【作者】 刘岳龙；

【导师】 崔治中； 秦爱建；

【作者基本信息】 扬州大学 ， 传染病与预防兽医学， 2001， 博士

【摘要】 鸡贫血病毒（Chicken anaemia virus，CAV）是引起雏鸡再生障碍性贫血、淋巴组织萎缩和成年鸡免疫抑制的鸡贫血病的病原。尽管各地分离毒株之间的序列同源性很高，但也有差异。本文对中国分离株的基因组DNA进行了PCR扩增和序列测定比较。首先对中国5株不同来源的CAV疑似病料（YZ9903、HA9805、JN0007、JL0010、KM9801株）用CAV全基因组核酸探针进行了斑点杂交，验证为阳性后，将病料组织DNA和病料感染细胞（MSB1细胞）DNA进行CAV基因组细胞凋亡素基因片段（0.35kb）的PCR扩增和序列测定，结果发现5个中国毒株的VP3基因序列完全相同，也与美国CIA-1株序列完全一致；与德国CUX-1株相比有一个核苷酸的差异（第674位C→T），但和其它参考毒株的这一位置的核苷酸一致，都为T；与日本TR20株、澳大利亚CAU269-7株序列的同源性也很高。所有毒株VP3序列有一个明显的特点，即核苷酸的差异仅出现于VP3编码基因（366个核苷酸）的前158个核苷酸区域，后208个核苷酸则没有变化。目前已知的CAV VP3的编码氨基酸（121个）有共同的氨基酸序列特征，即含有2个脯氨酸（Pro）丰富区域和2个强碱性氨基酸—精氨酸（Arg）和赖氨酸（Lys）的密集区域。上述CAV毒株之间VP3基因区域核苷酸变异所导致的氨基酸变异都没有引起这些重要结构区域的变化，这也再次证明了VP3与功能密切相关的二类氨基酸区域的保守性。 在体外扩增中国分离株HA9805株和JN0007株的全基因组时，将完整的基因组序列分为1.0kb、0.6kb、和0.8kb三段分别扩增出来，并成功地对CAV基因组1.5kb（1.0kb、0.6kb二段）测定了核酸序列。HA9805株和JN0007株1.5kb序列和已知的参考毒株（CUX-1株、CIA-1株、TR20株、CAU269-7株）相应序列相比，同源性很高（95％以上）。HA9805毒株和CUX-1毒株序列同源性最高（98.6％），JN0007毒株与TR20毒株序列同源性最高（98.5％），CAU269-7毒株与其它所有的毒株的同源性都相对较低。在已测定的基因序列中，编码区核苷酸变异很少，尤其是VP2编码区域的后半部分极为保守。核苷酸变异在其余的区域则是随机分布。基因编码区内的大多数的核苷酸变化并没有导致氨基酸的改变，即为无义突变，只有少量的核苷酸的变异导致了相应氨基酸的改变，这些氨基酸的改变对CAV的编码蛋白高级结构的影响有多大还不清楚。但Randall等（1996）发现的VP1高变区（第139-1位氨基酸）在HA9805株和JN0007株VP1编码区也出现了。在二株中国株CAV基因组的非编码区也同样出现其它CAV共同的病毒核酸复制和基因转译所需的重要调控元件。 在前期研究中，本实验室己获得CAV病毒全基因组的体外克隆、但在病毒非编码区（也是病毒复制和转译调控区）存在不完整的EC叩1 识别序列（5’－GACTTC－3’）、因而是不能自身环化的重组子（pCAVZ．4，5．okb人 在此基础上，本研究通过 PCR定向点突变法，将 pCAVZ．4的病毒非编码区的不完整的 EcoR序列（5’－GACTTC－3’）突变为完整的 ECOR识别序列（5’－GAATTC－3’），从而有利于 CAV基因组的体外环化。将此重组子命名为 pCAVE”（5．okb人 将 pCAVE“大量扩增，用 E。觎 l切出 CAV基因组 2．3kb片段，在体外自身连接。用绿色荧光素报告基因（PEGFP）预转染 MS盯细胞，确定转染 DNA最佳的用量条件为 1．0 u g、最佳的转染细胞密度为 lx10‘个／ml。将上述 2．3kb的自身连接产物在转染剂Effectene的作用下转染CAV的宿主细胞系 （MSBIL 经过 7－8 代的连续传代，获得了明显的具复制活性的 CAV病毒。将此病毒经肌肉途径感染1日龄SPF易感雏鸡，在14＊ 天能出现典型的病变。将 pCAVE”中的病毒基因组（2．3kb）插入 pCAVZ．4的单一E。叩I位点，构建了含有双拷贝CAV全基因组同方向串联的重组子（命名为PCAVZE”，7．3kb人 将此PCAVZE”环型质粒大量扩增并直接转染MSBI 细胞，同样获得了可复制性的病毒。本研究在病毒的非编码区特定位点进行点突变，构建了含有单拷贝CAV全基因组和双拷贝全基因组同向串联的重组子，并在MSBI 细胞成功转染为具复制活性的完整病毒。本研究思路和实验方法为将来在基因组水平体外构建低或无致病性的CAV活病毒的研究打下了基础。 本研究将 CAV基因组的三个编码蛋白衣壳蛋白（VPI＊ VPZ、细胞凋亡素（或称 VP3）的编码基因，按正确的插入方向、译读框架分别插入在原核表达性载体 PGEX－SX－3 中谷脱甘肽转移酶（GST）编码基因之后，经过酶切分析、序列测定证实了所构建的重组子的正确性。完整的VPI基因、及VPI大片段（缺失VPI的N－端21个氨基酸和C－端4个氨基酸的VPI）插入后未获得明显表达。将VPI大片段再分为VPI片段 1（672hP）和片段2（608hPL 分别插入载体后获得了表达。完整的仟 和细胞凋亡素基因插入载体后也成功地获得了表达。将所表达的融合蛋白经过SDS－PAGE电泳后分别回收，直接多次免疫小鼠，制备了相应的鼠源抗血清，与CAV感染的MSBI 细胞作间接免?更多还原

【Abstract】 Chicken anaemia virus (CAV) is a virus causing anaemia accompanied by aplasia of the bone marrow and atrophy of the lymphoid organs in young chickens and a transient severe immunodeficiency in older chickens. In this study ,five Chinese CAV isolates were detected positively using dot-blot hybridization with CAV genomic DNA probe. The VP3 genes (0.35kb) of the five Chinese isolates were amplified and sequenced . The VP3 sequences were all identical to the VP3 sequence of CIA-I strain (U.S.A.), there was only 1 nucleotide different from CUX-I strain (Gremany ) (C棐~T at 674nt) , but same to TR2O strain (Japan) and CAU269-7 strain (Austrailia) .The sequence differences in VP3 gene were restricted to the 5?terminat (158bp) region, the 3?terminal sequences were conserved in different CAV isolates. VP3 was a protein of 121 animo acids containing two proline-rich stretches and two Arginine-rich basic regions. The changed nucleotides were not affect those important functional regions.Three fragments (1.Okb,0.8kb,0.6kb) which were included full length genomes were amplified by polymerase chain reaction (PCR) from the genomic DNA of two Chinese isolates ,HA9805 strain and JN0007 strain. The 1.5kb sequence (including 1.0kb and 0.6kb fragments ) wered compared between the different CAV strains. Two Chinese isolatesHA9805 and JN0007, demonstrated overall nucleotide sequence homologous to each of CUX-1,CIA-1,CAU269-7,and TR2O strains (more than 95%). A phylogenetic analysis results indicated that CAU269-7 wasLf~phylogenetically distinct from all other isolates ,HA9805 and JN0007 appeared to be most closely related to CUX-l and TR2O respectively. The sequence coding C-terminal of VP2 (also including VP3 gene) was very conserved. The variations of nucleotides were located on other regions randomly . The most nucleotide variations were synonymous. However the biological characterization of the non-synonymous nucleotide changes was still not clear. The noncoding region of genome of HA9805 and JN0007, also contained motifs considered to be involved in DNA replication ,translation and transcriptional activity which were reported in other CAV strainsA recombinant plasmid (pCAV 2.4,5.0kb) which contained a incomplete EcoRI site (-GACTTC-) in the non-coding region and couldn抰 be re-circlized. In order to obtain infectious clone, CAV genomic DNA was amplified by PCR using the pCAV2.4 plasmid DNA as template and cloned into pUC18 vector (pCAVE~,5.0kb). The incomplete EcoRL site (-GACTTC-) in pCAV2.4 was point-mutated into the complete EcoRI site (-GAATTC-) in pCAVE~. The 2.3kb CAV genomic DNA was extracted from the recombinant pCAVE~ digested with EcoRI, then re-ligated, and transfected the host cell line (MSBI). After 8 passages ,the cell culture medium didn抰 turn yellow. The supernatant was inoculated into 1 day old SPF chickens . Typical anaemia and thymus atrophy were shown after 14-18 days post infection. However the circular pCAVE~ plasmid DNA, which contained single copy of CAV RF genomic DNA ,couldn抰 produce reproductive virus after being directly transfected MSB1 cells.The 2.3kb CAV genomic DNA extracted from pCAVE~, when was inserted into the pCAV2.4 at EcoRJ site, a plasmid pCAV2E + (7.3kb ) was constructed, that contained cloned tandemly-repeated CAV genomic DNA. Infectious virus was recovered from MSB1 cells transfected with the circular pCAV2E~ plasmid DNA. These results indicated that the re-ligated genomic DNA (2.3kb) and the tandemly-repeated CAV RF fragments were all capable to produce infectious virus following transfection into MSB1 cells. These results will be very useful in the further study on construction low or non-pathogenicity virus by mutation on genome.Expression of three virus-encoding proteins in vitro were studied. The whole length VP1 gene and the large fragment of VPI without N-terminal 21 amino acids and C-terminal 4 amino acids ,which were inserted into pGEX-5X-3 vector following GST gene in correct orientation, were not expressed in E coli. by更多还原