【作者】 李长贵；

【导师】 王军志； 张英起；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 流感病毒RNA聚合酶缺少校正功能,导致基因组在复制过程中容易发生突变,人群中因以往感染产生的抗体不能有效地防止新毒株感染,因而每年需要注射流感疫苗,给社会带来沉重的经济负担。如果有一种通用型疫苗,经过全程免疫后,产生广谱性抗体,则能够预防各种变异病毒,或降低感染病毒后疾病的严重程度。甲型流感病毒囊膜上除血凝素(HA)和神经氨酸酶(NA)外,还含有一个分子量较小的基质蛋白M2,其胞外区(M2e)非常保守,是研制通用型疫苗的最佳靶位。由于M2e只含有24个氨基酸,需要以各种措施来增强其免疫原性。本论文拟将口服脊髓灰质炎疫苗(OPV)Sabin1株病毒改造为疫苗载体,采用多聚蛋白融合策略,插入流感M2e基因,将前者作为表达载体和免疫佐剂,提高人体针对M2e的免疫应答,构建一种能抵御各种甲型流感病毒的通用型疫苗。通过RT-PCR方法扩增了Sabin1株病毒基因组的三个片段f1、f2、f3并分别克隆,在f1和f2、f2和f3片段之间含重叠序列,内有基因组唯一的酶切位点。因此,三片段可以通过酶切和连接获得全基因组。将三个克隆分别测序,与GenBank中序列比较,发现在基因组7440bp中只有三个位点不同,其中26 A→G,355 C→T两个位点处于非编码区,6735 A→G导致聚合酶3D上250位氨基酸由赖氨酸变为精氨酸。说明Sabin1株基因组比较稳定,适合于作为疫苗载体。基因组起始密码子(nt743)位于pGEM-f1质粒上,通过重叠PCR方法在之前插入多克隆位点(EcoRI、XmaI、XhoI)和人工蛋白酶切位点(ALFQG)构成的功能盒(Cassette),形成pGEM-f1m质粒;同样,在位于pGEM-f2质粒上P1/P2结合处引入类似序列,构建为pGEM-f2m质粒。连接为基因组后可形成两种基本型载体PV1和PV2。多克隆位点内插入的外源基因随多聚蛋白表达后,在病毒编码的蛋白酶切割后释放外源蛋白。为提高载体RNA的转染效果,另外在载体上进行两项改造:基因组前加入锤头型核酶(Rz)基序,合成RNA后发挥核酶功能,去除多余核苷酸,产生PV基因组真正的5’-末端;在基因组后导入poly(A)40结构,后者是PV RNA感染的必要基序。两个基序均以PCR引物延伸方法产生,并通过基因置换方式导入到pGEM-f1m和pGEM-f3中,经测序证明引入的基序与设计序列一致。将改造前和改造后的质粒进行三片段连接,形成八种载体,分别含有不同的功能盒及基序组合。将携带各种PV载体的质粒线性化后,以T7 RNA聚合酶体外转录法合成RNA并纯化,通过电泳发现RNA完整性、均一性良好,适合于转染细胞。对含有Rz序列的RNA进行体外切割,经PAGE电泳检测反应进程,确定在合适的缓冲液中作用1.5小时获得最佳切割效果。采用电穿孔、脂质体转染法和磷酸钙转染法将提取的PV Sabin1 RNA转染入Vero细胞,以形成的噬斑数目比较转染效率,筛选转染大分子RNA的最佳方法。实验结果证明脂质体Tfx-20的转染效率最佳,达到1.1~1.5×103感染性克隆/μg RNA,对细胞毒性不明显。以Tfx-20转染八种载体RNA,只有同时含有poly(A) 40和Rz基序的载体pPV1RzA和pPV2RzA能有效产生活病毒,约500~800克隆/μg RNA。感染性病毒产生时间与转染RNA量有关,当以10μgRNA转染时,60小时就可以观察到细胞病变;而以1μgRNA转染时,需要4天产生病变效应。只含有poly(A)40基序的载体RNA也可以成功转染,但效率低,当RNA量为1μg时无法得到活病毒。不含两个基序或只含Rz基序的RNA在Vero细胞上无感染性,即使将RNA量增加到30μg,培养10天亦无活病毒产生。从霍乱弧菌中扩增霍乱毒素B亚单位(CTB)基因,测序证明正确后,以PCR引物延伸法构建CTB和M2e的融合基因,并在两端分别添加EcoRI和XhoI位点,通过双酶切将融合基因导入到pPV1RzA中,保持正常的编码框架,以建立的方法转染Vero细胞,获得了重组病毒rPV-CTBM2e。另外,扩增SARS-冠状病毒S蛋白的受体结合区基因S-RBD后,以同样方法获得了重组病毒rPV-S-RBD。提取重组病毒RNA,以RT-PCR方法扩增涵盖插入序列的片段并测序,证明两个重组病毒均携带预期片段。将重组病毒传代多次,通过RT-PCR方法考察重组病毒的稳定性。rPV-CTBM2e病毒传代12次后仍保持完整的外源基因,而rPV-S-RBD病毒在传代过程中丢失外源基因。rPV-CTBM2e病毒一步生长曲线证明其繁殖有延迟现象,但最终能得到和Sabin1病毒同样的滴度。将M2e合成多肽和KLH载体蛋白偶联后免疫Balb/c小鼠,制备了针对M2e多肽的单克隆抗体,以ELISA方法筛选得到7株有活性的单抗。以单抗上清检测感染Vero细胞的流感病毒,发现其中5株能与细胞表面的天然M2e结合,其中两株为强阳性,可用于重组病毒M2e表达的检测。将重组病毒感染Vero细胞,通过免疫荧光方法,分别以抗M2e单抗和抗SARS-病毒免疫血清检测,发现在细胞中出现明显的荧光信号,并集中在细胞质中,说明外源蛋白在细胞质中成功表达,与预期结果一致。本研究为研制流感通用型疫苗和SARS-CoV疫苗奠定了基础。更多还原

【Abstract】 The primary means of controlling influenza virus at the present time is vaccination. The compositions of vaccine are changed yearly according to WHO recommendation. This is because RNA polymerase of influenza virus are less faithful than are DNA polymerases and are not able to correct errors by a proof- reading mechanism, thus mutations occur enough to provide ample source for the selection of naturally accumulated variants. The antibodies exist in human body induced by vaccination or exposure to previously circulating virus could not efficiently prevent the infection of mutated strains. A universal vaccine,which protects effectively against all influenza A strains and provides long- lasting immunity, will definitely be an attractive solution to overcome the threat of this promiscuous virus. It involves a vaccine based on the extra- cellular domain of the influenza M2 protein (M2e), which has remained nearly invariable since the virus was isolated in 1933. However, M2e is less immuno- genic since it is a small peptide of 24 amino acids. In this study, we aim to modify the Sabin 1 poliovirus as a vaccine vector and rescue the recombinant virus carrying the fusion gene of Cholera Toxin B subunit and M2e. Our strategy is to use the live attenuated virus to boost the immunogenicity of this peptide.Three fragments of Sabin1 poliovirus were amplified by RT-PCR and cloned into pGEM vectors. The overlapping sequences between neighboring fragments contain unique restriction enzyme sites within the genome. Thus, the 3 fragments could be joined together to form the whole genome by enzyme digestion and ligation. The sequencing results show that three nucleotides are different from the genome sequence in GenBank. Only one mutation, 6735 A→G, results in the change from 250Lysine to 250Arginine on polymerase 3D.In order to construct the gonome as a vector, a cassette containing EcoRI, XmaI, XhoI sites and artificial protease site was introduced adjacent before the initial codon of PV ORF. A similar cassette was also inserted into the P1/P2 joint site. Thus, two kinds of basic PV vectors could be derived based on the different insertion sites of extrageneous genes. The modification were carried out on different plasmids and accomplished by overlapping PCR methods.To improve the transfection efficiency of vector-derived RNA, further modifications were made on the vectors. A hammer-headed Ribozyme (Rz) motif was added just before the 5’-terminal of genome, thus the transcribed RNA could cleavage itself in vitro, releasing the additional nucleotides derived from plasmid and leaving PV RNA with the authentic 5’-end. A poly(A)40 stretch was patched to the 3’-terminal of genome to stabilize the transcribed RNA. Two modifications were carried out on target plasmids by primer extension PCR and fragment replacement, respectively. The original and derived plasmids were assembled into 8 kinds of vaccine vectors with different combination of various cassettes and motifs.The plasmid carrying vectors were linearized by enzyme digestion and were used as templates to synthesize RNAs by T7 RNA polymerase in vitro. Agrose electrophoresis result demonstrated that the dedived RNA are homogenous and intact in size, thus are suitable for transfection. The RNAs carrying Rz motif were subject to self-cleavage in reaction buffer and small Rz released were visulized by PAGE. Rz fragments accumulate as time progress and reach to platform at 1.5 h.To optimize the transfection condition, RNAs extracted from Sabin1 virus were transfected into Vero cells through different methods and reagents. The plaques achieved by 1μg RNA at different methods were calculated. The results suggested that liposome Tfx-20 has the highest efficiency with 1.1~1.5×103 infectious clones/μg RNA and exhibits the minimum toxicity on cells. RNAs transcribed from 8 kinds of vectors were transfected into Vero cell using establish- ed method. Only vectors containing both the Rz and poly(A)40 motifs can transfect cells with high efficiency, with 500~800 clones/μg RNA. When 10μg RNAs were transfected, CPE could appear as early as 60 h post- transfection. The RNAs carrying only poly(A)40 can also produce infectious viruses with lower efficiency, while RNA from vectors bearing only Rz or neither of the motifs failed to rescue live viruses on Vero cells.Cholera Toxin B subunit (CTB) gene was amplified by PCR and fused to M2e by primer extension PCR. EcoRI and XhoI sites were designed to 5’-and 3’- end of fusion gene, respectively. The gene was inserted into the polylinker site of pPV1RzA by double digestion and re-ligation. Rescued recombinant virus carrying CTBM2e gene, rPV-CTBM2e, was obtained by the established mthod. Similarly, a recombinant virus carrying the receptor binding domain gene from SARS-CoV Spike protein, rPV-S-RBD, was also prepared using the same vector. The foreign inserts were confirmed by RT-PCR and sequencing. rPV-CTBM2e was very stable during the 12 round of passage in Vero cell, while rPV-S-RBD lost the foreign insert quickly in serial passages.Monoclonal antibodies against M2e were prepapred by immunizing Balb/c mice with M2e peptide coupling to KLH carrier proteins. 7 clones of McAb were screened by ELISA, and 5 of them can bind to natural M2e displayed on Vero cells, which were infected with influenza virus.Vero cells infected with rPV-CTBM2e and rPV-S-RBD were treated with paraformaldehyde and permeated with Triton X-100. The expressed proteins were detected by McAb agsinst M2e and polyclonal antibodies against SARS-CoV and visualized by FITC-labeled secondary antibodies.The fluorescence signals appear mainly in the cytoplasm, thus confirmed the expression of foreign proteins. Our study involves in construction of Sabin1 poliovirus as a vaccine vector with high efficiency and provides a powerful tool for development of universal influenza vaccine and SARS-CoV vaccine.更多还原