【作者】 王键义；

【导师】 郭万柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 本实验提取了四川农业大学动物生物技术中心保存的Fa株、疫苗株783株、Bartha株和SA215株,四川野毒株SN株、SS株、SQ株、SL株和SCZ株,以及由韶关学院英东生物工程学院娄高明教授惠赠的北京BJ株、广东DG株、湖北HS株共计12株猪伪狂犬病病毒株的DNA并作为模板,应用PCR技术分别扩增得到了12条伪狂犬病毒完整的gC基因的片段。回收PCR产物,成功连接转化到感受态细胞大肠杆菌DH5α中,并通过菌落PCR和酶切鉴定正确后,送生物公司进行测序。将所得12条序列连同GenBank中登录的6条gC基因全序列共18条基因序列,使用生物软件对gC基因核苷酸序列的同源性、突变区域的定位、遗传进化关系、酶切位点的差异、密码子偏爱性、氨基酸序列的同源性、蛋白质亲水性、抗原表位分析、二级结构和三级结构预测等生物信息学内容进行分析。结果表明:PRV-gC基因的开放阅读框的核苷酸长度在1437bp～1464bp之间,氨基酸长度在478～487个之间,核苷酸同源性在94.9%～100%之间,氨基酸的同源性在90.2%～100%之间;在核酸175～222位存在高变缺失区,其中共有8个碱基突变和21个碱基的插入或缺失;在遗传进化关系上,四川地区流行的毒株与日本、欧美毒株有较近的亲缘关系,而我国其它地区的毒株间的遗传相关性较高,但与日本、欧美洲毒株亲缘关系较远;在18条PRV-gC基因核苷酸序列中均没有发现HindⅢ酶切位点,同时Fa株、DG株、SA215株、BJ株、Min A株和Ea株也没有BamHⅠ位点,Yamagata S-81株则没有SalⅠ位点,其它酶切位点出现的次数和位置都有细小差异;而不同PRV毒株gC基因均对G、C有明显的偏爱性。在蛋白质疏水性、抗原表位和二、三级结构的预测方面,不同毒株间具有相似性,但仍表现出一定程度的差异。更多还原

【Abstract】 We got DNA of Pseudorabies virus Fa strain, vaccine strain 783, Bartha and SA215, Sichuan wild strain SN, SS, SQ, SL and SCZ preservatived by Animal Biotechnology center of Sichuan Agriculture University, and Beijing BJ strain, Guangdong DG strain, Hubei HS strain donated by prof. Lou Gaoming of Shao Guan Institute Ying Dong Bioengineering College. DNA of the 12 Pseudorabies virus strains served as template, 12 strips of gC gene fragments of PRV were amplified by PCR. Reclaimed the product of PCR, ligated and allaxised into competent cell of E.coli DH5α, then sent to biology company to sequencing after accredited by colony PC R and biocatalyst cutting.The 12 strips gC gene sequences of different PRV strains, as well as 6 strips gC genes sequences which were downloaded from GenBank were analyzed by bioinformatics software. Analyzed and predicted that, such as nucleotide homology, location of mutation area, relationship of heredity and evolution, discrepancy of biocatalyst cutting site, preference of codon, homology of amino acid sequence, protein hydrophilicity, epitope, secondary structure and tertiary structure. The results showed that the length of open reading frame of gC gene was between 1437bp and1464bp, the number of amino acid encoded correspondingly was between 478 and 487. The ribonucleotide autoploidy was between 94.9% and 100%, the deduced autoploidy of amino acid sequence was between 90.2 % and 100%.There was a concentrated mutation area and a nucleotide deletion area on the 175-222 location segment of the nucleotide sequence, 8 basic groups mutated and 21 basic groups inset or absented. To analyze on the heredity and evolution relationship, the genetic relationship of the pseudorabies vims strains prevalent in Sichuan were closer to the pseudorabies vims strains isolated in Japan, Europe and America, but distant to the strains isolated in some region of our country. There was no HindIII biocatalyst cutting site in all 18 strips of gC gene, and there was no BamHI biocatalyst cutting site in Fa strain, DG strain ,SA215 strain ,BJ strain ,Min A strain and Ea strain either.Yamagata S-81 strain had no SalI site. There were tiny discrepancy on the occurrence frequency and situs of other biocatalyst cutting sites, and the gC gene of all strains had apparente preference to G and C. There was resemblance in proteo-hydrophobicity, epitope and higher structure prediction among those strains, but also had some changes and difference.更多还原