突变型乙型肝炎病毒前C-C DNA疫苗的基础研究

发布时间：2018-04-22 07:26

本文选题：乙型肝炎 + DNA疫苗　；参考：《中国人民解放军军医进修学院》2007年博士论文

【摘要】： 慢性乙型肝炎严重危害人类健康，目前尚无特效治疗手段。乙肝病毒(HBV)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应答，不能清除体内病毒。由于DNA疫苗在诱导机体特异性细胞免疫方面具有独特优势，已作为针对慢性乙肝具有潜力的免疫治疗手段被广泛研究。目前乙肝DNA疫苗研究的焦点在于如何诱导足够强的特异性细胞免疫，以达到抑制或消灭病毒、治疗乙肝的目的。HBcAg是抗原性最强的HBV结构蛋白，其基因常用于构建DNA疫苗，能引发针对病毒核心蛋白的特异性细胞免疫反应。HBeAg与HBcAg具共线性，是乙肝病毒核心抗原的分泌型。研究表明分泌型HBeAg(P18)诱导免疫耐受，而其胞内前体(P20、P22)则有可能诱导较HBcAg更强的细胞免疫。本研究课题首先以乙肝病毒前C(Pre-C)-C基因和自身复制调控元件增强子Ⅱ(EHNⅡ)为目的基因构建疫苗，采用常规PCR法从adr亚型HBV全基因DNA序列中扩增出EHNⅡ-前C-C基因片段，重组到载体VR1012中，构建出重组乙肝EHNⅡ/前C-C基因疫苗(VEC)，然后在此DNA疫苗基础上设计使前C基因蛋白产物HBeAg前体151、154、164、167位氨基酸点突变的4对引物，依次加入VEC中，采用单引物二次PCR法进行基因点突变，分别得到151点突变(VE1)、151+154点突变(VE2)、151+154+164点突变(VE3)、151+154+164+167点突变(VE4)4种变异体，使前C蛋白水解位点发生变异，生成前体产物P22、P20后无法被细胞蛋白酶水解切割产生成熟的分泌型HBeAg。然后将VEC、VE2、VE4分别转染入HepG2细胞，，ELISA法检测细胞裂解液、细胞培养上清液，结果均有目的蛋白表达。细胞裂解液的HBeAg含量VE4、VE2高于VEC，上清中则反之，VEC细胞裂解液的HBeAg含量低于上清，VE2、VE4细胞裂解液的HBeAg含量高于上清；酶免疫细胞染色定位见目的蛋白主要为胞浆内表达；蛋白免疫印记检测表达产物的分子量，结果表明VEC、VE2、VE4的表达产物分别为P18、P20、P22左右。分别用VEC、VE2、VE4于0、7、21天肌注方式免疫BALB/C小鼠，同时设VEC、VE2、VE4分别联合使用插入干扰素γ基因的VR1012质粒(VMγ)组和VEC、VE4各联合急性乙肝S基因的DNA疫苗(VAS)免疫动物组，ELISA法检测小鼠抗HBc、抗HBeIgG，发现免疫接种后第14天即可检测到抗体，第28天滴度更高，VE2、VE4组抗HBe水平高于VEC组，抗HBc水平无显著性差异。免疫结束后(第28天)取小鼠脾细胞用ELISpot方法和CTL杀伤试验检测特异性细胞免疫功能，证实3种质粒均能引发特异性细胞免疫反应，VE4、VE2强于VEC；联用VMγ免疫后能使VEC、VE2、VE4特异性细胞免疫反应增强，联用VAS后VE4、VEC特异性细胞、体液免疫反应无增强。本研究结果为治疗型HBVDNA疫苗的进一步临床试验提供了依据，奠定了基础。
[Abstract]:Chronic hepatitis B is a serious harm to human health. There is no special therapeutic method at present. The main cause of chronic hepatitis B virus (HBV) infection is the lack of effective specific cellular immune response and the ability to remove the virus in the body. The DNA vaccine has unique advantages in inducing the specific cell immunity of the body. At present, the focus of the study on hepatitis B DNA vaccine is how to induce strong specific cell immunity to inhibit or eliminate the virus..HBcAg is the most antigenic HBV structural protein for the purpose of treating hepatitis B. Its gene is often used to construct DNA vaccine and can be triggered. The specific cellular immune response to virus core protein (.HBeAg) is co linear with HBcAg, which is the secretory type of HBV core antigen. The study shows that secretory HBeAg (P18) induces immune tolerance, and its intracellular precursor (P20, P22) may induce stronger cellular immunity than HBcAg.
First of all, the vaccine was constructed with the pre C (Pre-C) -C gene of hepatitis B virus (HBV) and the self replicating element enhancer II (EHN II) as the target gene, and the EHN II pre C-C gene fragment was amplified from the adr subtype HBV whole gene DNA sequence by the conventional PCR method, and the recombinant hepatitis B EHN II / former gene vaccine was constructed. On the basis of this DNA vaccine, 4 pairs of primers that mutated the 151154164167 amino acid points of the precursor of the precursor of the C gene protein product were designed and added to the VEC, and the single primer two PCR methods were used for gene point mutation, and 151 point mutation (VE1), 151+154 point mutation (VE2), 151+154+164 point mutation (VE3), 151+154+164+167 point mutation (VE4) 4 species were obtained respectively. Variant, the mutation of the hydrolysis site of the former C protein, the production of the precursor product P22, and the production of the mature secretory HBeAg. after P20, and then transfected into HepG2 cells by VEC, VE2 and VE4 respectively, and the cell lysate is detected by ELISA method and the cell culture supernatant has the target protein expression. HBeAg content of the cell lysate is contained. The amount of VE4, VE2 was higher than that of VEC, whereas in the supernatant, the HBeAg content of the VEC cell lysate was lower than that of the supernatant, and the HBeAg content of the VE2, VE4 cell lysate was higher than that of the supernatant; the enzyme immunocell staining localization showed that the target protein was mainly in the cytoplasm, and the protein immuno imprint was used to detect the molecular weight of the expression products, and the results showed that the VEC, VE2, VE4 expression products were respectively. For P18, P20, and P22, VEC, VE2, VE4 were used to immunize BALB/C mice in 0,7,21 days, respectively, and VEC, VE2, and VE4 were combined to insert the interferon gamma gene into the VR1012 plasmids (gamma) group, respectively.
And VEC, VE4 each combined with the DNA vaccine (VAS) of acute hepatitis B S gene vaccine (VAS), the ELISA method was used to detect the anti HBc, anti HBeIgG, and the antibody was detected at fourteenth days after immunization, the titer of the twenty-eighth day was higher, the HBe level of the VE4 group was higher than that of the VEC group, and there was no significant difference. The spleen cells of mice were taken after the end of immunization (twenty-eighth days). The specific cellular immune function was detected by method and CTL killing test. It was confirmed that 3 plasmids could induce specific cellular immune responses, VE4 and VE2 were stronger than VEC, and VM gamma immunization could enhance the immune response of VEC, VE2, VE4 specific cells, combined with VAS VE4, VEC specific cells, and no enhancement in humoral immune response. The results of this study were therapeutic HBVDNA Further clinical trials of the vaccine provided a basis and laid the foundation.

【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】：2007
【分类号】：R392

【共引文献】