【作者】 刘志华；

【导师】 骆抗先；

【作者基本信息】 第一军医大学 ， 内科学， 2002， 博士

【摘要】 国内外的研究发现HBV／C区变异与病变重度相关，但两者之间的因果关系尚未确定。本研究以重组腺病毒为载体，将含有变异位点的HBV基因组转入HepG2细胞和BALB／C小鼠，从体内和体外研究变异位点的生物学意义，旨在阐明C区变异对宿主细胞免疫和体液免疫的影响。 1．重组腺病毒的构建 用定点诱变技术，以含有1.2拷贝HBV基因组的p3.8Ⅱ质粒为模板，构建含L60V和I97L的突变株。采用AdEasy系统构建重组腺病毒，以分子克隆手段将HBV基因组克隆入腺病毒穿梭质粒pAdTrack上，重组穿梭质粒与腺病毒骨架质粒共转化大肠杆菌BJ5183细胞，通过同源重组获得重组腺病毒质粒，用Pac Ⅰ线性化后，转染293包装细胞，获得含野生型和变异型HBV基因的3种重组腺病毒。 2．重组腺病毒生物学特征的鉴定 电镜下观察到了重组腺病毒具有典型的腺病毒形态，直径约70nm。用PCR和Southern blot证实HBV DNA已整合入腺病毒基因组，有遗传稳定性：用RT-PCR检出HBV特异的mRNA，用ELISA在感染了重组腺病毒的293细胞的培养上清中检出了HBeAg。 3．HBV基因在HepG2细胞中的表达 用重组腺病毒感染HepG2细胞，分别用ELISA法和Western blot从细胞上清和细胞裂解物中检出HBsAg、HBeAg和HBcAg的表达。以不同传染剂量的重组腺病毒传染HepG2细胞，结果表明，传染剂量越大，分泌性抗原的表达越强，此外，还比较了相同传染剂量的各种重组腺病毒感染HepG2细胞后其抗原表达的动态变化，发现AdHBV-WT的HBeAg表达量始终高于2个变异株；AdHBV-WT和AdHBV-L60V的HBsAg的表达量接近，而AdHBV-I97L的HBsAg表达极弱。 4．重组腺病毒诱导BALB／C小鼠的免疫应答 用AdHBV-WT、AdHBV-L60V和AdHBV-I97L以滴鼻途径传染BALB／C小鼠，以接种野生5型腺病毒和生理盐水的小鼠为对照，观察HBV基因在小鼠体内诱发的体液兔疫和细胞免疫应答。重组腺病毒感染的3组小鼠均能有效地产生抗．HBC抗体，抗体水平无明显差别，所有小鼠血清中抗－HBs均阴性，仅有部分小鼠产生了较弱的抗HBe抗体。检测小鼠血清中抗－HBc IgG抗体的亚类，结果表明 3个实验组小鼠血清中 IgG和 IgGZa两种亚类抗体均为阳性，以 IgGZa占优势，说明腺病毒诱导的对HBCAg的应答以Thl类免疫为主。比较3组实验小鼠IgGZa／IgGI的比值，未发现有显著性差异，表明 L60V和 I97L变异可能不影响 Thl几hZ的分化方向。分离小鼠脾淋巴细胞，体外用重组HBCAg刺激，以’H－TdR摄入法检测小鼠T淋巴细胞的增殖反应，发现AdHBVWT感染组小鼠强于另2组小鼠，说明AdHBVWT诱导小鼠产生HBCAg特异性T细胞反应的能力比AdHBVL60V和AdHBV－197L的强。 结论：C区的2个热点变异L60V和I97L与野生型相比，诱发小鼠特异性T细胞应答的能力较弱，变异位点可能通过改变免疫细胞识别表位而引起T细胞应答降低，从而逃避宿主的兔疫反应。因此，我们推测L60V和I97L可能是免疫逃避株。更多还原

【Abstract】 Although the relationship between mutations in HBV/C region and disease severity has been demonstrated in many epidemiological studies, the causality between them remains unclear. In this study, the adenovirus was employed as vector to transfer 1.2 copies of HBV genome containing L60V and I97L mutations into HepG2 cells and BALB/C mice, to investigate the biological implications of the two mutations in vitro and in vivo. We intended to determine whether the two mutations could change the immunogenity of HBcAg and had influence on the humoral and cellular immune response in mice.1. Construction of L60V and I97L mutants and recombinant adenovirusesBy site-directed mutagenesis technique, p3.8llplasmid, which contains 1.2 copies of HBV genome, was used as template to produce the two desired mutations. The HBV genome was cloned into the shuttle vector pAdTrack; the resultant constructs were cotransformed intoE.coli strain BJ5183, and the recombinant adenoviral plasmids were cleaved with Pac I to expose their inverted terminal repeats and transfected into 293 packaging cell to generate viruses. Three strains of recombinant adenoviruses carrying wild type or mutant HBV genomes were obtained.2. Biological characteristics of recombinant adenovirusesThe resultant recombinant adenoviruses showed typical adenovirus morphology with a diameter about 70 nm under electron microscope. The integration of HBV DNA into the adenovirus genome was identified by PCR and Southern blot assay. The viral genomes were stable when tested by PCR during the passage in 293 cells. HBV/C specific mRNA in 293 cells infected by recombinant adenoviruses was detected by RT-PCR and HBeAg in its culture medium by ELISA.3. Expression of HBV genes in HepG2 cellsBy ELISA and Western blot analysis, the expressions of HBV antigens were detected in cell lysate or culture medium of HepG2 cells infected by each of three strains of recombinant adenoviruses, respectively. When HepG2 cells were infected with AdHBV-WT at various multiplicity of infection (MOD , the expressions of HBsAg and HBeAg increased with MOI. Additionally, we infected HepG2 cells with the three strains of recombinant adenoviruses at same MOI. The results showed that the expression of HBeAg in HepG2 cells infected with AdHBV-WT was stronger than that of AdHBV-L60V and AdHBV- I97L during the whole test period. As for expression of HBsAg, AdHBV-WT was similar to AdHBV-L60V, however, the expression of HBsAg by AdHBV-I97L was so weak that it can be seldom detected.4. The immune response of BALB/C mice induced by recombinant adenoviruses Three of five groups of BALB/C mice were inrranasally inoculated withAdHBV-WT, AdHBV-L60V and AdHBV-I97L, and the other two groups with wild Ad5 and saline as control, respectively. Samples of mice serum were taken and tested for antibodies to HBV antigens by ELISA. The mice in experimental groups producedanti-HBc efficiently, and the levels had no significant difference, indicating that L60V3and I97L mutation may have no influence on the humoral immune response to HBcAg. All samples of mice serum were tested negative for anti-HBs. IgGl and IgG2a specific for HBcAg were positive in serum samples, but the level of IgG2a predominated in all samples, which reflected the type of immune response to HBcAg was inclined to Th1-like. The result implicated that L60V and I97L mutation might not change Th1/Th2 polarity. Lymphocytes were isolated from spleen of mice and stimulated with recombinant HBcAg in vitro. The proliferation response was assessed by 3H-TdR uptake. We found that T cell proliferation response in mice infected by AdHBV-WT was stronger than AdHBV-L60V and AdHBV-I97L. The results suggested that L60V and I97L mutation might downregulate T response to HBcAg.In this study, HBV genome was transferred and expressed successfully in HepG2 cells and BALB/C mice. Based on these models, the biological effects of the two hot-spot mutations L60V and I97L were studied. The results suggested that these mutations m更多还原