【作者】 胡建和；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2003， 博士

【摘要】 中国猪瘟兔化弱毒（脾淋毒）全长感染性cDNA的克隆和构建，可以使我们得到纯粹的CSFV C-株RNA病毒基因组，在DNA水平上研究和利用CSFV的基因突变、缺失、插入和替换。为进一步研究CSFV的复制机理、毒力及其决定因素、致弱机制、致病机理、基因产物的功能、特异性诊断、宿主嗜性以及DNA疫苗开发、标记疫苗的生产等方面的研究，打下了坚实的基础，并为众多病毒分子生物学研究工作者提供极为有价值的研究工具。主要研究内容包括： 1．中国猪瘟兔化毒（脾淋毒）基因组cDNA文库的构建、序列分析：根据已发表的猪瘟病毒（CSFV）核苷酸序列，借助计算机软件分析，选择高保守区段和基因组中的单一限制性酶切位点，利用RT-PCR及nested-PCR和Helf-nested PCR技术，成功地扩增出了覆盖C-株全基因组的7个cDNA重叠片段F1～F7，分别克隆到pMD-18T或pGEM-T Easy载体进行测序后，拼接出了其核苷酸序列。与国内外已发表的CSFV Shimen株、Berscia株、Cap株、Eystrup株、F114株、Glentorf株、Riems株、Russia C株和Alfort株进行了序列比较。 2．中国猪瘟兔化毒（脾淋毒）全长-cDNA的分子克隆：通过基因改造，将T7 RNA聚合酶启动子和目的片段连接过程中起辅助作用的限制性内切酶识别位点各自导入相应的cDNA重叠片段末端。将F3、F4 PCR产物，采用Acc Ⅲ酶切后与pMD-18T载体置同一体系中连接，获得连接片段F3-F4的重组质粒pMD-18T／F34。再将重组质粒pMD-18T／F1采用NotⅠ和PvuⅠ双酶切，重组质粒pGEM-T Easy／F2采用PvuⅠ和NsiⅠ双酶切，重组质粒pMD-18T／F34采用NsiⅠ和SalⅠ双酶切，载体pGEM-5zf（+）采用NotⅠ和SalⅠ双酶切，分别回收各目的片段，于同一体系中连接，获得5′半长cDNA重组质粒pGEM-5zf（+）／F1-4，长度为6517bp。将重组质粒pMD-18T／F5采用NotⅠ和FspⅠ双酶切，重组质粒pMD-18T／F6采用FspⅠ和Csp45Ⅰ双酶切，重组质粒pMD-18T／F7采用Csp45Ⅰ和SalⅠ双酶切，载体pGEM-5zf（+）采用NotⅠ和SalⅠ双酶切，分别回收各目的片段，于同一体系中连接，获得3′半长cDNA重组质粒pGEM-5zf（+）／F5-7，长度为5940bp。最后，将重组质粒pGEM-5zf（+）／F1-4采用NotⅠ和SplⅠ双酶切，重组质粒pGEM-5zf（+）／F5-7采用SplⅠ和SalⅠ双酶切，载体pGEM-5zf（+）采用NotⅠ和SalⅠ双酶切，分别回收各目的片段，于同一体系中连接，得到基因组全长cDNA重组质粒pGEM-5zf（+）／F1-7。T-A克隆的巧妙运用和多片段一步连接法的成功运用是全长cDNA得以快速构建的主要原因。 3．中国猪瘟兔化毒（脾淋毒）全长cDNA感染性的初步鉴定：利用限制性内切酶SrfⅠ将重组质粒pGEM-5zf（+）／F1-7线性化，以产生精确的基因组3′末端。以线性化的全长cDNA为模板，体外转录得到了CSFV基因组RNA。采用TransFast^TM Reagent脂质体转染试剂将基因组RNA转染到SK-6贴壁细胞。连续传代5代以后，收集各W摘要代细胞至第10代，采用RT一PCR、直接荧光抗体染色和夹心ELISA技术进行鉴定，结果均为阳性。表明利用反向遗传技术，可以从猪瘟病毒C一株的基因组RNA获得C一株重组病毒。这一技术为众多病毒分子生物学研究领域提供了极为有价值的研究工具。更多还原

【Abstract】 Successfully cloned and constructed infectious full-length cDNA of attenuated lapinized CSFV Chinese-strain (derived from spleen) could make us get pure RNA virus genome of CSFV C-strain, and further study and utilize mutation, deletion, insertion and substitution of CSFV gene on DNA molecular level. It has made the strong basis for further studying mechanism of replication, virulence and determinant, attenuation, pathogenesis, functions of genetic products, specific diagnosis, cell and host tropism, development of DNA vaccine and marker vaccine of CSFV, and provided an excellent tool for molecular virology. Main research contents include:Based on published nucleotide sequences of CSFV and by the help of computer analysis software, high conservative regions and single restriction enzyme sites of genome were selected. Utilizing RT-PCR and nested-PCR techniques, 7 overlapping cDNA fragments covering the full genome of CSFV C-strain were successfully amplified. After 7 fragments were cloned into pMD-18T or pGEM-T Easy vectors and transformed into E.coli JM109 respectively, genomic cDNA library were constructed. Genomic nucleotide sequence was spliced after 7 fragments were sequenced. Sequence comparison with other strains of CSFV revealed that the overall nucleotide sequence homologies of self-sequenced C-strain are 95.3% with Shimen, 94.9% with Berscia, 95.1% with Cap, 96.0% with Eystrup, 95.6% with F114, 95.2% with Glentorf, 98.9% with Riems, 93.6% with Russia C-strain and 85.4% with Alfort, the overall deduced amino acid sequence homologies of self-sequenced C-strain are 96.6% with Shimen, 96.7% with Berscia, 96.2% with Cap, 97.1% with Eystrup, 97.0% with F114, 96.1% with Glentorf, 98.9% with Riems, 95.5% with Russia C-strain and 92.4% with Alfort.T7 RNA polymerase promoter and helper restriction enzyme site were introduced into 7 overlapping cDNA fragments severally using gene modification techniques. Recombinant plasmid pMD-18T/F34 of connected fragment F3-F4 had been obtained after PCR amplified fragments F3 and F4 were both digested by restriction enzyme Ace III and further connected and cloned into pMD18-T vector by T4 DNA ligase. Recovery fragments from recombinant plasmid pMD-18T/Fl double-digested by Not I & Pvu I, pGEM-T Easy/F2 by Pvu I & Nsi I, pMD-18T/F34 by Nsi I & Sal I, and pGEM-5zf(+) by Not I & Sal I were put into one ligation system to construct 5’half-length cDNA pGEM-5zf(+)/Fl-4 whose length is 6 517bp. Recovery fragments from recombinant plasmid pMD-18T/F5 double-digested by Not I & Fsp I, pMD18T-18T/F6 by Fsp I & Csp45 I, pMD-18T/F7 by Csp45 I & Sal I, and pGEM-5zf(+) by Not I & Sal I were put into one ligation system to construct 3’half-length cDNA pGEM-5zf(+)/F5-7 whose lengthis 5 940bp. Finally recovery fragments from pGEM-5zf(+)/F1-4 double-digested by Not I & Spl I, pGEM-5zf(+)/F5-7 double-digested by Spl I & Sal I and pGEM-5zf(+) by Not I & Sal I were put into one ligation system to construct full-length cDNA pGEM-5zf(+)/F1-7. artificially using T-A cloning techniques and initiated one-step ligation of multi-fragments method were the main factors in the process of full-length cDNA construction.Constructed full-length cDNA pGEM-5zf(+)/Fl-7 was linearized by Srf I so as to obtain the exact 3’terminal of genome. Linearized full-length cDNA was used as template then genomic RNA of CSFV was in vitro transcriped by T7 RNA polymerase. Genomic RNA was transfected into plating cells SK-6 by liposome transfection reagent and transfected cells were passaged 10 times continuously. Cells were collected after being passaged 5 times and used to identify infection by RT-PCR, direct fluorescent antibody, sandwich ELISA. Results indicated that integrated particles of CSFV could be obtained from constructed full-length cDNA. Successful construction of infectious full-length cDNA of CSFV C-strain has provided an excellent tool for further study CSFV C-strain on level of molecular biology.更多还原