【作者】 程坚；

【导师】 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2001， 博士

【摘要】 H9亚型禽流感病毒（AIV）近年在我国部分地区广泛流行，给养禽业造成了严重的经济损失。油乳剂全病毒灭活苗虽对该亚型禽流感的控制发挥了重要作用，但由于其存在干扰免疫监测及成本较高等缺点，使用受到了限制。为了研制新型禽流感疫苗以克服上述缺点，我们对H9亚型AIV中国分离株的血凝素（HA）基因和鸡Ⅱ型干扰素基因（IFN-Ⅱ）进行了克隆、测序和分析，构建了表达HA基因及共表达HA基因和鸡IFN-Ⅱ基因的两种重组鸡痘病毒（rFPV-HA、rFPV-HA-IFN-Ⅱ），并测定了它们的免疫效力。1 H9亚型AIV中国分离株HA基因cDNA的克隆及序列初步分析 以酚-SDS法提取H9N2亚型AIV中国分离株A/Chicken/China/F/1998（简称F株）的基因组RNA，以此为模板，采用cDNA合成法及反转录聚合酶链式反应（RT-PCR），扩增到长度分别为1.1Kb和0.8Kb的两段HA基因片段。测序结果表明，这两个片段覆盖了HA基因的完整阅读框架，且在它们的重叠区有单一的PstⅠ酶切位点。利用这一酶切位点，将这两个片段连接成插入在质粒pUC18的HindⅢ和SalⅠ位点之间的全长HA基因，得重组质粒pUCHA。测定pUCHA位于HindⅢ和SalⅠ位点之间外源片段的核苷酸序列。 本研究获得的HA基因片段共有1708个核苷酸，包含了HA完整的阅读区的1680个核苷酸，推测其编码560个氨基酸，其中前18个氨基酸为信号肽序列，其余542个氨基酸组成的前体蛋白在第320位氨基酸处被切割为HA1和HA2多肽。F株HA裂解位点的氨基酸序列为RSSR↓GLF，不含连续的碱性氨基酸，具有低致病性AIV HA裂解位点的特征。F株与H9亚型AIV原型毒株A/Turkey/Wisconsin/1/66（H9N2）的HA基因的同源性较低，氨基酸和核苷酸的同源性分别为84.1％和82.9％。n 扬州大学博士学位论文 以 HAI CDNA的 55－1004 n酸为基元，建立 1992－1999年间 HgNZ亚型 AIV中国及绷边国家和地区分离株的遗传发生树。结果表明，近年来在这一地区流行的HgNZ亚型AJV招可敞为三憎系，同一谱系内，毒株间HA艄酸的同源性在95％以上。F株与髓地区的部分俯株及94年北京分离株的亲龈系较近，形成谱系卜 香港地区的另一些分离株和巴基斯坦分离株形成谱系2，它们与某些南美分离株的亲缘关系很近；与谱系1的亲缘关系也较近，它们之间HA核昔酸的同源性在 gi％R右；韩国分离株形成的谱系 3，该谱系与谱系 1、二的亲缘关系很远，它们之间HA核昔酸的同源性只有83％左右。2 表达HA辜因的直组鸡痘病霉的构巨 以 Hnd皿及 Sal消化PUCHA，得到 1．7Kb的 HA基因片段，将n向插人FPV插入载体 11 75的 Hnd Ill和 Sal位点之间，使之处于痘苗病商启动子 P7．5的下游，得到转移删 11 75HA。按常规的B眠脓染方法将其转染至已感染 FPV中国疫酣282E4（Wt郴）的鸡胚成纤测胞（CEF），通过筛选蓝色的病毒腑获得并纯化重组鸡痘病毒uV－HA。以PCR ＄＄1；ill实，dq，v－HA基因组中插有HA基因：以间接兔疫荧光肌实，fPV－HA感染的C扭釉了HA。3 表达鸡IFN－H羹因的匣组鸡痘病羹的钩 来达产物的生物活性 根据已发表的鸡 WN0基因序列，设计一对5！物，以 RTPCR方法从经 Con A刺激的鸡脾脏细胞中扩增出全长的鸡IFN二基因的cDNA序列。将扩增的基因片段定向插人质粒 AF09的 Sal和 Hnd IH位点之间，使其位于痘苗病毒早晚期启动子PEI的下游。 根据启动子 P肌的序列设计上游引物，鸡 IFNl基因的 cDNA 3 M列设计．下游5！物，以PCR法扩增包含痘苗病毒早晚期启动子PM在内的鸡IFN刁的基因片段。经一系列步骤，将扩增片段定向插人 FPV JWA载体 11 75中，得到转移载体1175IFN，用以转染已感染tFPV的CEF，并按上述重组病毒的构建和纯化方法，获得插谰 rw－n基因的靴重赈毒 ev－r：w－11。在 rrnv－lrx－11中，rw－11基因处于PDe的转录调控下。 以细胞病鲫制W侧－IFNI感染的CEF越了具有抗水泡性口炎病毒（VSV）活性的鸡IFN刁。fPV－mI（M．O．H．m）感染CEF 72小时后，培养上清中 IFN刁的表达量为 1280 U／ITl。动物硼表明，接种 rFPV－IFN刁 天或14天后，其表达的 IFNJ可显著减轻载体鸡痘病毒对 1日龄 SPF tills｛＄重增长的 程坚：表达和共表达Hg亚型肖流感病毒HA基因和鸡！FN坝基因的重组鸡痘病毒 ＊ 抑制作用。 4A表达HA墓因和鸡HN－11基因的直组鸡扈清羹的构建 经一系列步骤，将IFN－11的基因及其上游的启动子PM插人含全长HAcDNA 的质粒pUCm中，得重组质粒PHAIFN。在PHAJFN中，HA基因和IFN刁基因 的3’糊邻。将串联在一起的HA基因和IFNJ基因（包含上游启动子Py）片 段切下，将其定向插人 FPV e载体 11 75中，得到转移狲 11 75HAIFN。在 11 75HAff：N中，HA基因和 IFN。11基因分别处于启动子 P7．5和 PWh的转录控制下， 且转录方向正好相对。在IFN－11基因3’端有一痘苗病毒转录终止子，可避免这两 个基?更多还原

【Abstract】 H9 subtype avian influenza virus (MV) is widely distzibuted and has resulted in considerable economic loss to poultry industiy in China in recent years. In order to protect poultry against this low pathogenic subtype of API, inactivated vaccines in oil emulsion have been developed and proved to be effective in reducing severity of disease and spread of MV in field situation. However, the use of inactivated vaccines was restricted because of the disadvantages they inherited, including higher cost and interference of routine surveillance by serological test Here, we describe the construction of recombinant fowlpox viruses, rFP V-HA and rFPV-HA-IFN- II, expressing hemagglutinin (HA) from subtype H9N2 of API and coexpressmg the HA and chicken type II interferon(IFN- II) respectively, and their protective efficacies against homologous challenge in chickens for the development of better vaccines.1.Cloning and analyzing the HA cDNAThe genomic RNA of H9N2 subtype AIV AlChickenlCbina/F11998 (F strain for abbreviation), was extracted by phenol-SDS. When the purified genomic RNA was used as template for cDNA s~thesis and RT-PCR with two pairs of primers, two overlapping partial segments covering the complete HA cDNA, having the length of 1.1Kb and 0.8Kb nucleotides respectively, were obtained. Sequencs of the two eDNA segments showed that there was a unique Pst I site in the overlapping region of these two segments. By use of the unique Pst I site, we ligated these two segments into the complete HA cDNA, which was cloned in the recombinant plasmid pUCHA.vi~H )~f#眫{~The cloned HA gene was 1708 nucleotides long with a single open reading frame of 1680 nucleotides by sequence analysis. The open reading frame encoded a polypeptide of 560 amino acids, which included the signal peptide of the initial 18 amino acids at the N-terminal and the remaining segment of 542 amino acids. The latter was cleaved into two peptides, HAl and HA2, at the 320 amino acid site. The amino acids ofF strain at the cleavage site were RSSR ~ CILF, revealing the typical cleavage site features of low pathogenic AIV Compared with the H9 prototype AJV strain, A]Turkey/Wisconsin/1/66, the HA gene ofF strain shared 82.90/a sequence homology with that of it.According to the phylogenetic tree established for the H9N2 AJV isolates in China and neighboring countries and regions from 1992 to 1999, these isolates formed three lineages. F strain, the isolate from Beijing in 1994 and some 9 isolates from Hong Kong in recent years formed the first phylogenetic lineage, which showed significant phylogenetic difference with another lineage consisting of Korea isolates. The third lineage, also formed by isolates from Hong Kong, bad close phylogenetic relationship with the first lineage.2.Construction of the rFPV expressing HA (rFP V-HA)For the construction of rFPV-HA, the HA gene, removed from pUCI-IA as Sal I-Hind III fragment, was directionally inserted into the plasmid 1175, resulting in the transferring vector 11 75HA, in which the HA gene was under the transcriptional control of the vaccinia promoter P7.5. Then the transferring vector 11 75HA was used to transfect the chicken embryo fibroblast cells (CEF) pre-infected with wild type FPV (wt-FPV). The recombinant viruses were obtained and purified by blue plaque selection. The presence of HA gene in the genome of the purified recombinant virus was assayed by PCR and the expression of HA in the rFPV-HA-infected CEF was detected by indirect immunofluorescence.3.Cloning and expressing the chicken IFN- II geneA pair of primers were designed on the published data to amplify chicken WN- II gene with the Con A-stimulated chicken spleen cells. The cloned WN- II gene was then directionally inserted into the Sal I-Hind LII site of the plasmid pAFO9, resulting in the recombinant plasmid AWN, in which the IFN- II gene was under the control of theH9 *5~~ HA ~ I N-ll~ Iffl~1~ viivaccinia promoter PE/L.For the amplification更多还原