口服沙门菌递送治疗性HBV-HSP70融合DNA疫苗的实验研究

发布时间：2018-04-15 01:01

本文选题：乙型肝炎 + 热休克蛋白　；参考：《中国人民解放军军医进修学院》2011年博士论文

【摘要】：慢性乙型肝炎(CHB)严重危害人类健康,临床上尚无理想治疗手段,目前主要用核苷类药物和干扰素进行治疗,但是并不能完全治愈乙型肝炎,且长期使用会使HBV产生耐药突变并具有较大的副作用。乙型肝炎病毒感染人体后发生慢性化的主要原因是被感染者缺乏有效的特异性细胞免疫,无法清除体内的病毒。DNA疫苗由于在诱导机体特异性细胞免疫应答方面的优势,被广泛关注。但是经过多年来的研究,DNA疫苗仍未走进临床,重要原因之一就是随着受试动物体形的增大,DNA疫苗诱导免疫应答的能力迅速下降；此外缺乏满意的递送方式也是DNA疫苗发展的主要阻碍。因此,如何在安全的前提下,采取有效的递送方式和加强诱导的免疫应答,特别是加强细胞免疫应答水平,是目前研制有效DNA疫苗的关键问题。使用分枝杆菌热休克蛋白70羧基端(HSP70c)或T细胞刺激表位(HSP70t)基因构建融合基因疫苗可以起到佐剂的作用,同时减少机体针对HSP70的免疫应答。利用携带DNA质粒的减毒伤寒沙门菌口服,可直接将DNA质粒递送至抗原呈提细胞,极大提高DNA疫苗的利用率,同时口服的免疫方式安全方便,易于为患者接受。使用DNA疫苗初次免疫-痘苗病毒Ankara株(MVA)载体疫苗加强免疫的策略也可以有效增强机体的免疫应答。采取多种免疫增强策略综合应用,可以更好的优化DNA疫苗的免疫效果。本研究通过HBV基因与HSP70c或HSP70t基因构建融合基因疫苗、使用口服减毒伤寒沙门菌递送DNA疫苗和利用MVA载体疫苗强化免疫等多种手段,探索提高HBV DNA疫苗诱导特异性免疫应答的更佳优化条件。首先,将乙肝病毒preC/C基因或preS2/S基因、HSP70c基因或HSP70t基因分别先后插入VR1012载体,获得含HBV preC/C-HSP70c、HBV preC/C-HSP70t、HBV preS2/S-HSP70c和HBV preS2/S-HSP70t融合基因的DNA疫苗。再将构建好的重组DNA疫苗转染HepG2细胞,ELISA法和化学发光法检测目的抗原,证实重组DNA疫苗可以正确表达目的蛋白并具有抗原性。然后,将重组DNA疫苗电转化减毒伤寒沙门菌SL7207,获得稳定的携带重组DNA疫苗的重组减毒沙门菌。最后,分别采用HBV DNA疫苗肌肉注射初次免疫+MVA载体疫苗腹腔注射加强免疫、HBV-HSP70融合DNA疫苗肌肉注射初次免疫+MVA载体疫苗腹腔注射加强免疫、携带HBV DNA疫苗的重组减毒沙门菌口服初次免疫+MVA载体疫苗腹腔注射加强免疫和携带HBV-HSP70融合DNA疫苗的重组减毒沙门菌口服初次免疫+MVA载体疫苗腹腔注射加强免疫的方案,免疫Balb/c小鼠,收集血清和脾淋巴细胞检测特异性免疫应答。体液免疫方面,采用ELISA检测血清特异性抗体,结果显示各实验组均可诱导小鼠产生特异性抗体,抗体产生的时间和水平的差异无统计学意义。细胞免疫方面通过流式细胞术检测CD3+CD4+/CD3+CD8+T细胞比值评估非特异性细胞免疫水平,ELISpot检测肽刺激脾淋巴细胞分泌IFN-γ评估特异性细胞免疫效果,结果显示无论是特异性还是非特异性细胞免疫,HBV-HSP70融合DNA疫苗优于普通DNA疫苗,HBV-HSP70t融合DNA疫苗优于HBV-HSP70c融合DNA疫苗。使用减毒沙门菌口服递送DNA疫苗与裸DNA疫苗肌肉注射所诱导的细胞免疫应答水平相当。实验结果为治疗性HBV DNA疫苗的应用研究打下了基础。
[Abstract]:Chronic hepatitis B is a serious harm to human health . There is no ideal treatment in clinic , but it can not cure hepatitis B completely , but it can ' t cure hepatitis B completely . The main reason for chronic infection after hepatitis B virus infection is the lack of effective specific cellular immunity . The DNA vaccine has not yet entered the clinic because of its superiority in inducing body specific cellular immune response . One of the important reasons is that the ability of DNA vaccine to induce immune response is rapidly decreased with the increase of the body shape of the animal .
In addition , the lack of satisfactory delivery methods is the main obstacle to the development of DNA vaccine . Therefore , it is a key problem to develop effective DNA vaccine under the precondition of safety , effective delivery methods and enhanced immune response , especially the level of cellular immune response .

By taking the attenuated Salmonella typhimurium carrying the DNA plasmid to take orally , the DNA plasmid can be directly delivered to the antigen presenting cell , so that the utilization rate of the DNA vaccine can be greatly improved , the oral immunization mode is safe and convenient , and the immune response of the organism can be effectively enhanced .

In this study , a fusion gene vaccine was constructed by combining HBV gene and HSP70t gene , DNA vaccine was delivered by oral attenuated Salmonella typhimurium , and immunity was enhanced by using MVA vector vaccine . The optimal conditions for improving the specific immune response of HBV DNA vaccine were explored .

The recombinant attenuated Salmonella typhimurium SL7207 containing HBV preC / C - HSP70t , HBV preS2 / S - HSP70t , HBV preS2 / S - HSP7070 and HBV preS2 / S - HSP70t fusion gene was obtained .

In the aspect of humoral immunity , the serum - specific antibody was detected by ELISA . The results showed that all experimental groups could induce specific antibody and antibody production in mice . The results showed that the specific cell immunity was assessed by flow cytometry . The results showed that HBV - HSP70 fusion DNA vaccine was superior to common DNA vaccine . The results showed that the DNA vaccine was superior to HBV - HSP70t fusion DNA vaccine .

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

【参考文献】