was about 31.5 ng/g DW seeds. It could self-assemble into virus-like particles structure with diameter of about 22±2 nm and a sedimentation coefficient of 1.25 g.cm^-3. Western blot analysis revealed that the recombinant protein have both S and preS1 antigenicity. Animal experiments showed that recombinant protein in mice could provoke antibody against S and preS1. These results will encourage us to further evaluate the oral immunogenicity of this recombinant protein in the subsequent clinical study.3, In order to further improve the expression level of the recombinant protein SS1 in plant, the chloroplast expression system was tried firstly. An increasing number of studies show that plant chloroplast expression system has many advantages, of which high expression level of foreign proteins was most striking （the highest expression achieved to 46.7%TSP）. Therefore, chloroplast expression system used for producing hepatitis B surface major antigen was another strategy to enhance the expression level. However, there is no application of this expression system for the expression of hepatitis B major antigen to date. Many review literature showed that the protein expressed in the chloroplast could not be glycosylated, but there were no specific studies related to the reports to prove it. Hepatitis B surface antigen is a glycoprotein, and its glycosylation-degree directly relates to its antigenicity. In this study, a tobacco chloroplast expression vector was constructed. As a model plant, tobacco was firstly used for tentative expression of hepatitis B surface antigen. The result showed that foreign gene was correctly integrated into the predetermined site of the chloroplast genome. After four rounds of selection, the transgenic plants were not homoplastic yet. The recombinant protein was detected to be expressed in the transgenic plant by hepatitis B surface antigen detection kit, but the expression level was very low. This may relates to the unhomoplastic and unglycosylation. Related research is still continuing.4, To achieve a good immunological effection, an immune adjuvant protein CTB （cholera toxin B subunit） was also expressed in E.coli. in this study. The CTB gene encoding the cholera toxin B subunit without the signal peptide-coding sequence was cloned, and constructed into the E. coli expression vector pETCTB for its expression. The result showed that the recombinant protein was expressed in the form of inclusion bodies, accounting for about 25% of the total protein. Through the denaturation and refolding of inclusion bodies, the purity of recombinant protein CTB reached to 98%. The result of bioactivity detection in vitro showed that the purified recombinant protein was mainly presented in form of pentamer. The result of western blot showed that the protein in form of pentamer were about 66.8% of total purified protein.This established expression system of oral adjuvant will help the further oral experiments of this recombinant protein.更多还原