【作者】 郭晓雅；

【导师】 史皆然； 师长宏；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 硕士

【摘要】 背景重组活菌疫苗是以某些减毒或者无毒力的活菌为载体,将病原体的保护性抗原基因插入细菌的基因组或者质粒DNA中并使之高效表达的一种新型疫苗。重组卡介苗(Recombinant Bacillus Calmette-Guerin,rBCG)就是重组活菌疫苗中具有代表性的新型疫苗。它是以BCG为工程菌,借助分子生物学技术对其进行基因改造,利用其活疫苗特性,在机体内表达各种疾病的相关抗原,从而达到预防和治疗多种疾病的目的。本研究小组曾成功将屋尘螨抗原Der p2基因转入BCG中表达,制备出抗原Der p2-rBCG。经静脉和腹腔注射接种后,在小鼠体内诱导了Der p2特异性的Th1优势应答。因此我们推测可以通过抗原rBCG疫苗来治疗对某些特定抗原过敏的疾病。目前分枝杆菌几乎全部是以皮下注射的方式接种。但是研究发现,如果短期内重复皮下注射接种rBCG会引起局部严重的迟发性变态反应(DTH),因此我们考虑采用口服途径接种rBCG。而且,我们前期的研究发现给小鼠口服Der p2-rBCG,同样可以诱导抗原特异性的Th1应答[1, 2]。然而,BCG分枝杆菌并不是肠道定植菌,与肠黏膜的亲和力低,大量口服还会引起肠道正常菌群失调及口咽部感染,从而影响免疫效果。文献回顾发现,沙门菌外膜蛋白Omp D介导了沙门菌与肠黏膜上皮的黏附,因此我们设想此外膜蛋白能够介导rBCG与肠道黏膜的高亲和力结合,从而制备出具有肠道高亲和力的rBCG口服疫苗。目的利用分子生物学及基因工程技术,构建能以串联和并联形式、在细菌表面表达Der p2和Omp D抗原的两种rBCG口服疫苗,为体外实验和临床应用提供基础。实验方法和结果1. Omp D蛋白的表达及纯化以鼠伤寒沙门菌Salmonella typhimurium基因组为模板,通过PCR来扩增Omp D基因,与pMD-18T克隆载体连接后测序,结果与Genbank公布的Omp D基因序列完全一致。将Omp D基因克隆入原核表达载体pET-28a(+),经PCR和酶切鉴定后,阳性质粒命名为pET28a(+)-Omp D,并经IPTG进行诱导表达。SDS-PAGE分析证实成功表达了与预期分子量一致的Omp D蛋白。Western-blot证实该蛋白可与抗6×His-mAb发生特异性反应。可溶性分析显示该蛋白以包涵体形式存在于沉淀中,经Ni+-NTA亲和色谱法纯化获得了目的蛋白,且纯度达90%以上。2.兔Omp D多克隆抗体的制备及鉴定重组蛋白经变性、复性后,按100μg/kg的纯化蛋白加等量弗氏完全佐剂(FCA)乳化后接种于兔颈背部皮内,间隔2w后再进行第二、三次免疫;第三次免疫结束2w后以等量重组蛋白追加免疫1次,1w后收集血液,分离血清后ELISA检测特异性抗体效价达到1:10 000以上。Western-blot检测表明,该抗体能与Omp D蛋白发生特异性结合,其特异性与敏感性均较好。3.两种rBCG口服疫苗的构建与鉴定为了获得Der p2-Omp D融合基因,我们重新设计引物,经PCR法分别扩增获得新的Der p2和Omp D基因,测序正确后将两者克隆入原核表达载体pProEX HTb,获得pProEX HTb-Der p2-Omp D质粒。①串联表达:将Der p2-Omp D融合基因亚克隆入穿梭胞壁表达载体pCW,经酶切鉴定阳性命名为pCW-Der p2-Omp D质粒。将此重组质粒电穿导入BCG感受态细胞,构建可胞壁串联表达Der p2-Omp D融合蛋白的rBCG;②并联表达:构建pCW-Der p2与pCW-Omp D两种质粒,并将二者同时电穿导入BCG感受态细胞,以构建胞壁并联表达Der p2和Omp D蛋白的rBCG。经潮霉素抗性筛选的阳性克隆,均采用以下三种方式进行鉴定:PCR特异性扩增目的基因片段;用兔抗Der p2多克隆抗体与兔抗Omp D多克隆抗体对阳性克隆分别进行斑点免疫杂交法和间接免疫荧光法鉴定。证实两种rBCG口服疫苗构建成功,并能与特异性抗体发生反应。结论1.在E.coli表达系统中成功表达并纯化出Omp D蛋白。该蛋白能够与6×His-mAb发生特异性反应,免疫新西兰兔后获得Omp D多克隆抗体,通过ELISA检测抗体效价达到1:10 000以上。该抗体能与Omp D蛋白发生特异性结合,其特异性与敏感性均较好。2.采用基因工程手段制备出以胞壁形式串联和并联表达Der p2和Omp D蛋白的两种rBCG口服疫苗,并通过PCR、斑点免疫杂交法及间接免疫荧光法分别进行鉴定。证实了两种rBCG口服疫苗构建成功。更多还原

【Abstract】 BACKGROUUND:Live recombinant vaccines are based on the use of a live microorganism (virus or bacteria) that acts as a vector for the expression of genes from another organism. The new recombinant microorganism can be used as a vaccine for both organisms. Recombinant Bacillus Calmette-Guerin (BCG) is a typical vaccine of live recombinant vaccines, that insets foreign genes into BCG to construct multivalent vaccines by using molecular biology techniques, then induces long-term cellular and humoral immune responses. Recombinant BCG (rBCG) can prevent and cure many kinds of diseases by expressing associated antigens in vivo. We have constructed rBCG by transferring Dermatophagoides pteronyssinus antigen Der p2 into BCG in our lab. The recombinant Der p2-BCG could stimulate Th1 predominant immune response in mice when injected intraperitoneally or subcutaneously. The results indicated that rBCG vaccines could be used to treat some diseases which were hypersensitive to some specific antigens. However, if rBCG vaccines were injected repeatedly in the short term, serious local delayed-type hypersensitivity could be induced. Then, we considered that oral vaccines were administered to mice, and results showed that it could also induce antigen specific Th1 predominant immune response. Nevertheless, BCG doesn’t inhabit in the intestinal tract because of its low affinity to intestinal epithelia. Furthermore, a large dosis of BCG could cause dysbacteria and oropharynx adenitis, thus the immune efficacy is limited. Therefore, in order to improve the affinity of rBCG to intestinal epithelia, the protein Omp D, one of the major adhesion molecules, was used to prepare the rBCG oral vaccine.AIM:To construct two oral rBCG vaccines expressing Der p2 and Omp D (in form of series connection and parallel connection) antigens on its cell wall.METHODS AND RESULTS:1. Expression and purification of Omp D protein The Omp D gene amplification was encoded by PCR from genomic DNA of Salmonella Typhimurium, and cloned into vector pMD-18T. The DNA sequence of Omp D was identical with that of Genbank reported. After confirmed by sequencing, the target gene was inserted into expression plasmid pET-28a (+) to construct recombinant plasmid pET28a (+)-Omp D, which was identified by PCR and restrictive enzyme digestion. The recombinant plasmid was transformed into E. coli BL21(DE3) which then expressed Omp D under IPTG induction. The analysis of SDS-PAGE showed that there was a specific protein expression at 40 kDa molecular marker, and the protein was further identified by Western-blot using anti-6×His mAb. Then, it was purified by Ni+-NTA purification system under denaturing condition and its purity was about ninety percent. 2. Preparation of Omp D polyclonal antibody in rabbitA rabbit was immunized with Omp D protein according to the following procedure. The reannealed Omp D protein dissolved in PBS was emulsified with an equal volume of Freund’s complete adjuvant. On days 0, 14 and 28, 2 ml of the prepared mixture was intracutaneously injected into the rabbit. On days 42, 2 ml of the Omp D protein dissolved in PBS was injected into the rabbit. On days 49, antibody titers of Omp D protein was measured by ELISA and attained to 1: 10 000. The specificity and sensitivity of Omp D antibody were also analyzed by Western-blot.3. Preparation and identification of rBCG oral vaccineThe genes of Der p2 and Omp D were first amplified by PCR, and then were ligated into pProEX HTb vector respectively, and gained Der p2-Omp D fused gene.①Expressing in form of series connection: The Der p2-Omp D fused gene was subcloned into the shuttle plasmid pCW. The positive recombinant plasmid was named pCW-Der p2-Omp D, and was transformed into BCG competent cells to construct rBCG oral vaccine , which could express the Der p2-Omp D fused protein on its cell wall in the form of series connection.②Expressing in form of parallel connection: The pCW-Der p2 and pCW-Omp D plasmids were transformed into BCG competent cells together to construct the another rBCG oral vaccine, which could express Der p2 and Omp D proteins on its cell wall in the form of parallel connection. The two rBCG oral vaccines were selected by hygromycine, and the positive rBCG were identified by PCR, spot immunifaction and indirect immunofluorescence. The PCR result showed that there was specific amplification of the target gene. Spot immunifaction showed that specific banding of rBCG expressing product with Omp D pAb and Der p2 pAb was observed, indicating that rBCG possesses immunological function. Indirect immunofluorescence showed that specifc green immunofluorescences were observed, indicating that rBCG could express the goal protein on its surface. CONCLUSIONS:1. Omp D protein was purified successfully in E.coli DH5α. Omp D polyclonal antibody in rabbit was obtained, whose antibody titer attained to 1: 10 000 detected by ELISA. Its high specificity and sensitivity were analyzed by Western-blot.2. The two rBCG oral vaccines expressing Der p2 and Omp D proteins on its cell wall in the forms of series connection and parallel connection were preparated by genetic engineering, and then identified by PCR, spot immunifaction and indirect immunofluorescence successfully.更多还原