【作者】 黄莹；

【导师】 扈荣良；

【作者基本信息】 吉林大学 ， 预防兽医学， 2009， 博士

【摘要】 反向遗传学是对遗传信息为RNA的生命体而言,在获得生物体基因组全部序列的基础上,通过对靶基因进行必要的加工和修饰再按基因组组成顺序构建含有生物体必需元件的修饰基因组,让其装配出具有生命活性的个体。本研究对建立中国疫苗株狂犬病病毒反向遗传系统的成功探索,为进一步研究狂犬病病毒基因组结构与功能的关系,开发新型疫苗提供技术平台。本研究建立了中国人用疫苗株(CTN)狂犬病病毒反向遗传系统,其中主要元件包括:两端分别带有锤头状核酶(HamRz)和丁型肝炎核酶(HdvRz)并在G、L基因之间插入绿色荧光蛋白(GFP)基因的全长基因组cDNA重组真核表达质粒,以及核蛋白(N)、磷酸化蛋白(P)、RNA依赖的RNA聚合酶蛋白(L) 3个参与病毒转录和复制的辅助质粒。方法是将上述4个质粒共转染BHK-21细胞,在细胞RNA聚合酶II的作用下启动真核表达载体pVAX1的CMV启动子起始转录各个目的基因,成功拯救出CTN株重组狂犬病病毒(CTN-GFP),通过感染重组病毒的细胞传代实验证明此病毒能够稳定表达GFP。重组病毒CTN-GFP分别脑内接种1日龄乳鼠和成年小鼠,结果导致乳鼠全部发病死亡,并在发病死亡的乳鼠脑内检测到绿色荧光蛋白的表达,而对成年鼠不致病,提示此病毒具有与亲代CTN株病毒相似的致病特性。更多还原

【Abstract】 Reverse genetics is an approach to discovering the function of a gene that proceeds in the opposite direction of so called forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find the possible phenotypes that may derive from a specific genetic sequence by point mutations, gene insertion and gene deletions. Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism.Reverse genetics refers to manipulate the genome of RNA virus at DNA level by construction of the infectious molecular clone. The replication and expression process of RNA virus is not experienced DNA stage. In order to obtain infectious molecular clone, the genome of RNA virus should be transformed into cDNA, and then be cloned into an appropriate expression vector under control of a powerful promoter. The RNA polymerase transcription system was utilized to synthesize virus RNA, and the virus particles were packaged in host cells. Currently, reverse genetics has become one of the most important means in molecular virology research. With the development of reverse genetics techniques, it can be utilized to understand the relationship between RNA viruses’genome structure and function, the interactions between viruses and hosts, the mechanism of virus transcription and replication. Moreover, it can also be used to explore new genetic engineering vaccines.Rabies is a lethal zoonotic infectious disease induced by rabies virus, the case fatality ratio (CFR) is 100 percent approximately. So far, it is an acute infection disease of supreme case-fatality rate in human history. The number of dead people infected rabies virus is 55000 in the world every year. Ninety-nine percent cases are happened in Asia and Africa. The dead cases are about 35000-40000 every year in Asia. China is one of the most severe regions of rabies, and the number of rabies cases is increasing consecutively during recent years.The genome of rabies virus encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L). The N, P, L proteins and the viral genomic RNA compose a ribonucleoprotein complex (RNP), which constitute the template for viral gene expression and replication by the virus polymerase. Rabies virus is a non-segmented negative-strand RNA virus and the viral genomic RNA is not infectious, which made it different from positive-strand RNA virus. Only RNP, not free RNA, could initiate viral gene replication and expression. In order to obtain infectious molecular clone of rabies virus, the full-length genome plasmid is essential, other three helper plasmids carrying N, P, L gene involved in viral transcription and expression should be also supplied in the reverse genetic of rabies virus.Although there are many successful cases in developed countries, few successful infectious molecular clone of rabies virus were constructed independently in China. In this thesis, successful reverse genetics system of CTN strain rabies virus was not only a technique basis for explore new rabies vaccine, but also a technical platform for rabies virus to be a new type virus vector candidate.The whole genome of CTN strain rabies virus was divided into four fragments to amply by the unique restriction enzyme sites, and the pseudogene was replaced with a green fluorescent protein (GFP) gene. The cDNA sequences of Hammerhead ribozyme (HamRz), delta hepatitis ribozyme (HdvRz) and a linker contained nine unique restriction enzyme sites were synthesized and introduced into the multiple cloning sties of pVAX1 vector, the modified pVAX1 vector was named pVAX-R. The four gene fragments and GFP gene were subcloned into the pVAX-R vector in tunes by restriction and ligation. The recombinant full-length genome plasmid carrying a GFP gene (pCTN-GFP) and flanked by HamRz and HdvRz was constructed, and was subject to sequencing. The results of sequencing showed that the nucleotide sequence of the recombinant genome was consistent with that of rabies virus of CTN strain and the GFP gene published in GenBank, which demonstrated that the construction of the full-length genome plasmid was successful, and it is ready for virus rescue.A set of specific primers were synthesized and used to amplify N, P, L gene, RT-PCR products of each gene were cloned into pVAX1 vector to form three helper plasmids (pVAX-N, pVAX-P, pVAX-L) involved in viral transcription and replication. The helper plasmids were subject to sequencing. The results of sequencing showed that there were no mutations in these three genes compared with its parental virus. pVAX-N was transfected into BHK-21 cells, at 3 days post-transfection, cells were detected by direct immunofluorescence assay (DFA) using FITC-labeled rabies virus N protein-specific monoclonal antibody. The results showed that transient expression of N protein was detected in cells and pVAX-N plasmid could be used for virus rescue.BHK-21 Cells were transfected with pCTN-GFP, pVAX-N, pVAX-P, pVAX-L using liposome, after 4 days, MNA cells were inoculated with the supernatants and cells from co-culturing of the transfected BHK-21 cells for virus propagation, 3 days later, cells were detected under a fluorescence microscope. The results showed that the expression of GFP was detected in infected MNA cells. Total RNA and DNA were extracted from the MNA supernatants, respectively. RT-PCR and PCR were performed with a set of specific primers spanning GFP and L gene. The results showed that RT-PCR using genomic RNA of the rescued virus as a template amplified a fragment with the expected size of 890bp, and PCR using the same primers without RT step failed to produce any products, indicating that the recombinant rabies virus CTN-GFP strain was recovered from the four cloned cDNA plasmids, and the DNA band did not originate from the full-length genome plasmid used for transfection. Moreover, the recombinant virus can express GFP stably after three passages on MNA cells.10 1-day-old sucking mice and 10 4-week-old adult mice were intracellular inoculation with 104ffu/ml the recombinant rabies virus CTN-GFP strain at 20μl/per-mouse. The results showed that all the sulking mice inoculated with CTN-GFP virus developed neurological symptoms, and died. Rabies virus antigen was detected in tissue from one dead sulking mouse brain by the DFA test. However, the adult mice were not dead until 15 days post-inoculation, the result of DFA test of tissue from one adult mouse brain is negative. All the results above indicated that the recombinant rabies virus CTN-GFP strain has the similar biological property with the parental virus, which is pathogenic to sulking mice and nonlethal to adult mice.更多还原