【作者】 姜延龙；

【导师】 孔宪刚；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2009， 博士

【摘要】 反向遗传技术是近10年来迅速发展起来的一种病毒学研究方法,在研究病毒基因功能和研制新型疫苗方面有广阔的应用空间。新城疫病毒反向遗传系统的成功建立始于1999年,迄今为止已成功拯救的病毒株包括La Sota、Clone30、BC、ZJ1等,但V4株的反向遗传拯救系统尚未见报道。新城疫V4株是1966年澳大利亚学者Simmons分离到的自然弱毒,该毒株是自然无毒株,主要介导细胞免疫,最大优势在于其独特的耐热性,更适合在发展中国家推广使用。本实验首先对实验室保存的V4株进行了三轮的蚀斑纯化,由于V4株属无毒株,其在CEF细胞中很难形成可见的蚀斑,因此我们采用了用胰酶处理V4病毒,并在上层胶中添加尿囊液及Mg2+,这样可以偶尔产生可见的蚀斑,但实验结果重复性很差。得到的各蚀斑克隆株经56℃水浴耐热实验及-20℃反复冻融实验,比较它们的血凝性变化趋势,选择耐热及耐冻融性状更好的克隆株;实验表明,各克隆株之间耐热性存在明显差异,说明保存的V4毒是由不同的克隆株混合在一起的混合体,选择耐热能力最强的11号毒株进行后续实验。同时,对各克隆株F基因内部47-435bp的片段进行扩增、测序,比较序列后表明完全相同,说明保存的V4毒并不存在杂毒污染。设计了13对引物对全基因进行了扩增及测序,并最终获得了V4株全基因序列。序列分析表明V4株基因组全长15186nt,3’端和5’分别存在55nt的leader区和114nt的trailer区,中间部分包括NP、P、M、F、HN及L基因,各基因又由两端的非编码区和中间部分的开放性阅读框(open reading frame,ORF)组成。对各结构蛋白的基因进行了同源性分析及进化树分析,在参比的各毒株中NDV V4株NP、P、F和HN基因都是与QV4株的同源性为最高,分别为99.9%、96.7%、100%、99.8%,M基因与HB92株同源性最高,为99.4%,与QV4株为96.8%;L基因与已知的毒株比较,与HB92同源性最高,为99.4%。设计10对引物分别扩增各个cDNA片段,并最终连接为全长cDNA质粒pFT7-final,使得cDNA片段连接于T7启动子之后;同时构建了polII启动子及NDV特异性核酶控制下的全长cDNA克隆pFpolII-V4-final。在基因组3166bp处设计引物,利用融合PCR技术插入PmeI酶切位点,并在该位点插入了传染性法氏囊病病毒(IBDV)的主要抗原蛋白VP2的开放阅读框,构建了T7启动子控制下的拟表达VP2抗原的V4株全长cDNA克隆pBRT7-VP2。设计引物扩增了V4株3’leader区及5’trailer区,经融合PCR进行融合后与原始载体pPolII-BDV通过NgoMIV、SfiI酶切位点连接为pBR-LT;扩增了绿色荧光蛋白(EGFP)的开放阅读框并利用两末端的ClaI、SphI酶切位点,将其反向插入到pBR-LT中,构建了V4株微基因组pBR-EGFP。pBR-EGFP与辅助蛋白pCIneo-NP、pCIneo-P、pCIneo-NP以及负责产生T7聚合酶的质粒pCAGGS-T7共转染BHK21细胞,可见很强的绿色荧光表达,说明辅助质粒及pCAGGS-T7均可正常工作;通过与pEGFP-N1对照做了对比实验,多质粒共转染的效率约10%左右;而在pEGFP-N1单质粒转染组,转染效率可达到约40-50%,说明共转染的效率较低;以La Sota、V4及F48E9为辅助病毒进行了病毒包装实验,结果表明依靠微基因组两末端的非编码区序列,微基因组可被辅助病毒包装为缺陷型病毒,说明末端序列在病毒的包装过程中发挥了一定作用。虽然最终经过多次重复拯救实验,我们没能获得V4株的救获病毒,但目前已完成的工作已为V4株反向遗传体系的建立打下了坚实基础,在拯救体系及条件上进行改进后,有望于近期拯救成功。本实验首先通过蚀斑纯化的方法对保存的V4毒株进行了三轮纯化,比较了不同克隆株间耐热性及耐冻融性的差异,选择了综合性状更优良的克隆株;完成了该克隆株的全基因组序列的测定,分析了主要结构蛋白NP、P、M、F、HN和L的基因序列,比较了与其他毒株的核苷酸、氨基酸同源性及进化树分类结果,表明保存的V4株是QV4株的传代毒。构建了三个全长cDNA克隆,分别为T7启动子控制下的pFT7-final,II型启动子控制下的pFpolII-V4-final,以及在pFT7-final中插入了IBDV的VP2基因的嵌合cDNA克隆pBRT7-VP2;构建了V4株微基因组pBR-EGFP,共转染实验表明拯救体系及辅助质粒均正常工作;辅助病毒包装实验表明V4株3’leader区及5’trailer区在病毒的包装过程中发挥了一定作用。更多还原

【Abstract】 Reverse genetics technology, explored widely in virus genetic function research and chimeric vaccine , has got dramatic development in the last 10 years. The first recovery system for Newcastle disease virus(NDV) was constructed successfully in 1999, and nowadays a few rescue systems including La Sota,Clone30,BC,ZJ1 have been reported, but not V4 strain. V4 strain was firstly isolated by Australian scientist Simmons in 1966, which was natural avirulent strain and mediated cell immunity primely, the superiority of its unique thermostable characteristic makes it possible to be vaccined in developing countries.Firstly , the V4 stock was purified three times by plaque formation. It’s difficult to see clear plaques in CEF cells due to the avirulent characteristic , so we treated the V4 stock with trypsin and then allantoic Fluid and Mg2+ were added in the layer gel. In this case, we could see small plaques occasionally, but not stably. Then we treated the single clone strains with 56℃water bath and -20℃freezing and thawing, and heattolerant and coldtolerant strain was selected for further experiments. The results showed that there were some differences among these clones ,which indicated that our V4 stock was a mixture virus, and we selected the number 11 clone . Meanwhile, the 47-435bp fragment of different clone strains were amplified and sequenced ,the results indicated that the V4 stock was relatively purified.The whole genome sequences of V4 strain were obtained using 13 pairs primers.The results showed that the genome length was 15186nt,including 55nt 3’leader region ,114nt trailer region and NP,P,M,F,HN,L gene, each gene was composed of non-coding sequences and open reading frame(ORF) individually. The analysis of homologous genes and phylogenetic tree suggested that NP,P,F and HN gene was most homogenous with QV4 strain , 99.9%,96.7%,100%,99.8% individually, and M and L gene were most homogenous with HB92 strain both 99.4%.10 pairs primers were designed for the construction of complete cDNA clone pFT7-final, in which the cDNA fragment was under control by T7 promoter. Meanwhile we constructed additional plasmid in which cDNA was controlled by polII promoter. Besides, fusion PCR was explored to introduce a PmeI restriction enzyme site into the whole cDNA fragment in 3166bp, and the VP2 ORF of IBDV was inserted into pFT7-final by PmeI site, named pBRT7-VP2.The 3’leader and 5’trailer region was amplified and fused together ,then was linked with pPolII-BDV by NgoMIV and SfiI site to construct pBR-LT; the ORF of EGFP was amplified and inserted into pBR-LT to construct minigenome plasmid pBR-EGFP. After that, BHK21 cells were transfected by pBR-EGFP、pCIneo-NP、pCIneo-P、pCIneo-NP and pCAGGS-T7, the expression of EGFP indicated that our rescue system and plasmids worked well.In order to compare the transfection efficiency of co-transfection with single plasmid transfection, we selected pEGFP-N1 as a positive control.The results showed that the transfection efficiency of each group was about 10% and 40-50% respectively. The packaging experiments with helper virus La Sota,V4 and F48E9 strain showed that the minigenome could be packaged by these virus to generate defficienct virus particles, indicating the leader and trailer region played an important role in packaging process.Although we have tried several times to rescue V4 strain in both BHK21 and BSR T7-5 cells, we still couldn’t get rescued viruses. What we have finished nowadays should have based some reliable prime work for the successful recovery in future days.Our experiments purified V4 stock by plaque formation firstly three times ,and then compared the thermostable characteristic of different clone for future experiments. We sequenced the whole genome sequences of V4 clone and analysed the gene composition including NP,P,M,F,HN and L,then we compared them with other NDV strains for homogenous and phylogenetic tree, the results indicated that our V4 strain was mostly closed to QV4 strain genetically. Three complete cDNA clones named pFT7-final、pFpolII-V4-final and pBRT7-VP2 were constructed respectively; We also constructed the minigenome system for V4 strain named pBR-EGFP, further co-transfection suggested that our rescue system work well at the same time; Packaging experiments mediated by helper virus indicated that 3’leader and 5’trailer region were important in packaging progress.更多还原