【作者】 郝瑞峰；

【导师】 关平原； 张七斤；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 本试验参考禽呼肠孤病毒S1133株L1基因序列(GenBank),禽呼肠孤病毒1733株L3基因序列(GenBank)分别设计3对特异性引物,以禽呼肠孤病毒内蒙古分离株C-98株和天津分离株T-98株提取的总RNA为模板,采用RT-PCR方法分别扩增L1基因和L3基因的cDNA ,获得的cDNA连接到PMD19-T载体中,并进行克隆,经PCR和限制性内切酶切鉴定后的阳性重组质粒送宝生物(大连)有限公司进行测序,对测定的序列进行拼接,结果显示:L1基因cDNA全长3959bp,包含一个由3882bp组成的完整开放阅读框,共编码1293个氨基酸,且在5′端和3′端分别有21bp和56bp组成的非编码区;L3基因cDNA全长3907bp,包含一个由3858bp组成的完整开放阅读框,共编码1285个氨基酸,在5′端和3′端分别有12bp和37bp组成的非编码区,且序列已递交于GenBank数据库中,登录号分别为C-98(L1基因:EU616735;L3基因:EU616737);T-98(L1基因:EU616739;L3基因:EU616738)。序列同源性比较分析结果表明:C-98与T-98在L1和L3基因核苷酸及其推导的氨基酸都有着很高的同源性,为99.8%,99.9%;C-98和T-98与其它12株禽呼肠孤病毒分离株相比较,L1基因核苷酸同源性在85.4%～99.8%,推导的氨基酸序列同源性在96.4%～99.8%;L3基因核苷酸同源性在72.6%～99.9%,推导的氨基酸序列同源性在83.1%～99.8%。C-98和T-98 L1基因与3株哺乳动物呼肠孤病毒分离株L3基因核苷酸同源性在43.9%～44.5%,其推导的氨基酸序列同源性在43.0%～43.2%;C-98和T-98 L3基因与3株哺乳动物呼肠孤病毒L2基因核苷酸同源性在4.7%～5.0%,其推导的氨基酸序列同源性在26.6%～27.4%。进化树分析显示:L1基因分出4个不同的谱系,L3基因分出3个不同的谱系,且各谱系之间与致病性、血清型和分离地区没有相关性,但是C-98、T-98、S1133、1733、2408、919和T6始终在L1基因和L3基因进化树的同一个谱系中。氨基酸序列分析表明:C-98和T-98及12株参考毒株在L1基因靠近N末端有一个独特的亲水区,位于1aa～110aa,在亲水区内还存在一个可变区,位于19aa～51aa,在肽链的182aa～202aa位存在一个锌指结构基序C2H2;L3基因在169aa与188aa位点有鸟苷酸转移酶(即加帽酶)所必需的赖氨酸(Lys)残基,822aa-830aa具有SAM(S-腺苷-L-甲硫氨酸)结合位点,379aa-386aa存在ATP/GTP结合基序A。更多还原

【Abstract】 Three pairs of primers were designed for gene L1 and L3 according to the sequences of avian reovirus strain S1133 and strain 1733 in GenBank respectively, and the cDNA of the two genes were amplified by RT-PCR with the total extracted RNA of the Inner Mongolia isolates C-98 and the Tianjing isolates T-98. The PCR product were ligated with PMD19-T vector and cloned, the recombinant plasmid obtained were certified by PCR, restriction endonucleases analysis and sequenced. The result showed that the cDNA of L1 gene consists of 3959 bp including a whole open reading frame which codes 1293 amino acids with 3882 bp and two non-coding regions, 21bp and 56bp, at 5′end and 3′end respectively; and the cDNA of L3 gene, 3907bp, also includes a whole open reading frame, 3858 bp, which codes 1285 amino acids and a 12bp and 37bp non-coding region at 5′end and 3′end respectively. The nucleotide sequences of L1 and L3 genes reported in this study have been submitted to the GenBank database, the accession numbers of L1 and L3 genes include:C-98 L1(EU616735);C-98 L3(EU616737);T-98 L1(EU616739);T-98L3(EU61673 8).The analysis of the sequence homology showed that there were high homology both in nucleotide sequences and the deduced amino acids sequence of L1 and L3 gene between C-98 and T-98, 99.8% and 99.9% respectively; compared with other 12 ARV isolates, the homologies of the nucleotide sequence of L1 gene ranged from 85.4% to 99.8% and the deduced amino acids sequences ranged from 96.4% to 99.8%; the homologies of L3 gene were between 72.6% and 99.9% and the amino acids sequence were between 83.1% and 99.8%. The nucleotide sequence homologies of the gene L1 of C-98 and T-98 and L3 gene of other 3 MRV isolates were between 43.9% and 44.5% and the amino acid sequence were between 43.0% and 43.2%; while the nucleotide sequence homologies of the L3 gene of C-98 and T-98 and the L2 gene of other 3 MRV isolates were between 4.7% and 5.0% and the amino acid sequence were between 26.6% and 27.4%. Phylogenetic analysis of L1 and L3 genes revealed that L1 genes have diverged into four distinct lineages and L3 genes have diverged into three distinct lineages, and that there was no correlation between lineages and viral serotypes, pathotypes and origins, but C-98, T-98, S1133, 1733, 2408, 919 and T6 contained the L1 and L3 genes which were consistently located in the same respective lineages, the remaining virus strains demonstrated reassortment of either one of L1 or L3 genes. Analysis of amino acid sequence of L1 and L3 gene of C-98, T-98 and 12 virus strains showed that the amino acid sequence of L1 gene possesses a variable region (residues 19～51) located within a conserved hydrophilic region (residues 1～110) and a C2H2 zinc-binding motif(residues 182～202), and the amino acid sequence of L3 gene possesses two conserved K residues at positions 169 and 188 indicative of guanylyltrans- ferase activity, a conserved S-adenosyl-L-methionine-binding motif (residues 822-830), and an ATP/GTP-binding site motif A (residues 379～386).更多还原