幽门螺杆菌分子内佐剂多亚单位融合疫苗的实验研究
发布时间：2018-08-16 13:35
【摘要】： 幽门螺杆菌(Helicobacter pylori,Hp)是一种定植于人类胃粘膜的革兰氏染色阴性、螺杆状、微需氧菌;在全世界范围内广泛感染,对人类健康构成严重危害。Hp的抗感染治疗存在较大问题,使用单剂药物根除率低,而使用多联药物费用昂贵,治愈率不高且不能有效阻止Hp的再感染和复发,此外,耐药菌株的增加也使抗Hp治疗面临越来越复杂的难题,因此研制Hp疫苗和其有效接种可能是预防Hp感染最有前景的手段。 本研究依托实验室长期研究Hp疫苗的结果,拟在已完成的Hp三亚单位融合蛋白HspA-HpaA-UreB414(rHHU)的基础上,将该基因片段hhu与大肠杆菌不耐热肠毒素B亚单位基因ltB构建融合基因;并通过原核表达系统得到带分子内佐剂LTB的多亚单位蛋白疫苗;纯化目的蛋白后对沙鼠进行免疫后攻毒保护实验,评价该疫苗的免疫保护效率,并与其它疫苗方案的保护效果进行比较,为新型分子内佐剂多亚单位Hp疫苗的研制提供一定的实验依据。 本实验完成了以下几个方面的工作: 1.采用PCR技术分别扩增目的基因片段hhu和ltB; PCR重叠延伸拼接法将ltB基因融合于hhu基因的头部,构建lhhu融合基因形式;NcoⅠ、XhoⅠ双酶切后将融合基因构建至原核表达载体pET-28a(+)中;经酶切、测序鉴定后,阳性重组子分别转化宿主菌E.coli BL21(DE3)、E.coli JM109(DH3)。 2.工程菌分别在25℃、37℃及42℃条件经IPTG诱导表达;SDS-PAGE,兔抗LTB、兔抗Hp血清和小鼠抗His_6单抗免疫印迹检测,证实重组融合蛋白rLHHU在不同温度条件及不同宿主菌环境中均表达为两条分子量接近的蛋白带,显示目的蛋白发生了降解。 3.重新构建融合基因hhul形式,将ltB融合于hhu的尾部,并在两片段间引入8氨基酸柔性linker GGGSGGGS;融合基因构建至原核表达载体pET-28a(+)中获得表达质粒phhul;经酶切、测序鉴定后,阳性重组子转化宿主菌E.coli BL21(DE3);IPTG诱导工程菌表达,SDS-PAGE及免疫印迹检测,证实为目的蛋白的表达,分子量约为55.2KD;UVP扫描证实融合蛋白表达约占总蛋白的20%。 4.重组蛋白rHHUL提交PridictProtein(http://cubic.bioc.columbia.edu/predictprotein/)
[Abstract]:Helicobacter pylori (Helicobacter) is a Gram-negative, screw shaped, microaerobic bacterium that is colonized in human gastric mucosa. It is widely infected worldwide and has great problems in anti-infective treatment of H.pylori, which is a serious hazard to human health. The eradication rate of single drug is low, while the cost of multidrug is expensive, the cure rate is not high and can not effectively prevent the reinfection and recurrence of HP. In addition, the increase of drug-resistant strains also makes the treatment of anti-HP more and more complicated. Therefore, the development of HP vaccine and its effective vaccination may be the most promising means to prevent HP infection. Based on the results of a long term study of HP vaccine in laboratory, this study intends to construct the fusion gene based on the completed HP S3 subunit fusion protein HspA-HpaA-UreB414 (rHHU) and the E. coli heat-labile enterotoxin B subunit gene ltB. The multisubunit protein vaccine with intramolecular adjuvant LTB was obtained by prokaryotic expression system. Compared with other vaccine schemes, it provides some experimental basis for the development of novel intramolecular adjuvant multisubunit HP vaccine. This experiment has completed the following aspects of the work: 1. The target gene fragments hhu and ltBwere amplified by PCR, the ltB gene was fused into the head of hhu gene by PCR overlapping extension splicing method, and the lhhu fusion gene form was constructed by double enzyme digestion of NCO 鈪，
本文编号：2186137
[Abstract]:Helicobacter pylori (Helicobacter) is a Gram-negative, screw shaped, microaerobic bacterium that is colonized in human gastric mucosa. It is widely infected worldwide and has great problems in anti-infective treatment of H.pylori, which is a serious hazard to human health. The eradication rate of single drug is low, while the cost of multidrug is expensive, the cure rate is not high and can not effectively prevent the reinfection and recurrence of HP. In addition, the increase of drug-resistant strains also makes the treatment of anti-HP more and more complicated. Therefore, the development of HP vaccine and its effective vaccination may be the most promising means to prevent HP infection. Based on the results of a long term study of HP vaccine in laboratory, this study intends to construct the fusion gene based on the completed HP S3 subunit fusion protein HspA-HpaA-UreB414 (rHHU) and the E. coli heat-labile enterotoxin B subunit gene ltB. The multisubunit protein vaccine with intramolecular adjuvant LTB was obtained by prokaryotic expression system. Compared with other vaccine schemes, it provides some experimental basis for the development of novel intramolecular adjuvant multisubunit HP vaccine. This experiment has completed the following aspects of the work: 1. The target gene fragments hhu and ltBwere amplified by PCR, the ltB gene was fused into the head of hhu gene by PCR overlapping extension splicing method, and the lhhu fusion gene form was constructed by double enzyme digestion of NCO 鈪，
本文编号：2186137
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